Fermented foods: consensus statement and reviewing them

23rd March 2021

Translated from the original article in Catalan

The term “fermented foods” has been widely used but so far has not had a clear definition, there are inconsistencies related to the use of the term “fermented” and is sometimes used including more or less related products, such as probiotics. Although these foods have been consumed for thousands of years, they have recently received increasing attention among biologists, nutritionists, other scientists, and consumers.

In order to develop a definition and describe the role of fermented foods in the human diet, the International Scientific Association for Probiotics and Prebiotics (ISAPPconvened in September 2019 a group of experts to get a consensus on it. ISAPP is a non-profit organization, led by scientists and academics, and although it is funded by companies, its activities are not stipulated by industry. Its mission is to provide objective and scientific information on probiotics, prebiotics and other topics related to nutrition and health.

Figure 1. Website homepage of the International Scientific Association of Probiotics and Prebiotics (ISAPP)

These ISAPP experts have been a total of 13, from the USA, Ireland, Canada, Belgium and South Korea. Most of them are from universities and some companies, and their findings have been published recently (Marco et al 2021).

One of the main conclusions of the consensus they have reached is the definition of fermented foods and beverages: they are those made using the desired microbial growth, which involves enzymatic conversions of food components. In this work, in addition to reviewing what they are, the distinction between fermented and probiotic foods is defined, and the current knowledge about the safety, risks and benefits of these foods is revised. Finally, regulation of fermented foods and the possibility of including them in the dietary guidelines of different countries are reviewed.

What are fermented foods ?

Humans learned a few thousand years ago how to consume and make fermented foods, probably in parallel with the development of agriculture and livestock. See for example my article on fermented cheeses and milks made 7000 years ago.

The consumption of fermented foods spread and promoted from the prehistory to all civilizations especially because it is one of the most effective ways to preserve food, due to the formation of compounds that inhibit other harmful microbes and/or pathogens. These compounds produced by the microorganisms fermenting foods include organic acids (such as lactic or acetic acid), ethanol or bacteriocins. Just remember on the one hand all dairy products that can be stored longer than milk thanks to lactic acid produced by bacteria in cheeses, yogurts, etc. And on the other hand, the consumption of beers or wines as a good hygienic alternative in places and times where uncontaminated running water was not available. The greater sustainability of fermented foods is still very important today in poor regions of the world where there is not enough food security or where there is not access to electricity, refrigeration or clean water.

Other methods of preserving food are decreasing aqueous activity (aw) by: 1) adding salt or sugar or drying; 2) adding inhibitory compounds (e.g. spices or smoking); 3) vacuuming; and 4) heat treatments (cold or heat), among others.

The other reason for the consumption of fermented foods is the appearance of new organoleptic qualities, such as pleasant and different tastes, smells or textures, due to the biochemical transformations of microorganisms in the composition of the food.

Fermented foods are an important part of the human diet, even in developed regions where food safety and food preservation are well controlled. It has been estimated that more than 5,000 varieties of fermented foods (and beverages) are currently produced and consumed globally (Tamang et al. 2016).

Thus, in the above-mentioned consensus definition of ISAPP, it is very clear that fermented foods are those prepared in a way intended by humans where the activity of microorganisms is required and where they carry out a series of enzymatic reactions of the food components.

Although endogenous or exogenous enzymes, from plants, animals or other sources, may also be present in these foods, this activity is not sufficient to be considered a fermented food, as microbial activity is required.

On the other hand, the main difference with foods spoiled by microorganisms is that these foods are unwanted, and the fermented ones are deliberately made and controlled to generate desirable qualities.

It should also be pointed out that in fermented foods the action of microorganisms is not always through metabolism of fermentation. Indeed, from a biochemical point of view, “fermentation” is a type of metabolism where the energy source and both the electron donor and acceptor are organic compounds (especially carbohydrates), where ATPs are synthesized by phosphorylation of substrate (e.g. in glycolysis) and there is no ATP formation by oxidative phosphorylation with membrane-bound electron transport chains, as would be the case for respiration (Figure 2). Many classic fermented foods are made by fermentative metabolism such as lactic or alcoholic fermentations, but some are also considered fermented foods where microorganisms are doing aerobic respiration, such as acetic vinegar bacteria or molds from some cheeses. Therefore the term “fermented foods” has a broader view, independent of metabolism, where only the active and desired intervention of microorganisms is needed.

Figure 2. Main reactions of the basic mechanisms of ATP synthesis: Oxidative phosphorylation by electron transport chains (top) and Substrate-level phosphorylation (bottom).

Which are the fermented foods and the microorganisms involved ?

We find a good global relationship in the work of Tamang et al (2016), with the microorganisms involved, summarized in Table 1. Of course, this article is not an exhaustive relationship, as for example we miss the traditional Balearic “sobrassada”, although the Sicilian “soppressata” appears in the list, and probably both are related in origin. We see some of the best-known fermented foods in Figure 3.

Table 1. Types of fermented foods and the microorganisms involved (adapted from Tamang et al (2016)

Fermented milks and derivativesYoghurts, cheeses, buttermilk, kefir, kumis, leben, etc. They are from cows and also from many other mammals, such as sheep, goats, mares, camels, yaks and buffaloWorldwideLactic acid bacteria (LAB)
Some bifidobacteria
Some yeasts
Molds (Penicillium)
Fermented vegetable products (fruits, stems, bulbs, leaves, roots, legumes)Olives, sauerkraut, kimchi, various pickled vegetables (radishes, aubergines, onions, carrots), fermented cassava, soy products (sauce soy, miso, natto, tempeh).
Wines (see below)
Bacillus and other Firmicutes
Some molds (Rhizopus)
Other fermented vegetable derivativesVinegar
Fermented tea (i.e. kombucha) and fermented cocoa to make chocolate
WorldwideAcetic acid bacteria
Fermented meatSausages such as chorizo, pepper, sausages, sobrassadaWorldwide, especially EuropeLAB
Other Firmicutes
Some yeasts 
Fermented fish and saucesVery diverse, i.e., nuroc mamnam plaGarumEast and Southeast Asia
Ancient Rome
Other Firmicutes
Other bacteria
Alcoholic beverages from cereals, produced with fungal amylolytic culturesSakeJapanAspergillus oryzae
Alcoholic beverages from cereals, produced with human salivaChichaSouth AmericaSaliva
LAB and others
Alcoholic beverages from malt, germinated cereal grain (mainly barley, wheat)BeerWorldwideYeasts
Some lactic acid bacteria 
Alcoholic beverages from plant partsPulque from agaveMexicoLAB
Alcoholic beverages from fruitsVine winesAll regions of temperate climateYeasts
Oenococcus (malolactic fermentation)
Alcoholic beverages from honey Mead
Especially ancient world

As we see in Table 1, the main microorganisms in many fermented foods, from milk to meat and vegetables and others, are lactic acid bacteria (LAB). They are gram-positive bacteria of the phylum Firmicutes (DNA with low G+C), non-spore-forming, aerotolerant anaerobes, and are considered safe. Other bacteria responsible for some fermented foods are Bacillus (sporulated Firmicutes), other Firmicutes such as Staphylococcus, and bacteria of the phylum Actinobacteria (gram-positive with DNA of high G+C) such as BifidobacteriumPropionibacterium and Brevibacterium. Among the few gram-negative bacteria, the acetic acid bacteria (phylum Alphaproteobacteria) stand out and we must also mention Zymomonas, from the same phylum. You can see the phylogenetic location of all these bacteria in my post “Bacteria: 21 Main phyla, with 147 Important genera”.

In Table 1 we see that besides LAB, the other most important microorganisms in fermented foods are yeasts, especially Saccharomyces, unicellular ascomycete fungus. Other fungi acting in fermented foods are some filamentous ones, such as the ascomycetes Penicillium and Aspergillus, and the zygomycete Rhizopus.

Figure 3. Various fermented foods: sobrassada, cheeses, blue cheese, yogurt, olives, soy sauce, beer and wine.

Living or dead microorganisms in fermented foods ?

Microorganisms that have been actively involved in the processing of fermented foods may be present and viable, that is, alive, in some of these. However, they are absent in other fermented foods because they have been separated from the food or this has had a treatment, usually thermal, removing them (Marco et al 2021).

Among the fermented foods that contain living microorganisms we can mention yogurt, kefir and other fermented milks, most cheeses, miso, natto and tempeh, many of the fermented vegetable products that have not been heat treated such as olives, many of the sausages, kombucha, and some beers.

Fermented foods where microorganisms have been eliminated or removed are for instance bread, heat-treated fermented vegetable products, soy sauce, vinegar, wines, most beers, and coffee and cocoa beans once roasted.

In many uninoculated fermented foods, that is, with their own spontaneous microbiota, there is more than one microorganism responsible for the changes that take place from the original food to the fermented one. There is often a succession of types of microorganisms, depending on the composition of the food and the environmental conditions to which it is subjected: salt, temperature, dryness, etc. For example, in the fermentation of table olives, yeasts and other bacteria first predominate, and finally the LAB end up being imposed.

Differences between fermented foods and probiotics

Although sometimes fermented foods are labelled or named as “probiotics” or “contain probiotics,” it should be made clear that it is not the same in most cases. The term probiotic is only correct to use it when it has been shown to have some beneficial effect on the health of the consumer, and that this effect is due to a living and well-characterized microorganism. This health benefit is beyond the nutritional benefits of the food matrix that contains it. Therefore, the terms “fermented food” and “probiotic” cannot be used by each other.

In the case of fermented foods that may contain some probiotic microorganism, with proven effects on health, it should only be labelled with “contains probiotics” in the event that the probiotic microorganism is well characterized at the strain level and in quantities significant throughout the shelf life of the food.

Fermented foods and food safety

Fermented foods increase the safety of food for the consumer, in the sense that it is more difficult for harmful or pathogenic organisms to grow than with respect to the original foods before fermentation. Indeed, they often contain remarkable amounts of organic acids, such as lactic acid produced by LAB or acetic acid produced by homonymous bacteria. Many of these products at the same time have low water activity, and contain salt and other antimicrobials, making them safe (Adams & Mitchell 2002). Similarly, beverages containing > 4% ethanol or pH < 4.5 are also considered microbiologically safe.

In addition, many LAB, whether native or inoculated, produce bacteriocins that inhibit other undesirable bacteria, such as Listeria or Clostridium.

Some fermented foods also increase safety by removing toxic or antinutrient compounds from raw foods, as is the case with many fermentations of cereals, legumes, and tubers. For example, cassava contains cyanogenic glycosides which are eliminated in fermentation by Lactobacillus plantarum (Lei et al 1999). Also, in the fermentation of the sourdough some LAB facilitate the degradation, by the enzyme phytase (a phosphatase), of phytic acid present in cereals, which is a chelator of divalent cations (Ca, Mg, Fe, Zn) and therefore decreases its adsorption (López et al. 2001).

Furthermore, it can be stated that, with very few exceptions, the microorganisms that are the protagonists of fermented foods, basically LAB, yeasts and filamentous fungi, are not pathogenic nor produce toxic or harmful compounds. In fact, many of them, such as LAB themselves, but also many others (such as some Bacillus, Figure 4) are considered GRAS (generally recognized as safe) by the US FDA or QPS (qualified presumption of safety) by the European EFSA.

Figure 4. One of the last ingredients declared GRAS by the US FDA is precisely a Bacillus subtilis that can also be used in fermented foods. Source: US FDA Gras Notices.

However, as in any type of food, it is always necessary to be very careful, to make sure that the ingredients are fresh and safe, avoid any alteration, and have good controls throughout the production process and in the finished product, checking that there is no contamination of the usual food pathogens.

Some fermented foods contain compounds that can pose food safety risks if consumed in excess. This is the case with alcoholic beverages, which should be taken in moderation due to the effects of ethanol and should be avoided by people at risk. For a different reason and not related to microorganisms, it is also important not to overeat fermented foods that contain salt, such as soy sauce or kimchi.

Some of the few compounds produced by LAB that need to be controlled are biogenic amines, which can be found in small amounts in fermented foods such as cheese, sausages, some vegetables and wine. Biogenic amines can cause various health problems and especially migraines. Their production must be minimized by controlling potential producers species and inoculating non-producing strains.

Mycotoxins such as aflatoxins, ochratoxins and many others, are the main concern of foods fermented with filamentous fungi such as Aspergillus and Penicillium from fermented soybeans, cheeses and others (Sivamaruthi et al 2019). However, in most of these products selected strains are used, either by domestication over centuries or more recently by artificial selection, which do not produce toxins.

Benefits of eating fermented foods on human nutritional health

Beyond food preservation reasons and organoleptic qualities, there is some epidemiological evidence to suggest that diets rich in fermented foods may reduce the risk of disease and increase longevity, health, and quality of life. But these diets, such as the Mediterranean diet, include foods other than fermented ones, and therefore it is not certain that the positive effects are due exclusively to fermented foods. In addition, with the exception of yogurt and other fermented milks, few well-designed and controlled clinical studies have been conducted on the potential health benefits of fermented foods in terms of specific diseases (Marco et al 2021).

However, the indirect health effects of fermented foods are quite apparent when considering the nutritional aspects. Microbial activity leads to the enrichment and / or elimination of various compounds that affect and improve the nutritional composition of the final product.

First, microorganisms reduce the content of high-calorie sugarsid est, monosaccharides and disaccharides, present in milk, meat and vegetables. This reduces the glycemic index and reduces food intolerance, such as lactose in dairy products, wheat fructans, or raffinose and stachyose from legumes. Fermentation hydrolyses polysaccharides, proteins and fats, which facilitates digestion, and as mentioned, it removes various toxic or antinutrient compounds such as phytic acid.

In the case of foods containing polyphenols, lactobacilli have been shown to increase the bioavailability of flavonoids, tannins, and other bioactive compounds. The biosynthesis of vitamins, amino acid derivatives, organic acids such as lactic acid, peptides and cofactors by food-fermenting microbes is well known (Melini et al 2019).

It has been shown that many of the living microorganisms in fermented foods can survive gastric transit and reach the colon, as for example many LAB are tolerant of acidic pH and bile salts and have been shown to be able to maintain transiently in the colon (Elli et al. 2006). Although these microorganisms are unlikely to survive long, it has been shown that they may be metabolically active in the gastrointestinal tract, and that this short-term colonization would be sufficient to produce bioactive compounds, inhibit pathogens, and positively influence the immune system. These effects are increased if there is a daily and repeated consumption of fermented food.

Fermented foods, and the microorganisms they contain, have also been shown to influence the composition of the intestinal microbiota itself (Taylor et al 2020). See also González et al in 2019 and Le Roy et al in 2020. Another additional positive factor in the case of fermented vegetable foods is that many components of these are prebiotics and therefore favour the intestinal microbiota.

In addition, we must take into account the importance of what we eat, including fermented foods, in relation to the immune system. In humans and other mammals 70% of this system is in the gastrointestinal tract, and food is the main source of contact between external antigens and our body. This is particularly important in infants and the initial microbial colonization of the digestive tract. Ingestion of fermented foods during the early years of childhood has been associated with a reduced risk of childhood atopy (genetic predisposition to allergies) (Alm et al. 1999). For any age, it seems that the microorganisms of fermented foods and their components, such as glycopeptide, surface proteins, exopolysaccharides, lipoteichoic acid, or D-phenyl-lactic acid from LAB (Peters et al. 2019) are beneficial for the immune system, especially more demonstrated in fermented milks (Bourrie et al. 2016; Foligne et al. 2016).

In Figure 5 we see a diagram of the basic mechanisms of the possible benefits of fermented foods.

Figure 5. Basic mechanisms of the health benefits of fermented foods, especially from a nutritional point of view, with the transformations of food components into bioactive substances. SCFAs are short-chain fatty acids. Source: Marco et al 2021.

Finally, to conclude, it is necessary to remind that although fermented foods are consumed worldwide and account for approximately 1/3 of the human diet, they are usually absent as recommended foods in diet guidelines (Marco et al 2021). It is also a pity that most of the information that comes out in the media or in popular magazines or on social media about this type of food is exaggerated or wrong, often making them synonymous with probiotics.


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Origin of viruses and how they “invented” DNA

2nd November 2020

Translated from the original article in Catalan

These weeks or months we all are worried by a virus, the SAR-CoV-2 obviously. So, I thought it appropriate to do this small bibliographic search reviewing the intriguing topic of the origin of these organisms so unique and so different from other living beings.

Remember what viruses are

Viruses are non-cellular organisms, that is, they are not cells: neither prokaryotes like bacteria and archaea, nor eukaryotes like protists, fungi, plants and animals. Therefore, viruses do not have a complex internal structure with many components as cells have, and above all viruses do not have all the metabolic activity involved in the maintenance and reproduction of cellular organisms.

From a functional point of view, viruses are submicroscopic infectious agents, which can only reproduce within cells, of other host organisms of course. Therefore, they are intracellular parasites, and are present in all possible cellular organisms, from archaea and bacteria to all types of eukaryotes. Viruses are found in any ecosystem and they are the most abundant biological entities on Earth (Edwards & Rohwer 2005).

Cellular organisms have the characteristics of the definition of living things, such as having a biological cycle, metabolism, growing, adapting to the environment, responding to stimuli, reproducing and evolving. The concept of living thing or living being has also been defined as any autonomous system with evolutionary capabilities (Peretó 2005). In principle, viruses do not have all these traits, which is why it is sometimes questioned whether they can be called “living things.” However, they can reproduce, at the expense of others, and evolve, and being closely related in their biological cycle to cellular organisms, I do not see how they could be considered “non-living things”. It would be like saying that they are non-biological organisms, which is obviously not true.

In their extracellular phase viruses are inert particles, named virions, almost all measuring between 20 and 300 nm, smaller than most bacteria. The structure of virions is limited to a protective layer of protein, the capsid, and the genetic material inside, RNA or DNA. The capsid can be helical, polyhedral or spherical, and gives the morphology observed under the electron microscope. Additionally, the virions of some viruses (especially animals) have an external structure, a membrane-type envelope, with proteins and phospholipids. Other viruses have more complex structures, such as some bacteriophages (Figure 1).

Adhesion and/or entry of virions into the host cell takes place by various methods, so that their genetic material enters in. There the information of this genetic material will be expressed, thanks to the biosynthetic machinery of the cell and will make more copies of the virus, which once outside the cell, will be more virions that can infect other cells.

Figure 1. Some of the different morphological types of viruses (left to right): helical (e.g. tobacco mosaic virus), polyhedral (e.g. adenovirus), spherical (e.g. flu), and complexes such as bacteriophages.

The classification of viruses is mainly based on their kind of genetic material, i. e. the genome, whether it is DNA or RNA, whether it is single or double stranded, and the replication strategy of this genome and biosynthesis of the mRNA (Figure 2). Some examples of these 6 classes of viruses are (Madigan et al, 2019):

  • Class I              bacteriophages lambda and T4, animal herpes
  • Class II             bacteriophage fX174
  • Class III            gastrointestinal rotavirus
  • Class IV           poliovirus, coronavirus
  • Class V            flu, rabies
  • Class VI           retroviruses such as HIV
  • Class VII is sometimes added (in the Baltimore classification, the discoverer of retroviruses), which are partially double-stranded DNA viruses and make an intermediate RNA to replicate. Ex: hepatitis B.

Figure 2. The six types of viruses according to their genome (DNA or RNA, double or single strand) and the mRNA replication and generation system. By convention, mRNA is orientation (+) (from Madigan et al., 2019).

Possible theories of the origin of viruses

Their origin has always been somewhat enigmatic, given the characteristics of these non-cellular organisms. Although viruses are very diverse and therefore different points of origin can be thought of independently, the similarity of their structures and a protein capsid that envelops a nucleic acid suggest at least one common mechanism for explaining their origin.

The 3 most referenced hypotheses to explain how the viruses originated are:

a) They would be forms derived from parasitic unicellular organisms, that evolutionarily would have been reduced to the minimum.

b) They would be fragments of genetic material that would have escaped cellular control becoming parasites.

c) They would be relics of precellular forms, that is to say of the protobionts.

In fact, hypothesis a) has the argument in favour of the existence of intracellular parasites such as Mycoplasma(Tenericutes bacteria) or Microsporidia (eukaryotic fungi), but these microorganisms maintain some cellular characteristics, such as the synthesis of proteins. In addition, no intermediate stage between cells and viruses is known.

Hypothesis b) has in favour the existence of plasmids and transposons, which can be considered as viral precursors, and the fact that viruses can often integrate cellular genes. But it is difficult to explain how these released nucleic acids would have incorporated a protein envelope. In addition, evolutionary affinities between viruses and hosts in the same domain should then be expected. For example, bacteriophages (phages) and bacteria, so phages should have some evolutionary similarities to bacteria, and instead phage proteins (such as T4) are more similar to eukaryotic proteins than their bacterial counterparts (Gadelle et al, 2003). Moreover, most viral proteins have no cellular counterparts in any of the 3 domains (Forterre 2006).

Hypothesis c) has against the fact that all current viruses are obligate parasites and require a stage of intracellular development for their development. However, as we will see below, this is the hypothesis that is gaining more and more recognition.

Two clear arguments in favour of hypothesis c) and against the others are:

  • There are viruses from all groups of cellular organisms, which makes hypothesis b) difficult to explain.
  • There are DNA and RNA viruses, which makes hypothesis a) unlikely.

However, it should be noted that criticisms of any of these hypotheses are made in the context of the current biosphere, where “modern” viruses need “modern” cells to replicate, where current cells cannot revert to viral forms, or where free DNA cannot capture proteins from current cells to form capsids, and so on. But things could have been very different before the formation of “modern” cells of archaea, bacteria, and eukaryotes. In this sense, we are less constrained by the current reality when proposing new evolutionary scenarios for the origin of viruses (Forterre 2006).

Quick review of the origin of life

To see the possibilities of hypothesis c), origin of viruses from protocellular forms, we will review what is today the most likely hypothesis of the origin of living things on Earth. You can see some good reviews of all this in the books by Zubay (2000), Schopf (2002) and Ribas de Pouplana (2004), and in the article by Peretó (2005), among others.

Prebiotic chemistry was the set of reactions by which biological components originated by abiotic synthesis. As is well known, in a first phase, already postulated in 1920-1930 by Oparin (Miller et al., 1997) and Haldane (Tirard 2017), and for which the experiments of Urey and Miller in 1953 (Bada & Lazcano, 2003) provided experimental support, it is assumed that from the basic molecules of the primitive atmosphere (water, methane, ammonium, nitrogen and others) were synthesized organic monomers such as amino acids, monosaccharides and organic acids, with the sources of energy of the primitive Earth. The importance of the contribution of organic matter from comets and meteorites is also becoming increasingly evident (Oró 2001). In a second phase, biogenic macromolecules could have been formed by polymerization of the monomers, probably on an inorganic support.

Once there were enough prebiotic organic compounds (not to be confused with “prebiotics”, a nutrition term for substrates used by the microbiota that give health benefits), the pre-cell protobiont phases had to be related to the 3 basic properties of living things:

  • The establishment of wrapping structures, i.e. membranes
  • The transformation of nutrients and energy, i.e. a minimum metabolism
  • An inherited mechanism, i.e. the ability to replicate and transfer characteristics to offspring.

As we see in the diagram (Figure 3), the current hypothesis is that these phases were in this order, so that in structures with envelopes (amphiphilic vesicles) began to generate mechanisms of protobioenergetic reactions that became autonomous systems, which began to acquire hereditary characteristics based on RNA (the RNA world), and that later, more stable DNA would eventually replace RNA as a genome molecule. The role of RNA is supported by the variety of RNAs existing in cells and by the catalytic characteristics of some of these, ribozymes, in addition to be a gene molecule (Peretó 2005).

Figure 3. Scheme of the hypothetical transition from prebiotic chemistry to cells, without a time scale but which could have been around 4000 M years ago. These protobiont phases include (from left to right) the origin of autonomic systems with protometabolism without genetic material, the first protocells with pre-RNA and then RNA plus proteins (the RNA world, with “ribocytes”), and then the incorporation of DNA, until reaching the LUCA (last common universal ancestor) with known biological characteristics. B: bacteria; A: archaea; E: eukaryotes (from Peretó 2005).

The origin of viruses: relics of protobionts

As discussed above, this is the most likely hypothesis today.

Taking up what we are now discussing about the origin of life, in fact, in this world RNAs could be distinguished as two phases (Figure 4). The first would begin when RNA as such would have become the genetic material carrying information but as we see, before (the “pre-RNA” world) there could have been protobionts (or protocells) with other “genetic” molecules, whether nucleic acids or other molecules, and this first protobiont with RNA would have meant a bottleneck or breaking point, which would have begun to predominate over the previous ones, which would have become extinct. As we know, this phenomenon is very common in evolution. In a second phase of the RNA world, ribosomes would have appeared as protein synthesis machines, which would have allowed a rapid evolution towards more efficient cell forms, as opposed to the use of peptides or other less efficient ways of synthesizing proteins. (Forterre 2005). Finally in some of the protobiont lines, DNA would have ended up replacing RNA as genetic material, also being a breaking point in the evolutionary line.

Well, as we see (Figure 4), some of the lineages could survive by parasitizing successful individuals in the next phase: they would be viruses. At each stage there would be a critical point of origin (breaking points or bottlenecks, black lines) from a novel organism that would give rise to many lineages. Some of these, instead of becoming extinct, could survive as viral lineages (white lines) by parasitizing the successful protocellular lineages of the next phase (Forterre 2005).

Figure 4. Hypothesis of the phases of the protobionts evolution in the RNA world: description in the text (from Forterre 2005).

In both phases of the RNA world very diverse organisms (the lineages depicted in Figure 4) would have coexisted: prey, predators, free-living, and parasites. It is therefore likely that protocells and virus-like entities coexisted, and thus RNA viruses would have originated (Figure 5). We see how in the first phase different lineages of RNA protobionts would coexist, with various protein production mechanisms (the small crossed inner circles, Fig. 5A), including the ancestor of the current ribosome system (the 2 black subunits). This lineage (in blue, Fig. 5B) would have eliminated its competitors. Some lineages of this first phase (green and red) would have survived as intracellular parasites with an extracellular phase in their biological cycle. Eventually these parasites would have lost their own protein synthesis machinery and become RNA viruses (Fig. 5C).

In addition, there are currently single-stranded RNA and double-stranded RNA viruses with different ways of replicating (Figure 2), as should be the case in this RNA world with very diverse lineages. Moreover, double-stranded RNA viruses of bacteria and eukaryotes have similar structures and their RNA-polymerase-RNA-dependent are homologous. This model implies a polyphyletic origin for the different superfamilies of RNA viruses, and when the protobionts would have become DNA, the parasitism of these viruses would have been maintained, giving rise to all the various RNA viruses we currently observe in cellular organisms. This model can be accommodated to explain DNA viruses originated from lineages of DNA protobionts (Forterre 2005).

Figure 5. Hypothesis of the origin of RNA viruses: description in the text (from Forterre 2005).

DNA, invented by some viruses ?

It is known that DNA ended up replacing RNA as genetic material during these early stages of evolution for two reasons: a) DNA is more stable than RNA because the 2’O of ribose is very reactive, being able to break the phosphodiester bond; and b) The deamination of cytosine to uracil is a frequent spontaneous chemical reaction that can be repaired in DNA but not in RNA, for obvious reasons: uracil is native to RNA.

In today’s living things, the DNA precursors, deoxyribonucleotides (dNTPs), are formed mainly by ribonucleotide reductases that reduce ribonucleotide ribose (rNTPs) to deoxyribose. They are synthesized from them thanks to complex enzymes, which should appeared in the second phase of the RNA world, since DNA could not be formed only from RNAs, even if these were ribozymes.

But what about DNA thymine instead of RNA uracil ?

The fact that dTMP is currently produced from dUMP and not by reduction of TTP suggests that U-DNA could have been an intermediary in the transition from RNA to DNA and therefore there would have been a “U-DNA world”. So it turns out that some bacterial viruses have DNA with uracil, which is U-DNA instead of the usual T-DNA. In fact, in today’s viruses we can find quite a diversity of DNAs, some with U-DNA, many others with T-DNA and some others with hydroxymethylcytosine-DNA (Figure 6), and in addition DNA viruses have a wide variety of replication mechanisms, and enzymes to pass from one type to another. That would be what can be called a “virosphere”, which gives rise to think about its origin in these stages of protobionts. Therefore, DNA as we see it with thymine, could have been an invention of some viruses (Forterre 2006).

Figure 6. Scheme of the evolution of genomes from RNA to modified DNA genomes. All types are present in the “virosphere” but only T-DNA is present in cellular organisms. RNR: ribonucleotide reductase; TdS: thymidyl synthase; HmcT: hydroxymethylcytosine transferase. (from Forterre 2006).

Hypothesis of DNA transfer from DNA viruses to cellular organisms

A DNA virus (Figure 7A, red genome) could have infected an RNA (blue genome) protocell and could have co-evolved (Fig. 7B), such that RNA genes would have been progressively transferred to viral DNA by retrotranscription (Fig. 7C, white arrow) and the viral genome would have evolved into a DNA plasmid within an RNA protocell, but eventually (Fig. 7D) the plasmid DNA would predominate over the RNA genome, due to its greater genetic efficiency and would end being a prechromosome of DNA.

A similar mechanism (Figure 7 E-G) could explain the formation of DNA plasmids in DNA protocells. Anyway, these resulting protocells would be prokaryotes. Given all this, the formation of eukaryotic cells, in addition to the well-known theory of the symbiosis of archaea and bacteria, could also be hypothesized as the formation of the nucleus, in RNA protocells, from uptake of DNA viruses enveloped by the formation of intracellular membranes, giving rise to the nuclear membrane, similarly to the formation of animal virus envelopes (Forterre 2005, idem 2006).

Figure 7. Hypothetical models of DNA transfer from viruses to RNA protocells (left A-D) to form DNA cells, and of the formation of plasmids (right E-G) from DNA viruses: description in the text (from Forterre 2005).

There are other possible explanations for explaining the origin of viruses, with arguments in favour of the other hypotheses discussed above, but I think that this where viruses would be forms that come from protobionts, is gaining weight. Another curious hypothesis is that viruses would have originated from self-replicating proteins such as prions that would have coupled with RNA. See Lupi & Dadalti (2007) for more information.


As we have seen, viruses, with their great diversity and being present in all cellular organisms, would be the descendants or remnants or relics of these forms of protobionts from the earliest times of life on Earth. As in other living things, evolution driven by a set of factors of genetic variability, and especially natural selection, would have resulted in what we now see. Viruses have been evolving much longer than we have, are present in all ecosystems on the planet, and are the most abundant biological entities. So we could say that in principle they must be smarter than us. We must continue to learn to live with them, as we are seeing in 2020 with SARS-CoV-2, and all this despite the fact that we doubt about their condition of being living things.


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BACTERIA: 21 main phyla, with 147 important genera

16th April 2020

Translated from the original article in Catalan

If you want to go directly to the simplified phylogenetic tree (Figure 4) that I propose below, click here. Idem for the inventory of the 21 phyla, click here. Idem for the alphabetical list of bacterial genera, click here.

Purpose of this article

From time to time, students have told me that when in class or doing some bibliographic work, they come up with a name of a microbial genus that is not very familiar to them, they have no idea where to find which type of microorganism it is and what are its basic characteristics, besides using Wikipedia. Although there is plenty of bibliography, and for queries only for phylogenetic location of a particular genus I use the Taxonomy browser section of the National Center for Biotechnology Information (NCBI), it often happens that an easy and quick source of information is missing, and this source should not be too exhaustive.

This is the purpose of this post: to summarize the major bacterial phyla, with the list of the most important genera. These should be the ones that seem most relevant, especially for the environment or in food and other industrial applications, also some because of their characteristic metabolism, and also some of the more well-known pathogens.

Not to be too exhaustive, for the moment I limit myself to bacteria, and therefore are not considered archaea, eukaryotic microorganisms, or viruses.

Taxonomy and phylogeny of the Bacteria

Taxonomy is the science of naming, defining and classifying groups of living beings based on the characteristics they share. These groups are taxa, which are divided into hierarchical categories, which are (variable depending on the organisms): Domain, Kingdom, Phylum, Class, Order, Family, Gender and Species. The first taxonomic system was developed in the 18th century by Carl von Linné, who laid the foundations for binomial nomenclature (Genus + species).

After Linné, the taxonomy developed mainly thanks to Ernst Haeckel (19th century) and Robert Whittaker, who proposed the 5 kingdoms: 4 eukaryotes (Animals, Plants, Fungi and Protista) plus that of the Monera, prokaryotes, bacteria basically (Whittaker 1969).

Although taxonomic classifications could be established only on the basis of phenotypic characteristics (morphology, structures, metabolism, etc.), taxonomy is currently elaborated by looking at the kinship relationships between organisms and their evolutionary history, that is, phylogeny. Making phylogenetic trees, based on genetic similarities, can explain the evolution of organisms, both current and extinct ones.

Historically, before molecular knowledge, the bacterial classification or taxonomy presented many difficulties and mistakes, given their microscopic size and lack of easily distinguishable morphological features, unlike plants and animals. Classification was based on cell wall structure (Gram) and metabolism only, but a phylogenetic tree could not be performed.

This changed in the hands of Carl Richard Woese (1987), promoter of the molecular phylogenetic revolution, who classified all organisms (not just bacteria), based on ribosomal RNA sequences, first defining the Archaeabacteria (now Archaea), and thus introducing the concept of the three domains (Figure 1).

Fig 1 woese fig4

Figure 1. Universal phylogenetic tree of the 3 domains (Archaea, Bacteria and Eukaryotes), determined by comparing the sequences of rRNAs, where line lengths are proportional to the calculated distances for alignment (Woese 1987).

Regarding only the Bacteria, based on the rRNA, Carl Woese established 11 divisions (Figure 2).

Fig 2 woese fig11

Figure 2. Bacterial phylogenetic tree determined by comparing the sequences of 16S rRNAs, where line lengths are proportional to the calculated distances for 16S alignment, and the point of origin is an Archaea consensus sequence (Woese 1987).

Later on, after Woese’s phylogenetic tree, it has been modified, on the one hand, incorporating numerous groups of discovered bacteria, especially thermophiles, chemolithotrophs and others from extreme environments. On the other hand, the development of non-culture techniques has allowed numerous bacteria to be detected without isolating them. Among these techniques where the DNA of environmental samples can be directly analysed, we should point out the methods of metagenomics, which amplify and sequence fragments of the genes (16S or others) of all the bacteria present, and treat the data with bioinformatics programs to compare with others and deduce possible new species.

Coincidentally with Woese, the classification of all living beings was enhanced by Thomas Cavalier-Smith, especially at the protist level (Cavalier-Smith 1993). Some of the more complete recent bacterial trees have been based on comparing some more conserved genes, such as Lang et al (2016), which proposes different super branch models of 3000 prokaryotes sequenced on the basis of 24 genes.

Based on all this, one of the most recent phylogenetic trees is the one proposed by Hug et al. (2016), which has been compiled based on published sequences, including the genomic data of 1000 unknown and non-isolated organisms. This “tree of life” with the 3 domains of bacteria, archaea and eukaryotes reveals a predominance of bacterial diversification and underlines the importance of organisms of which there are no isolated representatives (Figure 3). For this tree, 30437 species genomes from the 3 domains available by September 2015 in the NCBI databases were used. Currently (March 2020) there are already 50159 species sequenced in the NCBI: 1724 archaea, 26467 bacteria, 4915 eukaryotes and 17053 viruses .

Fig 3 Hug-et-al-figure-1

Figure 3. Current view of the tree of life, encompassing the total diversity of sequenced genomes, with 92 bacterial phylum, 26 archaea, and the 5 eukaryotic supergroups (Hug et al. 2016).

By comparing the genetic sequences of many bacteria, we have seen the difficulty of producing evolutionary phylogenetic trees with branches as we always represent them, because horizontal gene transfer (HGT) is a common phenomenon in bacteria. By means of the mechanisms of transformation, viral transduction and conjugation, bacteria share many genes in their evolution and blur the branches, so that the representation should be more like a network. The representations of phyla in evolutionary branches should therefore be taken as a relative approximation.

The Bergey’s Manual has undoubtedly been the most important bibliographic resource for the determination, identification and systematization of all prokaryotic organisms, namely Bacteria and Archaea. Started in 1923 by David H. Bergey, it has logically had successive updates, while retaining the importance of being the Reference Manual for the description of all prokaryotic features. The latest paper version of the Bergey’s Manual of Systematic Bacteriology comprises 5 volumes in 7 books (2001-2012). More recently an online version has been published (Whitman, 2015).

Another valuable resource is the LPSN (List of Prokaryotic Names with Standing in Nomenclature) database (Parte 2014), which collects the online list of all prokaryotic names that have been validated by publication in the International Journal of Systematic and Evolutionary Microbiology, under the rules of the International Bacterial Nomenclature Code. The LPSN currently lists 15,974 taxa, distributed in 41 bacterial phyla plus 5 archaea ones. In addition, the LPSN includes the updated classification of prokaryotes, their nomenclature, and culture collections.

However, both Bergey and LPSN are either too exhaustive and impractical to search for a particular bacterial genus or to have a quick overview of phylogenetic relationships between various phyla.

My proposal of simplified phylogenetic tree of Bacteria

Based on the tree described by Hug et al (2016) (Figure 3), and limiting to bacteria, I dare to simplify it, doing without the almost not known phyla or the numerous branches without isolated representatives. With that said, here are the 21 main phyla we see in Figure 4.

As we see in Figure 4, Terrabacteria and Hydrobacteria are two higher taxonomic categories that comprise the vast majority of bacterial phyla, the 99% of bacteria. Terrabacteria would have evolved by acquiring resistance adaptations to terrestrial environmental conditions such as desiccation, UV radiation, high salinity, including a characteristic cell wall (Gram-positive), and others of them would have developed oxygen photosynthesis (cyanobacteria). Hydrobacteria would be the rest of the bacteria, most Gram-negative, that would have evolved in aqueous or humid environments, and which include the 2 superphyla FCB (Fibrobacter-Chlorobi-Bacteroides) and PVC (Planctomyces-Verrucomicrobia-Chlamydia), and the large group of Proteobacteria. The supertaxa Terrobacteria and Hydrobacteria would have diverged 3,000 million years ago, when the Terrabacteria would have begun to colonize the continents.

In the same Figure 4, I have indicated the protomitochondria and protochloroplasts, which emerged from the phyla Alfaproteobacteria and Cyanobacteria respectively, about 1500-2000 million years ago.

Fig 4 arbre bacteris

Figure 4. Simplified current view of the bacterial phylogenetic tree with 21 main phyla, based on the DNA sequence set of 16 ribosomal proteins (modified by Hug et al 2016). LUCA: Last Universal Common Ancestor.

The 21 main bacterial PHYLA: most important features and genera

I describe below in short, the 21 phyla (Figure 4), following the phylogenetic tree from right to left. For the most relevant taxa (class, order) that I comment in some phyla, I have followed the categories as they are in the NCBI (Taxonomy). I have summarized the descriptions based on the basic sources of Microbiology information, such as Brock (Madigan et al. 2017), Lengeler et al. (1999), Tortora et al. (2018), or the Prescott (Willey et al. 2017), and within the most common internet resources, in addition to WikipediaMicrobeWiki must be mentioned.

1. Aquificae

E.g., Aquifex or Hydrogenobacter, it is a phylum close to Thermotogae, and both are the closest bacteria to the archaea. They are gram-negative bacilli, hyperthermophiles, aerobic chemolithotrophs, they oxidize H2 to H2O, and are found in hot or volcanic waters.

2. Thermotogae

E.g., Thermotoga, it is a phylum close to Aquificae. They are hyperthermophilic, fermentative anaerobes, gram-negative bacilli with a “toga” type wrap, and are found in hot water and hydrothermal vents.

3. Deinococcus – Thermus

They are very resistant to extreme environments, therefore extremophiles, which includes 2 groups of which the most well-known genera are those giving the name to the phylum:

Deinococcus are gram-positive cocci with thick wall and a outer membrane, gamma-resistant, UV-resistant, and of pink color due to carotenoid deinoxanthin.

Thermus are hyperthermophilic gram-negative bacilli, found in hot springs, and also in composting. Th. aquaticus was isolated by Thomas D. Brock and H. Freeze, from Yellowstone geysers, and it is well known for Taq DNA polymerase, which is widely used in PCRs because it is not denatured at 95°C. Th. thermophilus, also with thermostable DNA-polymerases, is a model for genetic manipulation.

4. Cyanobacteria

Cyanobacteria were formerly known as “blue-green algae” or cyanophytes because they are filamentous and perform photosynthesis, such as algae and plants. Like these, they make non-cyclic photophosphorylation, with 2 photosystems and chlorophyll. In fact, they are the evolutionary origin of proto-chloroplasts, they “invented” oxygenic photosynthesis, are the only bacteria currently doing so, and they generated the atmosphere as we know it some 2700 million years ago. Fossil stromatolites made of cyanobacteria biofilms are the earliest signs of life on Earth. They are filamentous gram-negative, with inner membranes. Some attach N2 to thicker specialized cells (heterocysts), containing nitrogenase. They are found in many habitats, both terrestrial and aquatic, some are symbionts of plants, others make cyanotoxins, and they are the main cause of blooms in eutrophic waters. Some are edible (Spirulina), mainly used as a feed supplement. With very active secondary metabolism, they are also an interesting source of antiviral, antibiotic and antitumor agents. Other genera are: Anabaena, Chroococcus, Nostoc, Oscillatoria, Pleurocapsa and Synechococcus.

5. Firmicutes

They are a large phylum of gram-positive, bacilli or cocci, chemoheterotrophic, with a low G + C content in the DNA (most with <50%). It mainly includes 3 great classes, Bacilli, Clostridia and Negativicutes:

Bacilli with 2 orders, Bacillales and Lactobacillales:

Bacillales, that are aerobic or facultative, mainly with aerobic respiration. Important genera:

Bacillus, endospore-forming bacilli, ubiquitous in terrestrial environments, where together with Paenibacillus they favour plant crops (see my post). The most resistant sporulate is B. stearothermophilus, a model for thermal sterilization calculations. There are some pathogens such as B. anthracis (anthrax) and B. cereus (food poisoning). Other many species are of industrial interest: production of enzymes (such as amylase) or proteases (subtilisin of B. subtilis), peptide antibiotics, some are bird probiotics (see my post on Probiotic Bacillus), and B. thuringiensis is widely used as bioinsecticide for their Cry toxins and their genes incorporated in Bt genetically modified plants (cotton, corn and others).

Listeria, facultative non-endospore-forming anaerobic bacilli, saprophytes but also opportunistic pathogens (L. monocytogenes) and cold-resistant, are the leading cause of death among foodborne diseases.

Staphylococcus, cluster-shaped facultative anaerobic cocci, saprophytes living on the human skin and membranous mucosa. Some are pathogens due to coagulase formation.

Lactobacillales: they are the lactic acid bacteria (LAB). They are bacilli or cocci, aerotolerant anaerobes with a fermentative metabolism, producing mainly lactic acid from sugars. Not sporulated, they are present in decaying plants (mainly Lactobacillus) and dairy products (especially Lactococcus, Lactobacillus and Streptococcus). They are amongst the most important groups of microorganisms used in food industry: dairy and other lacto-fermented foods, such as vegetables, meats, fish, wines and beers, etc., where these bacteria contribute to conservation, by decrease of pH and production of bacteriocins, and give organoleptic qualities. They are generally considered GRAS (Generally Recognized as Safe). In addition, they also play a role in the healthy animal and human microbiota, both in the gastrointestinal tract and on the mucous surfaces. That is why some of them are the most common probiotics, especially Lactobacillus. On the other hand, Oenococcus is the exclusive genus of wines where malolactic fermentation takes place (here is a brief summary), a peculiar fermentation linked to ATPase. Other important BL genera are: Enterococcus (some may be pathogens and other probiotics), Leuconostoc, Pediococcus (present in beers, see my post), Weissella, Carnobacterium, Aerococcus, and Fructobacillus.


They are strict anaerobic bacilli that form endospores. They are saprophytes, mainly fermenting plant polysaccharides, and live mainly in soils. Some are opportunistic pathogens in the digestive (Clostridium difficile) or saprophytes that can cause gangrene (C. perfringens) and the worst produce some of the most dangerous toxins: C. tetani and C. botulinum. However, they are very abundant in the healthy gut microbiota (see my post) and therefore possible probiotics (Clostridium, Eubacterium, Coprococcus and Ruminococcus, producers of the beneficial butyrate and propionate, and especially Faecalibacterium prausnitzii or Christensenella, associated with low body index, and low fat.

Although of the same class as Clostridia and also sporulated anaerobes, the genus Heliobacteria are anoxygenic photoheterotrophs (with bacteriochlorophyll g, one photosystem and cyclic photophosphorylation), they are not grampositive and fix N2.


They are sporulated anaerobes, phylogenetically close to Clostridia, but gram-negative, as they have an outer membrane similar to that of proteobacteria (possible horizontal gene transfer). Selenomonas is crescent-shaped, motile, present in the rumen of ruminants. Veillonella are cocci in the human gut, beneficial because they ferment lactose, giving acetate and propionate. Phascolarctobacterium is a pleomorphic bacillus that also produces these short-chain fatty acids in the gut, and therefore also beneficial.

6. Tenericutes

Phylum closely related to Firmicutes, but they have no cell wall. Unique class: Mollicutes. They are very small of size (0.2-0.3 µm) and genome (0.6 Mbp), because they are intracellular parasites of animals and plants, saprophytes and/or pathogens. Having no cell wall, they are resistant to many antibiotics. Variable form, they can live without oxygen. Mycoplasma are human pathogens that can cause pneumonia or sexually transmitted infections.

7. Chloroflexi

E.g., Chloroflexus, they are also called green non-sulphur bacteria or chlorobacteria, they are filamentous or gliding, with inner membranes (chlorosomes). They are anoxygenic photoheterotrophs (with bacteriochlorophyll cs, one photosystem and cyclic photophosphorylation). They are gram-negative but without outer membrane. The class Thermomicrobia includes those that are thermophilic (Thermomicrobium), some with pink pigment.

8. Actinobacteria

Another large phylum of gram-positive, heterotrophs, aerobic and anaerobic, with a high content of G+C in DNA (most with >50%), irregular in shape and some filamentous. They have very versatile catabolism and ubiquitous in terrestrial and aquatic environments. Includes these main orders:

Actinomycetales, such as Actinomyces, are facultative anaerobes, can make endospores, are filamentous but some are bacilli. They are economically important microorganisms in soils, both agricultural and forestry. They decompose organic matter, along with the fungi, which they resemble because they form filamentous mycelium.

Bifidobacteriales, anaerobes, they ferment carbohydrates. They are irregular bacilli, especially bifid, e.g. Bifidobacterium. They are important in the gut microbiota of mammals, in particular child in humans, and used as probiotics.

Corynebacteriales, aerobes, bacilli more or less irregular, some in the form of a club and others sometimes form hyphae. Abundant in different terrestrial environments, some are industrially important as amino acid producers, such as glutamic and lysine (Corynebacterium glutamicum). Others are pathogens: C. diphtheriae, Mycobacterium tuberculosis (see my post), M. leprae, and some opportunists with low virulence such as Nocardia.

Frankiales, filamentous, such as Frankia, live symbiotically fixing N2 in root nodules of many types of angiosperms.

Micrococcales, with genera such as: Micrococcus, cocci present in water and soil, saprophytes and opportunists, useful for biodegradation of contaminants and some in meat products, have very resistant cysts (see my post on microbial persistence); Cellulomonas, cellulose-degrading bacilli of soil, by glucanases; Arthrobacter (synonymous Siderocapsa) are common aerobic bacilli and cocci in the soil, some used for glutamic production and for bioremediation, some nylon degraders have even been described, and their DNA is the most persistent of permafrost, more 300,000 years (see my post); Brevibacterium linens is located on human skin, produces thioesters, the typical stink of feet, and is also used in cheeses (Munster, Limburger, etc.).

Propionibacteriales, such as Propionibacterium, anaerobic bacilli that synthesize propionic from sugars and from lactic acid. They may also use fumarate, by a peculiar fermentation with ATPase. Present in gut microbiota and animal skin, some of them are the cause of human acne (reclassified as Cutibacterium acnes) (see also my post). Others are important for the production of Vitamin B12 and dairy products, especially Swiss cheeses with “eyes” (Emmental and others).

Streptomycetales, with the important genus Streptomyces, are the most well-known Actinobacteria, with more than 500 species. Aerobes, form a complex mycelium of well-developed hyphae and are dispersed with aerial spores from structures comparable to mycelial fungi, but prokaryotic. Abundant in the soil and decaying vegetation, they produce geosmin and 2-methylisoborneol, which give the characteristic “ground” odour, invertebrate-attracting compounds that help bacteria disperse their spores. They have a complex secondary metabolism, which is why they are so important in industry: antibacterial antibiotics (streptomycin, neomycin, tetracycline, etc.), antifungals (nystatin), antiparasitic, anticancer, and also for the heterologous expression of eukaryotic proteins.

9. Fibrobacteres

Within the Hydrobacteria in Figure 4, together with Chlorobi and Bacteroidetes they belong to the FCB superphylum, formerly called Sphingobacteria by Cavalier-Smith. They are strict anaerobic gram-negative bacilli. They include some of the main cellulolytic bacteria in the rumen, such as Fibrobacter. They degrade beta-glucans, producing formate, acetate and succinate.

10a. Chlorobi

They are considered a single phylum together with Bacteroidetes, in the FCB superphylum. They are mainly green sulphur bacteria, gram-negative bacilli or cocci, strict photoautotrophic anaerobes that perform anoxygenic photosynthesis, with bacteriochlorophylls located in chlorosomes and the plasma membrane. They have one photosystem, and they use sulphides as electron donor. They fix CO2 through the reverse citric acid cycle. They can produce sulphates or accumulate elemental S outside the cell. Chlorobium is found on the seabed and lakes, and it is abundant for ex. in the Black Sea.

10b. Bacteroidetes

Also from the FCB superphylum and the same phylum as Chlorobi, they are non-sporulated strictly anaerobic gram-negative (outer membrane) bacilli, exclusive from the gastrointestinal tract of animals, where they are among the most abundant bacteria, notably Bacteroides and also Prevotella. They metabolize carbohydrates (polysaccharides especially) and other compounds such as bile salts, producing short chain fatty acids, beneficial for the host (see my post on Bacteroides). However, some of them can be pathogenic if they go outside the digestive tract. It seems that the Prevotella/Bacteroides ratio in humans is higher in high fibre diets and lower body weight. Flavobacterium is a known Bacteroidetes fish pathogen.

11. Planctomycetes

Of the PVC superphylum, e.g. Planctomyces, they are anaerobic gram-negative bacteria very particular: ovoid with a pseudo-stem appendix terminated in a substrate-adherent structure, with membrane invaginations reminiscent of eukaryotic cell structures, and cell wall with almost no glycopeptide. They reproduce by budding and generate free flagellate forms that then will be sessile. They live in waters, both sweet and marine. Some such as Brocardia contain a membranous structure, anammoxosome, where an important metabolism for the nitrogen cycle occurs: anaerobic ammonium oxidation (Anammox) with nitrite, producing N2.

12a. Verrucomicrobia

Also of the superphylum PVC, and considered of the same phylum with Chlamydiae, there are few described species. They are similar in shape to warts and are anaerobic gram-negative, isolated from soil, water and human faeces. Akkermansia, an aerotolerant, present in the human gut microbiota, has been linked to lower obesity and lower incidence of related diseases, thanks to maintaining the mucin-degrading mucosa, contributing to barrier function.

12b. Chlamydiae

Also of the superphylum PVC and of the same phylum with Verrucomicrobia, they are gram-negative cocci, obligate intracellular of eukaryotes, many animal pathogens and some protozoa symbionts. They have two forms (like viruses): the extracellular, particulate or elemental body, only 0.3 μm, which generates the intracytoplasmic reticular form of 0.5 μm by endocytosis. Chlamydia infections are the most common sexually transmitted bacterial disease.

13. Acidobacteria

Such as Acidobacterium, they are aerobic or facultative or anaerobic gram-negative bacilli, heterotrophic, many of them oligotrophic, most are acidophilic (pH 3-6), and they have capsules with a lot of exopolysaccharide. Although poorly isolated in culture, they are ubiquitous, especially in soils, where up to 50% of the present bacteria, where many are symbiotic in the rhizosphere of plants. Some are biodegraders of aromatic compounds (Holophaga) and / or metal scavengers (Geothrix).

14. Nitrospirae

Monophyletic phylum, they are gram-negative aerobic helical- or comma-shaped (vibrions). Hard to isolate, they are present in marine ecosystems forming biofilms but also in wet land or in activated sludge from sewage treatment plants, biofilters and others. They are nitrifying bacteria, making nitrite oxidation, e.g. Nitrospira.


As we see in in Figure 4, most of the remaining phyla contain this term. They are the largest and most metabolically diverse group of bacteria, and they have in common to be gram-negative with lipopolysaccharide outer membrane. They are almost half of the sequenced prokaryotes and include both phototrophs and heterotrophs with a common evolutionary origin, which are supposed to be anoxygenic phototrophs such as the purple bacteria (e.g. Rhodospirillum). For this reason and for having a phylogenetic relationship based on 16S rRNA, Woese (1987) called them “Purple and related bacteria” and established the first alpha, beta, gamma and delta subdivisions. Shortly after, Stackebrandt et al (1988) proposed the vocable Proteobacteria, based on the Greek god Proteus, by the analogy that it could take multiple forms.

15a. Deltaproteobacteria

Considered common phylum with Thermodesulfobacteria, they include two groups:

Mixobacteria (order Myxococcales), aerobes that live in soils, heterotrophs consuming insoluble organic matter, that move by gliding. They have very large genomes with respect to other bacteria, 10 Mbp, some up to 16 Mbp. The biological cycle (e.g. Myxococcus) is complex: vegetative forms are gliding bacilli that are grouped into fruiting bodies (by quorum sensing of contact) of different shapes and colours, and that give resistant spherical mixospores. Some are antibiotic producers and others like Sorangium, produce anti-tumour drugs.

The other large group is the strict anaerobic sulphur bacteria. The disassimilating reduction of sulphates in marine and water purification environments accounts for 50% of the mineralization of organic matter. They include these two orders:

Desulfovibrionales, the main sulphate reducing bacteria: Desulfovibrio, Desulfobacter and others. They are flagellate rods or curved bacilli that live in aqueous environments, where they degrade organic matter, by anaerobic respiration using sulphate as an electron acceptor. They produce SH2, which, in addition to stinking, reacts with metals, corrodes them, and produces e.g. FeS. They are considered ones of the oldest microbes on Earth and are very important in the S cycle.

Desulforomonadales reduce elemental S, also by anaerobic respiration, but they can also use other inorganic compounds such as nitrate, Fe3+ and other metals. They also produce SH2. Geobacter is one of the main genera, used for biodegradation and bioremediation of pollutants, and it is being studied for the design of microbial cells that generate electricity thanks to the conductivity of the biofilms they form.

15b. Thermodesulfobacteria

Same phylum as Deltaproteobacteria. They are sulphate reducers, thermophilic and hyperthermophilic, bacilli isolated from thermal sources, seabed and hydrothermal vents. The most known, Thermodesulfobacterium, has a membrane lipid (phosphoaminopentanotetrol) found only in the archaea. Geothermobacterium, isolated in Yellowstone, has an optimum temperature of 85-90°C, the highest of bacteria, and reduces Fe3+.

16. Oligoflexia

Phylogenetically related to Deltaproteobacteria, this phylum includes Bdellovibrio and other predators of gram-negative bacteria. They are small (about 1 µm) aerobic curved rods (vibrions), with a polar flagellum that allows them to swim over 100 times their body-length per second. In its biological cycle, the motile free form attaches to a bacterium prey, penetrates it, forms a spherical complex within the host, uses hydrolases to digest proteins and DNA from the host, and grows filamentous, the host is lysed and the filament is separated in 3-6 free daughter cells, all in 4 h.

17. Spirochaetes

Spirochetes are gram-negative bacteria with an outer membrane, and characteristic spiral or helical shape. They are rather long (3 to 200 µm), due to the axial filament, set of flagella, located in the periplasmic space. This filament shrinks, allowing motility. They are anaerobic heterotrophs or facultative of diverse aquatic environments. Spirochaeta is free-living and non-pathogenic, but other genera are pathogenic, such as Leptospira (leptospirosis), Borrelia (tick-borne Lyme disease), or Treponema (syphilis and tropical diseases).

18. Epsilonproteobacteria

They are gram-negative heterotrophic, most of them microaerophilic, motile and curved, spiral or helical. The most well-known are symbionts or pathogens in the digestive tract of animals, including humans. Campylobacter is a pathogen mainly of poultry, and in humans from eating contaminated food. Helicobacter is very common in the stomach causing ulcers and gastritis. Arcobacter is an emerging pathogen that, in addition to being found in the digestive tract of animals, can also be a seafood contaminant.

The phylum also includes quite a few non-pathogenic isolates isolated from deep-sea vents and marine sediments, such as Sulfurimonas.

19. Alphaproteobacteria

Large and very diverse phylum of gram-negative with outer membrane in the cell wall. Together with betaproteobacteria and gammaproteobacteria, they constitute a clear monophyletic group, of common origin, which are the most typical Proteobacteria (Figure 4). The whole of these 3 phyla was named Rhodobacteria by Cavalier-Smith in 1987.

Alphaproteobacteria include the following main orders:

Rhodobacterales, such as Rhodobacter, a study model for bacterial anoxygenic photosynthesis. They are the so-called non-sulphur purple bacteria (due to their color resulting from bacteriochlorophylls plus carotenes), to differentiate them from sulphur purple bacteria (Chromatiales, within Gammaproteobacteria). They have a wide variety of metabolisms: photosynthesis, lithotrophy and aerobic and anaerobic respiration, and are present in all aqueous environments.

Rhodospirillales, which also includes other non-sulphur purple bacteria (Rhodospirillum) with broad metabolic capabilities and a spiral shape. Others are acetic bacteria (Acetobacter, Gluconobacter, Gluconacetobacter, Komagataeibacter and others), aerobic bacilli well known for respiratory oxidative metabolism, oxidizing sugars and ethanol to acetic acid, producing the vinegars. Another is Magnetospirillum, a spiral-shaped microaerophile containing magnetosomes, organelles with magnetite (Fe3O4), that allow them to orient themselves with the geomagnetic field.

Caulobacterales, such as Caulobacter, curved rods, are oligotrophic in freshwater. They have a characteristic cell cycle with two differentiated forms: one with a peduncle attached to a substrate, which splitting asymmetrically generates a flagella free form that ends up in a pedunculate form.

Magnetococcales, with Magnetococcus, marine cocci with characteristics similar to Magnetospirillum, including magnetosomes.

Rhizobiales, with Rhizobium, the well-known N2 fixers, endosymbionts in the leguminous root nodules. In the same order are: Agrobacterium, which causes plant tumours by transferring its DNA, and for that reason it is widely used in genetic engineering (A. tumefaciens); Rhodopseudomonas, another photosynthetic non-sulphur purple bacterium of water and soil; Brucella, small coccobacilli pathogens of humans and other animals; and Nitrobacter, nitrifying chemolithotroph bacilli, which oxidize nitrite to nitrate.

Ricketssiales are obligate endosymbionts of eukaryotic cells, many pathogens, such as Rickettsia, pleomorphic human pathogen (cocci, bacilli, etc.) transmitted by arthropods, and Wolbachia, which infects many arthropods and nematodes. As shown in Figure 4, the phylogenetic relationship suggests that mitochondria (endosymbionts) developed from this group.

Sphingomonadales, such as Sphingomonas, are strict aerobic bacilli, with glycosphingolipids in the outer membrane, instead of the lipopolysaccharides of other gram-negatives, and with typical yellow colonies. Present in many different environments, where they survive with low nutrient concentrations and a versatile ability to biodegrade compounds, including aromatics and other xenobiotics. That is why they are used for bioremediation, and their extracellular polymers (sphingans) are used in the food industry. Zymomonas are facultative anaerobic bacilli, with the unique feature of bacteria doing alcoholic fermentation, in Mexican pulque, or African palm wine, degrading sugars to pyruvate by the Entner-Doudoroff pathway.

20. Betaproteobacteria

A diverse phylum of aerobic or facultative groups, of varied forms, with metabolic versatility, both heterotrophic and chemolithotrophic, and some phototrophic. The main orders are:

Burkholderiales, most are motile aerobic bacilli: Burkholderia and Bordetella, human and other animal pathogens; Ralstonia and Achromobacter are common in soils and are opportunistic pathogens; Alkaligenes are also opportunistic pathogens, and some produce polyhydroxybutyrate, a biopolymer; Oxalobacter is exceptionally anaerobic, found in the human microbiota and rumen of ruminants, where it degrades oxalic, benefitting the host, by peculiar fermentation with ATPase; Sphaerotilus natans are filamentous (up to 0.5 mm) heterotrophic aerobic growing within a long, tubular sheath, are present in contaminated water and prevent active sludge flocculation; Acidovorax, known pathogen of cucurbitaceous crops (pumpkin, zucchini, cucumber, watermelon, etc.); Ideonella sakaiensis is a degrader of PET plastic (see my post).

Neisseriales, nonmobile aerobic diplococci, colonize the mucosa of many animals without causing damage, and only two species are human pathogens: Neisseria meningitidis and N. gonorrhoeae.

Nitrosomonadales, diverse order with a few bacilli aerobic chemolithotrophs, such as Nitrosomonas, the most well-known of nitrifiers, which oxidize ammonium to nitrite, or Thiobacillus, the known sulphur (colourless) oxidizing bacteria and also of Fe2+, and Gallionella, helical and filamentous rods, which oxidizes iron but is microaerophilic. Methylophilus are (with other bacteria and fungi) methylotrophs, which use C1 compounds as a substrate, such as methanol, methane, and therefore are environmentally beneficial. Spirillum are spiral-shaped heterotrophic microaerophiles present in freshwater containing organic matter.

Rhodocyclales, as Zoogloea, are motile aerobic bacilli, relevant to aerobic wastewater treatments, mainly in activated sludge process, where they degrade organic matter and help to form biological flocs that settle down.

21. Gammaproteobacteria

The last big phylum of Proteobacteria includes many important groups scientifically, medically and environmentally, with these main orders:

Xanthomonadales, aerobic rods, most are phytopathogens such as Xanthomonas species that affect the crops of citrus fruits, tomatoes, rice and others, and Xylella in the vine. Some are opportunistic pathogens for humans.

Chromatiales, are the purple sulphur bacteria (Chromatium, Thiocapsa), which perform anoxygenic photosynthesis from sulphides or thiosulphate, producing elemental S. They have inner membranes with bacteriochlorophyll and carotenes and are present in the anoxic areas of lakes and other aquatic habitats such as intertidal areas.

Methylococcales, such as Methylococcus, are another large group of methylotrophs, which use methane as their source of energy and carbon, and which is oxidized to formaldehyde, and then this is assimilated by the ribulose monophosphate cycle, on inner disk-shaped membranes. perpendicular to the cell wall.

Thiotricales, are mainly chemolithotrophs, with cocci shape clustered into filaments, which accumulate sulphur granules. Beggiatoa lives in waters containing H2S, and oxidizes it to sulphur, but is also heterotrophic. Thiomargarita namibiensis, found in marine sediments, is the largest bacterium ever found, up to 0.7 mm in diameter, and accumulates S in the periplasm and nitrate in vacuoles, as it is also nitrifying.

Legionellae, with Legionella, aerobic pleomorphic bacilli, known pneumonia-causing pathogens (legionellosis), and other respiratory diseases. Most infections are due to poorly maintained cooling towers.

Oceanospirillales are a metabolically diverse group but all prefer or need a high salt content, such as Halomonas, motile aerobic rods.

Pseudomonodales include many motile bacilli or motile coccobacilli (with polar flagella), strict aerobic heterotrophs and oxidase positive. Pseudomonas is one of the most ubiquitous bacterial genera in many terrestrial and aquatic habitats, with some plant pathogens and other opportunists in humans, and causes food spoilage. Some of them (P. syringae) facilitate the nucleation of ice crystals causing plant tissue freezing or cloud condensation or artificial snow formation (see my post). On the other hand, their large and diverse aerobic catabolic ability makes them useful for wastewater treatment and bioremediation of hydrocarbons and other complex organic compounds. Azotobacter are cocci or oval-shaped, motile, with thick-walled cysts and extracellular lime strength, free-living in soils, with a relevant role in the N-cycle as Nfixers. Acinetobacter is also common in soils, where they mineralize aromatic compounds, and some are opportunistic pathogens, especially in hospitals. Moraxella are similar, commensals of animal mucosa but also some pathogens.

Aeromonadales, such as Aeromonas, facultatively anaerobic bacilli, morphologically similar to Enterobacterales, they are present in aqueous environments, and are a common cause of gastroenteritis and other water-borne infections or contaminated food.

Vibrionales, are vibrions or coccobacilli, motile facultative anaerobic, present in aqueous media, among which there are quite a few pathogens of humans, such as Vibrio cholerae, and of other animals, especially fish. However, it also includes most bioluminescent bacteria: Photobacterium, Aliivibrio and many Vibrio, from marine environments, many symbiotics of fish and other animals. The light produced (490 nm, cyan, blue-green) is due to luciferase-linked lux-flavin chromophores, reacting with oxygen and fatty acid reducing reactions.

Pasteurellales, they are bacilli or pleomorphic, without flagella, facultative anaerobes and oxidase positive (unlike Enterobacteriales), commensals at the mucosa surfaces of birds and mammals, and some are pathogens. Pasteurella are pleomorphic and zoonotic pathogens. Many Haemophilus are human pathogens, and H. influenzae was the first genome to be sequenced, by Craig Venter group in 1995.

Enterobacterales, it includes most of the well-known gram-negatives with outer membrane, some of them pathogens. They include the so-called enterobacteria, family Enterobacteriaceae, among which there are symbionts and pathogens, especially in the animals’ gut. They are facultative anaerobes, perform the mixed-acid fermentation and other metabolisms, have no cytochrome c oxidase, and most are motile bacilli or coccobacilli with peritrichous flagella. Of particular note is Escherichia coli, probably the most well-known bacterium and model organism for biochemical, genetic and molecular knowledge. Some E. coli are pathogens, other opportunists, many commensals, and some even beneficial members of the gut microbiota, and used as probiotics. The following are also Enterobacteriaceae: Salmonella, the intracellular pathogen of many animals by endotoxins, causing typhoid fever humans, food-borne infections, and other pathogens; Shigella, a pathogen in humans and other primates, also with endotoxins, it is a major cause of diarrhea; Yersinia pestis, coccobacillus pathogen well known for epidemics; Klebsiella, ubiquitous in many environments, is usual commensal of human mucosa and gut; Enterobacter are thermotolerant faecal coliforms (they grow at 44.5°C), opportunistic pathogens, and some are useful in dairy products; Citrobacter can use citrate as their only source of C, are also ubiquitous in many environments, and most are non-pathogenic

Of the same order Enterobacterales but of other families: Erwinia, pathogen of plants; Hafnia, commensal of the human gastrointestinal tract, used as a lactic ferment, and possible probiotic; Proteus, opportunistic pathogen; and finally, Thorsellia, present in the gut microbiota of the Anopheles mosquito, which could be used genetically modified against the mosquito to prevent malaria transmission.

Alphabetical LIST of 147 bacterial GENERA, with the corresponding Phyla

(link by clicking on the Phylum)

This is not a static, fixed list:  please, if you notice any other genera that you consider important, please tell me and I’ll incorporate it.

Genus Phylum
Acetobacter 19. Alphaproteobacteria
Achromobacter 20. Betaproteobacteria
Acidobacterium 13. Acidobacteria
Acidovorax 20. Betaproteobacteria
Acinetobacter 21. Gammaproteobacteria
Actinomyces 8. Actinobacteria
Aerococcus 5. Firmicutes
Aeromonas 21. Gammaproteobacteria
Agrobacterium 19. Alphaproteobacteria
Akkermansia 12a. Verrucomicrobia
Alcaligenes 20. Betaproteobacteria
Allivibrio 21. Gammaproteobacteria
Anabaena 4. Cyanobacteria
Aquifex 1. Aquificae
Arcobacter 18. Epsilonproteobacteria
Arthrobacter 8. Actinobacteria
Azotobacter 21. Gammaproteobacteria
Bacillus 5. Firmicutes
Bacteroides 10b. Bacteroidetes
Bdellovibrio 16. Oligoflexia
Beggiatoa 21. Gammaproteobacteria
Bifidobacterium 8. Actinobacteria
Bordetella 20. Betaproteobacteria
Borrelia 17. Spirochaetes
Brevibacterium 8. Actinobacteria
Brevibacterium 8. Actinobacteria
Brocardia 11. Planctomycetes
Brucella 19. Alphaproteobacteria
Burkholderia 20. Betaproteobacteria
Campylobacter 18. Epsilonproteobacteria
Carnobacterium 5. Firmicutes
Caulobacter 19. Alphaproteobacteria
Cellulomonas 8. Actinobacteria
Chlamydia 12b. Chlamydiae
Chlorobium 10a. Chlorobi
Chloroflexus 7. Chloroflexi
Christensenella 5. Firmicutes
Chromatium 21. Gammaproteobacteria
Chroococcus 4. Cyanobacteria
Citrobacter 21. Gammaproteobacteria
Clostridium 5. Firmicutes
Coprococcus 5. Firmicutes
Corynebacterium 8. Actinobacteria
Cutibacterium 8. Actinobacteria
Deinococcus 3. Deinococcus – Thermus
Desulfobacter 15a. Deltaproteobacteria
Desulphovibrio 15a. Deltaproteobacteria
Enterobacter 21. Gammaproteobacteria
Enterococcus 5. Firmicutes
Erwinia 21. Gammaproteobacteria
Escherichia 21. Gammaproteobacteria
Eubacterium 5. Firmicutes
Faecalibacterium 5. Firmicutes
Fibrobacter 9. Fibrobacteres
Flavobacterium 10b. Bacteroidetes
Frankia 8. Actinobacteria
Fructobacillus 5. Firmicutes
Gallionella 20. Betaproteobacteria
Geobacter 15a. Deltaproteobacteria
Geothermobacterium 15b. Thermodesulfobacteria
Geothrix 13. Acidobacteria
Gluconacetobacter 19. Alphaproteobacteria
Gluconobacter 19. Alphaproteobacteria
Haemophilus 21. Gammaproteobacteria
Hafnia 21. Gammaproteobacteria
Halomonas 21. Gammaproteobacteria
Helicobacter 18. Epsilonproteobacteria
Heliobacteria 5. Firmicutes
Holophaga 13. Acidobacteria
Hydrogenobacter 1. Aquificae1. Aquificae
Ideonella 20. Betaproteobacteria
Klebsiella 21. Gammaproteobacteria
Komagataeibacter 19. Alphaproteobacteria
Lactobacillus 5. Firmicutes
Lactococcus 5. Firmicutes
Legionella 21. Gammaproteobacteria
Leptospira 17. Spirochaetes
Leuconostoc 5. Firmicutes
Listeria 5. Firmicutes
Magnetococcus 19. Alphaproteobacteria
Magnetospirillum 19. Alphaproteobacteria
Methylococcus 21. Gammaproteobacteria
Methylophilus 20. Betaproteobacteria
Micrococcus 8. Actinobacteria
Moraxella 21. Gammaproteobacteria
Mycobacterium 8. Actinobacteria
Mycoplasma 6. Tenericutes
Myxococcus 15a. Deltaproteobacteria
Neisseria 20. Betaproteobacteria
Nitrosomonas 20. Betaproteobacteria
Nitrobacter 19. Alphaproteobacteria
Nitrospira 14. Nitrospirae
Nocardia 8. Actinobacteria
Nostoc 4. Cyanobacteria
Oenococcus 5. Firmicutes
Oscillatoria 4. Cyanobacteria
Oxalobacter 20. Betaproteobacteria
Paenibacillus 5. Firmicutes
Pasteurella 21. Gammaproteobacteria
Pediococcus 5. Firmicutes
Phascolarctobacterium 5. Firmicutes
Photobacterium 21. Gammaproteobacteria
Planctomyces 11. Planctomycetes
Pleurocapsa 4. Cyanobacteria
Prevotella 10b. Bacteroidetes
Propionibacterium 8. Actinobacteria
Proteus 21. Gammaproteobacteria
Pseudomonas 21. Gammaproteobacteria
Ralstonia 20. Betaproteobacteria
Rhizobium 19. Alphaproteobacteria
Rhodobacter 19. Alphaproteobacteria
Rhodopseudomonas 19. Alphaproteobacteria
Rhodospirillum 19. Alphaproteobacteria
Rickettsia 19. Alphaproteobacteria
Ruminococcus 5. Firmicutes
Salmonella 21. Gammaproteobacteria
Selenomonas 5. Firmicutes
Shigella 21. Gammaproteobacteria
Sorangium 15a. Deltaproteobacteria
Sphaerothilus 20. Betaproteobacteria
Sphingomonas 19. Alphaproteobacteria
Spirillum 20. Betaproteobacteria
Spirochaeta 17. Spirochaetes
Spirulina 4. Cyanobacteria
Staphylococcus 5. Firmicutes
Streptococcus 5. Firmicutes
Streptomyces 8. Actinobacteria
Sulfurimonas 18. Epsilonproteobacteria
Synechococcus 4. Cyanobacteria
Thermodesulfobacterium 15b. Thermodesulfobacteria
Thermomicrobium 7. Chloroflexi
Thermotoga 2. Thermotogae
Thermus 3. Deinococcus – Thermus
Thiobacillus 20. Betaproteobacteria
Thiocapsa 21. Gammaproteobacteria
Thiomargarita 21. Gammaproteobacteria
Thorsellia 21. Gammaproteobacteria
Treponema 17. Spirochaetes
Veillonella 5. Firmicutes
Vibrio 21. Gammaproteobacteria
Weissella 5. Firmicutes
Wolbachia 19. Alphaproteobacteria
Xanthomonas 21. Gammaproteobacteria
Xylella 21. Gammaproteobacteria
Yersinia 21. Gammaproteobacteria
Zoogloea 20. Betaproteobacteria
Zymomonas 19. Alphaproteobacteria


Bacterial phyla, Wikipedia

Bacterial taxonomy, Wikipedia

Cavalier-Smith T (1993) Kingdom protozoa and its 18 phyla. Microbiol Reviews 57, 953-94

Hug LA et al. (2016) A new view of the tree of life. Nat Microbiol. 1, 16048

Lang JM, Darling AE, Eisen JA (2013) Phylogeny of bacterial and archaeal genomes using conserved genes: Supertrees and supermatrices. PLoS ONE 8(4): e62510

Lengeler JW, Drews G, Schlegel HG (1999) Biology of the Prokaryotes. Blackwell Science.

Madigan M, Bender KS, Buckley DH, Sattley WM, Stahl DA (2017) Brock Biology of Microorganisms, 15th ed. Pearson.

MicrobeWiki: https://microbewiki.kenyon.edu/index.php/MicrobeWiki

NCBI, National Center for Biotechnology Information: https://www.ncbi.nlm.nih.gov

Parte AC (2014) LPSN – list of prokaryotic names with standing in nomenclature. Nucleic Acids Research 42, D1, D613-D616.

Stackebrandt E, Murray RGE, Trüper HG (1988) Proteobacteria classis nov., a name for the phylogenetic taxon that includes the “Purple bacteria and their relatives”. Int J Syst Bact 38, 321-325

Tortora GJ, Funke BR, Case CL, Weber D, Bair W. (2018) Microbiology: an Introduction. 13th ed. Pearson.

Whitman WB, ed. (2015) Bergey’s manual of systematics of archaea and bacteria. ISBN 9781118960608.

Whittaker RH (1969) New concepts of Kingdoms of organisms. Science 163, 150-160.

Wiley J, Sherwood L, Woolverton CJ (2017) Prescott’s Microbiology, 10th ed. McGraw Hill Education.

Woese CR (1987). Bacterial evolution. Microbiological Reviews 51(2): 221–71

Yeasts 3000-years-old are alive and other histories of dormant cells

18th August 2019

Translated from the original article in Catalan

A few months ago -April 2019- my friend Jordi Diloli, Professor and Archaeologist, shared a very surprising article (Aouizerat et al 2019) with me. It was echoed on the internet (Borschel-Dan 2019), and I will comment here.

“Resurrected” yeasts from 3,000 years ago

The group of researchers led by Ronen Hazan of the Hebrew University of Jerusalem took samples of 21 clay containers from various sites in present-day Israel from 2500 to 5000 years ago, from the Persian, Philistine and Egyptian (this is the oldest) periods. Archaeologists believed that these vessels contained fermented beverages such as beer or mead (Figure 1). The authors submerged the containers in a rich YPD medium, specific for growing yeasts and other fungi, and incubated them at room temperature for 7 days. Then, samples of this medium were spread on agar plates with the specific medium, and the resulting colonies were isolated for subsequent analyses (Aouizerat et al 2019).

Fig 1 pottery Hazan

Figure 1. Clay vessels from where the yeasts were isolated (Image of Judah Ari Gross, Times of Israel).


The isolates that were found were 6 strains of different yeast species, and one of which was Saccharomyces cerevisiae, specifically from a Philistine site dated 3000 years ago. Obviously, it is very surprising that living yeasts of such ancient remains have been isolated. For this reason, the authors of the work carried out a series of experiments that could confirm this unique fact and that the isolates were not a product of contamination.

Firstly Aouizerat et al (2019) showed that it is possible to isolate yeasts from clay vessels that have contained beer or wine after a certain time. They did so with containers with unfiltered beer buried for 3 weeks, and also with another vessel that had repeatedly contained wine but not used last 2 years. With these samples they developed the isolation methodology and in both cases they were able to isolate yeasts. No isolates were obtained from a control sample with filtered beer, therefore without yeasts.

To demonstrate that the isolates were originals of the old vessels because these had contained the fermented beverage, authors applied the same protocol with samples of other ceramics that were surely not for this purpose, and also with sediments near the containers. The result was clearly negative for these samples: only 2 isolated yeasts from 110 samples, while the mentioned 6 yeast strains were isolated from the 21 initial samples. That is, yeasts would be significantly more abundant in containers of alcoholic fermented beverages than in other related archaeological vessels or sediments around them.

Another argument that supports the hypothesis of this work was the identification of these 6 yeasts. Total DNA was obtained and processed to sequence the genomes and compare them with the databases. Two of them, from the Egyptian period, were identified as Saccharomyces delphensis, a species that has been isolated from African dried figs and is not at all common on soil. Therefore, this suggests the use of figs in the alcoholic beverages of these containers. Another isolate was identified as Rhodotorula, common pollutant yeast in African beers. Another was identified as Debaryomyces, a frequent yeast in traditional African sorghum beers. As said before, another isolate was identified as Saccharomyces cerevisiae, the yeast most used to make wine, beer or bread (Figure 2). In spite of this, the genetic sequence of this S. cerevisiae was clearly different from the strains most commonly used today, as commercial or laboratory strains, and therefore the possibility of contamination is excluded. And finally, the other isolate was identified as Hypopichia burtonii, previously isolated yeast from Ethiopian mead.

These genetic data, together with the phenotypic characterization -fermentative kinetics and other biochemical characteristics carried out with the isolates by Aouizerat et al (2019)- suggest that these yeasts actually come from an environment related to alcoholic beverages. These authors even elaborated beer with these isolates and some of them, especially the Saccharomyces, gave a very good analytical and sensory result.

Fig 2 Saccharomyces_cerevisiae_SEM.jpg

Figure 2. Saccharomyces cerevisiae at the scanning electronic microscope (MD Murtey & P Ramasamy)


Aouizerat et al (2019) conclude that the isolates are descendants of yeasts that were originally used 3000 years ago, in large quantities and in repeated fermentations. This would have facilitated their survival in pore microenvironments of the ceramic matrix of these containers, and the microcolonies would have continued to grow minimally for millennia thanks to the humidity and residual nutrients. The authors make the analogy with some handmade beers where it is usual that the containers waste serve as starter for new productions.

Finally, the authors of this work speculate that it is possible to isolate microorganisms from archaeological remains, not only yeasts, and that in the case of bacteria it could even be easier, given the resistance characteristics of some of them, such as the sporulated ones.


Is there no previous similar work to that of Aouizerat et al (2019) ?

As we have seen, this is certainly a very surprising finding. Scientifically, the work is quite accurate and has been “approved” by the international community: the article is published in an open-access journal with prestige (mBio, high impact factor: 6.7), of the American Society for Microbiology, where all the articles are reviewed by a minimum of two experts, besides the editors. The results presented by the article seem very well worked, and the conclusions are well reasoned.

However, in my opinion it is still almost incredible, and it is strange that nothing like this has been found before. Maybe if someone else had previously tried to isolate such old microorganisms without getting them, perhaps it would not have been published ? Maybe nobody has previously tried to do something similar ? A “malicious” explanation might be that archaeologists have their own interests and microbiologists or molecular biologists have others, and that for this type of work the collaboration of both is needed. Well, it seems not being so, since there are a lot of studies on microorganisms from ancient remains, but they have been almost always focused on the detection and analysis of ancient DNA. These studies demonstrated the presence of certain microorganisms although they did not proceed to isolate them.


DNA gives evidence of microorganisms in ancient remains

In relation to yeasts, the oldest evidence is that ribosomal DNA of Saccharomyces cerevisiae has been obtained from residues found in Egyptian wine jars 5000 years old (Cavalieri et al 2003). It must be remembered that the oldest archaeological evidence of large-scale wine production has 7400 years, in north of the Zagros Mountains, in present-day Iran (McGovern et al 1986). As it is known, S. cerevisiae is also the bread and beer yeast, derived from cereals, but since neither S. cerevisiae nor its spores are aerial, surely the use of this yeast in fermented grape juice, as well as dates, figs or honey, historically preceded its use for brewing and bread (Cavalieri et al 2003). It is probable that the wine yeasts naturally occurring in damaged grapes (Mortimer & Polsinelli 1999) were used to ferment other cereal products such as cereals, and after centuries of selection for humans, they evolved into specific strains to ferment food and beverages from cereals.

The genomes of pathogenic microorganisms have also been studied in archaeological remains by means of new massive DNA sequencing techniques, in order to know to epidemic diseases of historical importance, such as black plague, tuberculosis, cholera or leprosy (Andam et al 2016). Logically, in these cases the archaeological remains are human ones, such as bones, teeth, coprolites or mummified tissues. In this way, for example, the phylogeny and evolution of Yersinia pestis strains causing the black plague have been recognized by remains of the Bronze Age (5000 years ago) and until the well-known epidemics of the 6th and 14th centuries (Bos et al 2011). Another well-known case is the Helicobacter pylori genome identified in the intestine of the Ötzi mummy, the iceman in the eastern Alps, 5300 years old (Maixner et al 2016).

DNA has also been isolated from specific bacteria of the human gut, such as Bifidobacterium and Bacteroides, to demonstrate the human presence in archaeological sediments 5000-12000 years old, in north east of Poland (Madeja et al 2009).

It should be remembered that DNA is degraded over time, and in fact it is more unstable than other cellular components. This macromolecule spontaneously suffers damage by oxidation, hydrolysis, and fragmentation in pieces that may be less than 100 bp. Most fossils or other biological remains of more than 100,000 years old no longer contain PCR-amplifiable DNA (Hofreiter et al 2001), although it seems that if the samples are extracted from frozen sediments, with constant temperatures below zero, DNA could be recovered from up to 400,000 years or a little longer (Willerslev et al 2003). In addition the tissues are colonized over time by fungi and bacteria that greatly reduce the relative amount of endogenous molecules and can contribute to giving false positives. The risk of contamination is very high and often this is not taken in account. Generally the DNA of the host that is analysed can be less than 1% of the total DNA found. All these factors complicate the DNA extraction, the construction of sequence libraries, the alignment of DNAs and the analysis of genomes (Andam et al 2016).

Surprisingly, there are a few published works where it is found old DNA of plants, animals and various microorganisms, some million years (My) old, even hundreds of My. The most remarkable are those obtained from amber samples of 20-40 My, and those obtained from a halite 250 My old. This would be comparable to the Jurassic Park fiction where almost non-degraded DNA from the dinosaurs of 100 My old “was recovered”.

Hebsgaard et al (2005) thoroughly reviewed all these more spectacular cases, with the conclusion that these works suffered from inadequate experimental approaches and inadequate authentication of the results. Therefore, there are great doubts as to whether DNA sequences and in some cases viable bacteria could survive such large geological times.

In addition, it is worrying that these works with so old DNA have not been replicated independently in order to confirm their authenticity, and that they did not show a relationship between the age of the sample and the persistence of DNA depending on the different types of bacteria (Willerslev et al 2004). In contrast, Willerslev et al studied the persistence of DNA in permafrost and they found a clear relationship of DNA degradation with time (Figure 3). As seen, DNA amount is very small beyond 100,000 years and it is hardly found beyond 1 My.

Fig 3 willerslev A

Figure 3. Persistence of not degraded bacterial DNA over time (kyr, thousands of years) maintained in permafrost, measured by fluorescence (Willerslev et al 2004).


When analysing the bacterial phyla of these DNA, Willerslev et al (2004) observed (Figure 4) that the most persistent are those of Arthrobacter, the main representative of Actinobacteria (high G+C gram-positive), followed by sporulated (Bacillaceae and Clostridiaceae), and finally the Gram-negative Proteobacteria.

Fig 4 willerslev D

Figure 4. Proportions of the main bacterial phyla (Actinobacteria in brown, sporulated in orange and Proteobacteria in blue) based on DNA obtained from permafrost samples, along time (kyr, thousands of years) (Willerslev et al 2004).


This increased persistence of non-sporulated Actinobacteria is surprising because sporulated bacteria have always been considered the most resistant of all types of cells. Although endospores have special adaptations such as proteins binding DNA to reduce the rate of genetic modifications, they do not have active metabolism or repair and their DNA will degrade over time. The mechanism of greater resistance of Actinobacteria is unknown, but there may be some activity and repair of DNA at temperatures below zero, and/or adaptations related to the dormant cells state (Willerslev et al 2004).

Anyway, the limit for PCR-amplifying the DNA would be between 400,000 years and 1.5 My for samples kept below zero, but this is much more unlikely in non-frozen materials, such as the amber of halite samples of million years, and much less likely to find viable cells from these samples so old (Willerslev et al 2004).


“Resurrected” bacteria

The same commented works where DNA of some millions of years (My) was found, are the most surprising cases of having “resurrected” microorganisms, basically bacteria: viable cells of the sporulated Bacillus from amber samples of 30 My (Cano & Borucki 1995), Staphylococcus also from amber of about 30 My (Lambert et al 1998), and the most spectacular case of Bacillus from an halite of 250 My (Vreeland et al 2000 ). This sporulated bacterium would have been in a hyper-saline environment of the last Permian and trapped in a salt crystal, surviving until now. In the case of Staphylococcus isolated from amber, in spite of not being sporulated, they are bacteria very resistant to extreme conditions, and which have been isolated also from ancient permafrost and very dry environments (Lambert et al 1998).

In spite of this, the revision of these cases by Hebsgaard et al (2005) concludes that none of them fulfilled the relative rate of molecular distance test, which is the probable rate of mutations calculated in comparison to related lineages. Therefore, these isolations are arguable and not reproduced. In addition, in the case of the 250 My Bacillus, it has been argued that the inclusion of bacteria in the halite could be the consequence of a subsequent recrystallization (Lowenstein et al 2011).

Another review on microorganism preservation records (Kennedy et al 1994) comments published cases up to 600 My, indicating that it is curious that there are several cases with more than 1 My, and also cases with less than 10,000 years ago, but there are very few cases of intermediate periods. These authors also point out the doubts raised by works with surviving bacteria so old, which would surely be artefacts or contaminations.

On the other hand, the most credible works are those of Abyzov et al (2006) and Soina et al (2004), which demonstrated the presence of several living microorganisms, both prokaryotes and eukaryotes (especially yeasts, but also some microalgae), in Antarctic ice samples that have some thousands of years. These authors combined classical microbiological methods, such as enrichment and isolation of colonies, together with epifluorescence microscopy, electronic microscopy, and molecular techniques. The bacteria found were Gram-positive (Micrococcus) and gram-negative (Arthrobacter), which are not sporulated, but they have cist-shaped dormant cells, which can survive while maintaining viability at temperatures below 0ºC for some thousands of years.

When geologically ancient DNA findings are published as well as viable cultures of ancient samples, the independent reproduction of the results by another laboratory is fundamental, to exclude any contamination from the same laboratory. In the case of having recovered living cells, it is necessary to demonstrate the reproducibility of the isolation, sequencing the genomes of the cultures obtained in independent laboratories from the same sample, and checking that in both cases the genomes coincide (Hebsgaard et al 2005).

From the remains of the Roman fort of Vindolanda, in the north of England, viable endospores of Thermoactinomyces, member of Bacillales (Unsworth et al 1977) have been recovered. They are about 1900 years old and the remains were a mixture of clay with straw and other vegetable materials. The authors propose to use these sporulated bacteria as indicators in archaeological studies.

Besides sporulated bacteria, there are several groups of non-sporulated ones for which anabiosis resistance abilities have been demonstrated. In particular, they have been isolated from permafrost and the tundra soil of Siberia of about 1 My (Suzina et al., 2006), in the limit of what we mentioned earlier (Willerslev et al 2004), which is quite difficult to believe. In order to study experimentally the formation of these anabiosis forms, Suzina et al incubated several gram-positive and gram-negative bacteria, and some archaea, in poor media with limiting nitrogen, and after a few months they obtained their dormant cells. They had cist structures, with capsule and a thickened cell wall, intramembranous particles and a condensed nucleoid (Figure 5). They also observed that these cysts did not have metabolic activity and supported stress factors such as lack of nutrients or heating.

Studying the permafrost isolates, they confirmed that there are cist structures very similar to those obtained in the laboratory, with multi-layer wall structures of up to 0.4 μm. In fact, these authors believe that most of the bacteria present in the permafrost and the tundra are in the form of a cyst (Suzina et al 2006).

Fig 5 fig2 modi Suzina

Figure 5. Sections of a vegetative cell (a) of Micrococcus luteus and of a cyst cell (b) of the same bacterium, obtained after 9 months of culture in a medium limiting in nitrogen. C, microcapsule; CW, cell wall; OL1, 2, 3, outer layers of the cell wall; IL, inner layer of the wall; CM, cytoplasmic membrane; N, nucleoid. The bar measures 0.3 μm (Suzina et al., 2006).


Other “resurrected” yeasts and fungi

Besides the surprising mentioned article by Aouizerat et al. (2019), there are other few published cases of yeasts and other “resurrected” fungi such as the following.

Chicha is a beer-like beverage from corn, yellowish and slightly effervescent, elaborated and consumed by Andean populations for some thousands of years, whose traditional process has the peculiarity of using amylase of saliva for convert the starch into fermentable sugars. Fermentation traditionally took place in clay containers called “pondos”. From the remains of the chicha pondos from the Hipia culture in Quito (2100-2800 years old), various yeast were isolated, especially Candida, Pichia and Cryptococcus (Gomes et al 2009). Interestingly, some of these yeasts have been confirmed molecularly as Candida theae, similar to those isolated from contaminated Asian tea (Chang et al 2012). It is worth mentioning the absence of Saccharomyces in these ancient chicha, although today it is the main yeast, coming probably from beer and wine fermentation that led the Spaniards (Gomes et al 2009).

From Greenland ice samples of about 100,000 years (Ma et al 1999), several microorganisms were revived, such as bacteria (Micrococcus, RhodotorulaSarcina) and yeasts (Candida, Cryptococcus) and other fungi (PenicilliumAspergillus). The authors also isolated the DNAs and demonstrated the phylogenetic relationship of the isolates. Once again, we see how ice provides a stable environment that facilitates the conservation of microorganisms and their DNA.

Raghukumar et al (2004) have recovered living Aspergillus (sporulated Ascomycota fungus) and other fungi from sediment samples of the deep-sea, about 5900 m deep in the Chagos trench, south of the Maldives, in the Indian Ocean. Based on the depth in the sediment and the present Radiolaria, authors estimated that they correspond to a minimum of 180,000 years, and up to 430,000 years in some samples. From the isolates identified as A. sydowii they obtained spores that germinated and grew in hydrostatic pressure equivalent to the depth of 5000 m, and at a temperature of 5ºC. With microscopy of epifluorescence and bright field, the fungal hypha and their relation to the particles of the sediment are clearly observed (Figure 6). It seems that this Aspergillus found in the deep-sea is the oldest fungus recovered alive so far. The authors suggest that preservation would have been possible thanks to high hydrostatic pressure, along with low temperature.

Fig 6 Raghukumar Aspergillus deepsea indian

Figure 6. Photomicrographies of deep-sea sediment (5900 m) of the Indian Ocean with hyphae of Aspergillus sydowii and sediment particles. (a) epifluorescence microscopy combined with that of bright field; (b) epifluorescence (Raghukumar et al 2004).


One of the most surprising works, and hard to believe, is that of Kochkina et al (2001), where a lot of fungi of all kinds and bacteria, especially actinobacteria, were isolated from samples of permafrost from Russia, Canada and Antarctica reaching 3 My old. The authors even suggested that there is no limit of years to recover viable microorganisms. This article has had very little echo, and it is not even mentioned by later articles as Raghukumar et al. (2004).



As we have seen, evidence of DNA from no-living yeasts in ancient remains related to winemaking dates back to around 5000 years in ancient Egypt (Cavalieri et al 2003). Regarding other microorganisms, taking into account the natural degradation of DNA over time, it seems that the oldest samples would be about 400,000 years at most, in particular actinobacteria in frozen sediments such as permafrost (Willerslev et al 2003 ). Publications of bacterial DNA recovered from several millions years (up to 600 My) have many scientific concerns about their credibility and reliability (Kennedy et al 1994).

With regard to living yeast as those of 3000 years apparently isolated by Aouizerat et al (2019), it seems that Candida and others were isolated from containers to elaborate chicha about 2800 years old (Gomes et al 2009), although this reference is a review and the original work does not appear to have been published. Other authors (Abyzov et al 2006; Soina et al 2004) also find alive yeasts, without specifying which ones, in Antarctic ice samples of some thousands of years. More surprising are the isolated isolations of yeast and other fungi and bacteria from Greenland ice samples 100,000 years old (Ma et al 1999), as well as those of Aspergillus from the Indian Ocean seabed of about 180,000 years (Raghukumar et to 2004).

Regarding other “resurrected” microorganisms, some of the most reliable are the several Antarctic ice bacteria of some thousands of years (Abyzov et al 2006) and Thermoactinomyces spores of Roman remains 1900 years old (Unsworth et to 1977). Of the oldest, perhaps the anabiosis forms of bacteria conserved in permafrost a million years old (Suzina et al., 2006) would have certain likelihood. Curiously, these bacteria would be non-sporulated but they would have a cyst structure, with multi-layer walls and other intracellular modifications. The other findings of “resurrected” bacteria from more millions of years of amber or halite, just like their DNA and also because of this, are very hard to believe (Hebsgaard et al 2005).

Thinking in the cellular forms of resistance and anabiosis, as the bacterial endospores and the mentioned cysts, it must be remembered that yeasts, like many other fungi, have the ability to produce spores, in particular ascospores as they are Ascomycetes. Although these ascospores have a greater capacity for resistance than vegetative cells in dry conditions or other inhospitable environments and have a persistence in time, apparently there is no work (or I have not been found) related to the recovery of yeasts ascospores from ancient remains.

The work of Aouizerat et al (2019) makes no mention of the yeast spores, neither as a possible explanation of the yeast survival in these ancient remains. In fact, they propose that the microcolonies of yeasts on ceramics pores would have continued to grow minimally during these 3000 years thanks to the humidity and residual nutrients. Well, we do not know, and neither if the yeast ascospores have had any role.

Finally, we can believe the finding of Aouizerat et al (2019) is truth, but obviously further investigation in other similar archaeological samples must be done. This research should be done not only for yeasts, but also for bacteria of other fermented products. Besides considering the sporulated ones, other bacteria should be considered, that could survive thanks to the cell cysts or other forms of anabiosis.



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Borschel-Dan A (2019) Israeli scientists brew groundbreaking “ancient beer” from 5,000-year-old yeast. The Times of Israel, 22nd may 2019.

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Cano, R.J. and Borucki, M.K. (1995) Revival and identification of bacterial spores in 25- to 40-million year-old Dominican amber. Science 268, 1060–1064

Cavalieri D et al (2003) Evidence for S. cerevisiae fermentation in ancient wine. J Mol Evol 57:S226-232

Chang CF et al (2012) Candida theae sp. nov., a new anamorphic beverage-associated member of the Lodderomyces clade. Int J Food Microbiol 153, 10-14.

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Bacteroides, our most abundant gram-negative bacteria

17th April 2019 

Translated from the original article in Catalan.

Update on this topic: see the work of the Yolanda Sanz group, IATA-CSIC, such as Gómez del Pulgar et al., 2020.

What are Bacteroides ?

Bacteroides is the best-known genus of the most abundant gram-negative bacterial group within us, specifically in the intestine. They are up to 8·1010 per gram of stool. They are strict anaerobes, non-sporulated, non-mobile, with a form of rod with rounded tips (Figure 1). They are resistant to bile salts, at the concentration of 20% of the small intestine, and they have a good ability to use polysaccharides.

Fig1 Gerard F2.large

Figure 1. Electronic micrograph of cells of Bacteroides sp. D8 (Gerard et al 2007)

First of all, it should be noted that there are excellent revisions of Bacteroides, such as that of Wexler (2007), describing their beneficial aspects in the intestinal microbiota, which we will comment on here, as well as the toxic aspects and other characteristics.

Bacteroides live exclusively in the gastrointestinal tract of animals, and therefore they show great flexibility to adapt to the nutritional conditions of the intestinal environment. As commensals and mutualists, they establish long-term partnerships with the guests and provide them with benefits. The adaptation of these bacteria includes making modifications to this environment. For instance, many Bacteroides code for cytochrome bd oxidase, which can reduce oxygen concentrations, making it easier for them to grow as strict anaerobes, and at the same time, other bacteria of the usual microbiota also benefit from this (Wexler, Goodman 2017).

The most common substrates of these bacteria are the vegetable polysaccharides of the diet and of host’s mucus (Wexler, Goodman 2017). These carbohydrates are degraded and fermented, producing mainly short-chain fatty acids (SCFA). Bacteroides are the main producers of propionate in intestinal tract, and this acid is one of the beneficial SCFA, together with acetate and butyrate, because they are an energy source for colonocytes and contribute to maintenance of the correct glucose homeostasis and lipid metabolism (Ríos-Covián et al 2017). Bacteroides also remove side chains from bile salts, facilitating the return of bile acids to liver circulation. On the other hand, another beneficial aspect is that they exclude other possible pathogens as they colonize the intestinal tract and do not let others settle.

Due to the fact that the animal’s intestinal tract is the main habitat and environmental reservoir of Bacteroides, it is thought that there has been a symbiotic evolutionary relationship between these bacteria and the hosts (Troy, Kasper 2010). As in many other evolutionary cases, this mutual commensalism between microorganisms and hosts is almost a symbiosis, where virtually each of the organisms cannot live without the other.

As habitual residents of the intestine, the vast majority of Bacteroides are not harmful, on the contrary. Nevertheless, in conditions of metabolic imbalances such as diabetes or surgical patients, some of them are opportunistic and can be pathogens, and some have a certain resistance to antibiotics. In fact, B. fragilis, the most abundant species in the microbiota of healthy people, can give in these cases very serious infections and is the most important anaerobic pathogen bacterium in humans (Mancuso et al 2005). The abundance of B. fragilis is evident even because their bacteriophages are used as tracers of human faecal matter in water (Jofre et al 1995).

What kind of bacteria is Bacteroides ?

As detailed in the NCBI Taxonomy section, the genus Bacteroides is a bacterium of the Fibrobacter-Chlorobi-Bacteroidetes superphylum. We can see its phylogenetic relationship with other bacterial groups in Figure 2. Bacteroidetes phylum also includes Cytophaga, Flavobacter and Sphingobacter, in addition to the Bacteroidia class, which mainly includes the Bacteroidales order. This includes 2 families: the Bacteroidaceae and the Prevotellaceae. Besides Bacteroides, Prevotella is another of the best-known genera, which in fact was previously known as B. melaninogenicus.

Fig2 Bern 12862_2004_Article_146_Fig1_HTML

Figure 2. Phylogenetic tree of the bacterial groups (Bern, Goldberg 2005).

Bacteroides, some of the predominant in the human intestinal microbiota

The human intestinal microbiota, and from mammals in general, is very complex, but surprisingly, there are few phyla that predominate. Specifically, 98% of identified bacteria in humans (Figure 3) belong to 4 phyla: 64% Firmicutes, 23% Bacteroidetes, 8% Proteobacteria and 3% Actinobacteria. Therefore, Bacteroidetes are one of the most predominant bacteria in the intestinal microbiota. In fact, since Firmicutes are such a large and diverse phylum, which includes microbes as diverse as clostridial and lactic acid bacteria, it can be considered that Bacteroidetes, as a much more homogeneous group, are practically the predominant ones.

Fig3 brock 767 modif

Figure 3. Bacterial composition of the human colon deduced from the 16S rRNA obtained from 17242 sequences of faecal samples (Madigan et al 2012)

To see in depth the predominant species of the intestinal microbiota, very recently, a metagenomic and functional study of 737 genomes sequenced from bacterial isolates of faecal samples from 20 British and American adults (Forster et al 2019) has been done. 273 bacterial species have been detected, of which 105 had not been found before. As we can see (Figure 4), among the 20 dominant species there are 8 Bacteroides, plus 2 Parabacteroides, that is 10 Bacteroidales, signalled in green. Therefore, they are half of the majority species. The other 10 are 6 clostridial (Firmicutes, in blue), 3 are Actinobacteria (in yellow) and 1 is Proteobacteria (in orange).

Fig4 Forster 2019 Fig4

Figure 4. Major species of the human intestinal microbiota, detected with metagenomic data analyses (Forster et al 2019).

Although the microbiota is different in each person, at the strain level the individual microbiota is very stable. In a study with 37 healthy people (Faith et al 2013) about 200 strains of 100 different species have been found, and 60% of the strains remain for each person in a period of 5 years. Of those that remain, those of Bacteroidetes and Actinobacteria are the most stable.

In the same study (Faith et al 2013), gut microbiota of 6 people in the same family have been compared and it has been found that among the 75 most common bacterial species in the 6 persons, 18 are Bacteroidetes (24%): 11 Bacteroides, 3 Parabacteroides, AlistipesBarnesiella, Odoribacter and Butyricimonas. The only species of the 75 found in everybody is a Bacteroides: B. vulgatus.

The microbiota that accompanies us is changing throughout life (Figure 5). In fact, there are relatively few Bacteroides in the babies. However, these bacteria are already present among the few microbes of the placenta, where Proteobacteria predominate (Aagard et al 2014). After the birth, Bacteroides are increasing over the first months and years, mainly with the weaning and diet changes, as microbial diversity increases. Then, in adults Bacteroides are ones of the most abundant microbes (Gómez-Gallego, Salminen 2016).

Fig5 GomezGallego fig 1

Figure 5. Changes in the human microbiota throughout life (Gómez-Gallego, Salminen 2016).

Solid food intake in children, between 4 months and 1 year, causes a significant increase in Bacteroidetes (Figure 6). We see the great difference in the microbial composition from 118 day to 370. It is a pity that in this study (Koenig et al 2011) no more intermediate samples were took between these days, where little by little children go from porridge and a bit of cereals, to the ingestion of peas and other legumes, carrots, potatoes, etc. This increase in Bacteroidetes with solid food is surely related to the fact that Bacteroidetes are specialists in the breakdown of complex polysaccharides, and at the same time these compounds promote their growth. At the same time, there is a clear increase in the levels of AGCC, an enrichment of microbial genes associated with the use of carbohydrates, a greater biosynthesis of vitamins, and also an increase of xenobiotic degradation. Therefore, the role of Bacteroidetes seems primordial in the establishment and maintenance of the adult’s microbiota. Even though there are differences between individuals, once adult, microbial composition is quite stable throughout life, with certain variations depending on changes in diet or habitat or medication.

Fig6 Koenig fig 3

Figure 6. Metagenomic analysis of DNA sequences extracted from faecal samples of children (Koenig et al 2011).


Bacteroides in other mammals

The intestinal microbiota is present in all animals with a more or less developed digestive system. Apart from the insects, whose microbiota has been deeply studied (Engel, Moran 2013), the most studied in this aspect are mammals, of course. Their composition has been studied (Ley et al 2008), specifically in faecal samples of 106 individuals of 60 species of 13 different taxa, including human, other primates, herbivores, carnivores and omnivores.

Of the 17 bacterial phyla found, 65% were Firmicutes, 16% Bacteroidetes, 8% Proteobacteria and 5% Actinobacteria, among others. Therefore, the relevance of the Bacteroides is evident, and the proportions are similar to those mentioned above for humans. Regarding the majority group of Firmicutes, it is a pity that this work, like others, does not distinguish between different groups, especially among lactic acid bacteria and Clostridiales. Curiously in this work there is a greater presence of Bacteroides in primates and omnivores in general, and also in some herbivores, than in carnivores (Figure 7). In these there are very few Bacteroides, and instead there are more gamma-Proteobacteria, probably enterobacteria (Ley et al 2008).

Fig7 Ley fig S1A

Figure 7. Percentage of faecal samples sequences of different mammals assigned to the main different bacterial phyla (Ley et al 2008)

Different Bacteroidales are biomarkers of lifestyles

In the search for microbial taxa that could be biomarkers of diets or lifestyles, it has been seen that the biomarker more clearly related with people from rich western countries is the genus Bacteroides, whereas to the sub-Saharan ones it is Prevotella, another one of the same phylum. These two genera, together with some from the clostridia group, are the most abundant ones.

If the long-term majority diet is rich in animal proteins and fats, as in Western countries, Bacteroides predominates, and if the diet is rich in carbohydrates like in sub-Saharan countries, Prevotella prevails (Gorvitovskaia et al 2016).

What about Bacteroides in cases of dysfunction?

The beneficial relevance of Bacteroides, or their group, Bacteroidetes,on health is obvious in cases of diseases or dysfunctions such as allergies or obesity (Figure 8), where the diversity of the microbiota is much lower, and the number of Bacteroidetes is low.

Fig8 GomezGallego fig 2

Figure 8. Changes in the microbiota in dysfunctional situations such as allergies and obesity. (Gómez-Gallego, Salminen 2016).

Bacteroides against obesity

Well-known experiments of intestinal microbiota in relation to obesity have been those carried out with mice without previous microbiota colonized with microbiota from human twins of which one was obese and the other lean (Ridaura et al 2013). The result was that the mice with obese twin microbiota (Ob) became obese, while those of lean twin microbiota remained lean (Ln) (Figure 9). In addition, in the lean mice a greater intestinal production of SCFA and a greater microbial transformation of the bile acids were observed, whereas in the obese there was a greater metabolism of branched amino acids.

As mentioned in the previous section, in the obese mice a reduction of 50% Bacteroidetes is observed, apart from an increase in Firmicutes and methanogens (Figure 10). And as we see the Archaea methanogens decrease the hydrogen, producing methane, and the lower level of hydrogen promotes fermentation of ingested food in excess by the Firmicutes.

Fig9 mice obese lean Kay Chersnush

Figure 9. Obese and lean mice resulting from colonization with gut microbiota from obese and lean human twins respectively (image of Kay Chernush / Getty Images).

Fig10 brock 768 modif

Figure 10. Differences in intestinal microbial communities between lean (left) and obese (right) mice (Madigan et al 2012).

The most surprising, however, of this work (Ridaura et al 2013) is the cohabitation experiment of the two types Ob and Ln mice, where it is observed that after 10 days of coexisting together, the obese have diminished their body fat (Figure 11), and when their microbiota have been studied by sequencing, a transfer of the microbiota from lean mice to obese is observed (Figure 12). As we can see, the main bacteria transferred are Bacteroidales, which strengthens the importance of these bacteria.

Fig11 ridaura change body

Figure 11. Adiposity (% body fat) of obese (Ob) and lean mice (Ln), and the same after 10 days of cohabitation in the same cage (Obch and Lnch) (Ridaura et al 2013).

Fig12 ridaura ob ln bacteroi

Figure 12. Demonstration of the transfer of Bacteroidales (7 species: 5 Bacteroides, 1 Parabacteroides and 1 Alistipes) of the intestinal microbiota of lean mice (Lnch)  to the obese (Obch) after 10 days of cohabitation in the same cage. Each column corresponds to a mouse (Ridaura et al 2013).

Bacteroides against cholesterol

It has been known for many years that the intestinal microbiota is able to convert cholesterol in its saturated form, coprostanol (Figure 13). In other mammals some Eubacterium (belonging to the clostridial group) have been found to be responsible, but in humans we did not know what microorganisms could do it. Recently Gérard et al (2007) have isolated a strain of human stool that is able to do it and has been identified as Bacteroides, probably a species close to B. vulgatus.

Fig13 Gerard colesterol

Figure 13. Formulas of cholesterol and coprostanol (Gerard et al 2007)

Glycans (polysaccharides), important for mutualism between Bacteroides and the human host

Most non-digested macromolecules that reach the colon are glycans (word virtually synonymous of polysaccharides), which are a very important part of the fibre. The only glycan that is practically digested previously in the small intestine is starch. The consortium of microorganisms that inhabit the colon produces a huge enzymatic repertoire with the ability to degrade a range of complex polysaccharides that the host cannot process. That’s why the intestinal microbiota is often referred to as a metabolic organ.

On the other hand, the abundant commensal microbes of the intestinal microbiota must resist the inhospitable conditions of the previous sections and to settle in the colon without affecting the host. Therefore, instead of interacting with the epithelial cells of the intestine, they remain in the external mucus layer on the epithelial surface. At the same time, this mucus protects resident microbes from attacks by other bacteria and bacteriophages, and it is a nutrient substrate. It has been shown that the ability to survive in this ecosystem is closely related to the use and production of glycans by resident bacteria (Comstock 2009).

Well, precisely this ability to interact with glycans is an important characteristic of Bacteroidales, which, as we have seen, are the most abundant microorganisms in the intestine, along with Firmicutes. In fact, Bacteroidales have an extensive enzymatic machinery to use the complex polysaccharides present in the colon, and use them as a source of carbon and energy. This great capacity has been proven by sequencing the genome of B. thetaiotaomicron (Xu et al 2003) where it has found containing more than 80 loci of polysaccharides that encode proteins related to the detection, importation and degradation of specific glycans of the colon.

As we can see (Figure 14), Bacteroides use both the glycans of the host’s diet and those produced by the intestinal epithelium, they metabolize them, and produce the beneficial SCFA, and on the other hand, they synthesize glycans that accumulate in the form of exopolysaccharide (EPS) contributing to form biofilms, and in capsules that give immune signals to the host (Comstock 2009). All in all, the relevance of the glycans in the mutual relations between Bacteroides and the human host is confirmed.

Fig14 Comstock F1

Figure 14. Use and production of glycans (polysaccharides) by Bacteroides. IM (inner membrane): cytoplasmic membrane; OM (outer membrane): external part of the gram-negative cell wall; EPS: exopolysaccharide of mucosal layers, not covalently linked, unlike the capsular polysaccharide (Comstock 2009).

In addition to the glycans produced by the host, some Bacteroides can also use those that produce other microorganisms of the microbiota, as shown by B. fragilis, the most frequent species on the surface of the intestinal mucosa, which can metabolize exopolysaccharides produced by bifidobacteria (Ríos-Covian et al 2016). EPS production for bifidobacteria is stimulated by bile. This ability of B. fragilis to use EPS of bifidobacteria gives them more survival capacity when nutrients are scarce. At the same time, the degradation of the EPS can affect the viability of the bifidobacteria, and therefore, Bacteroidales would have a regulatory role of the intestinal microbiota in general.

Some glycans produced by Bacteroidales have a beneficial effect on the host’s immune system. In particular, it has been seen that polysaccharide A (PSA) produced by B. fragilis is able to activate the immune response on T-cells dependent, which influences the development and homeostasis of the immune system (Troy, Kasper 2010). In fact, the colonization of germ-free mice (without microbiota) with B .fragilis is sufficient to correct the previous imbalance of cells Th1 and Th2 (T helper) (Figure 15). In addition, PSA can protect against colitis, such as those produced by Helicobacter, by repressing proinflammatory cytokines associated with another type of T cells -Th17- and other mechanisms (Mazmanian et al 2008).

Fig15 Troy Fig1 PSA B fragilis

Figure 15. Impact of polysaccharide A (PSA) of Bacteroides fragilis in the development of the immune system by recovering the balance of Th1/Th2 cells (Troy, Kasper 2010).

The diet can make Bacteroides contribute to a good metabolic balance

In relation to said above about glycans such as EPS, it has been seen that if in the environment there is little organic nitrogen and an easily fermentable carbon source such as glucose, Bacteroides produce more lactate and less propionate, and instead with more organic nitrogen (yeast extract) and polysaccharides, these bacteria produce more propionate (Ríos-Covián et al 2017). When EPS are present, as more complex carbohydrates and slowly fermented, the carbon of the amino acids can be incorporated at the level of pyruvate, and then the path to succinate and propionate is enhanced and the redox equilibrium is maintained. Since a higher propionate production is beneficial to the host, these authors conclude that in cases of host metabolic dysfunctions, a good diet design (complex carbohydrates with organic nitrogen) would help to modify metabolic activity of Bacteroides, and these would help promote healthy effects to the host, in addition to interacting with the other beneficial bacteria.

Bacteroides as probiotics?

EFSA (European Food Safety Authority) has not accepted virtually any claim of positive effects of probiotics on health due to the restrictive requirements of studies with humans. The mechanism of probiotics action is strain-dependent and often is not well known. In addition, it could be that the incorporated bacteria did not produce sufficient measurable changes in healthy individuals to obtain a claim of health effect. Further studies at the genetic level, antibiotic resistance profile and probiotic selection criteria are required.

Traditional probiotics are mostly Lactobacillus and Bifidobacterium, but also some strains of other lactic acid bacteria, and from Bacillus, E. coli and Saccharomyces. Besides these, the so-called “next generation” probiotics are being introduced, thanks mainly to new culture and sequencing techniques. Among these new possible probiotics, there are the verrucomicrobial Akkermansia muciniphila, and some clostridia (see my post), like Faecalibacterium prausnitzii, the main producer of butyrate, but also some Bacteroidales. These ones also have a clear advantage over clostridia and other Firmicutes, because are much more stable in the intestinal tract throughout the life of the person (Faith et al 2013).

As we have seen, being some of the most abundant microorganisms in our intestinal microbiota, Bacteroides generally have clear benefits for the host, such as fighting against obesity, or cholesterol. Transplants of faecal microbiota for diarrhea associated with Clostridium difficile infections are being successful (Van Nood et al 2013) and therefore there is a clear possibility of using some specific strain or several ones, and in this way the Bacteroides are clear candidates due to their abundance in the samples of faecal microbiota.

In addition to those mentioned, other benefits of Bacteroides are those related to the immune system, at the level of cytokines and T cells and development of antibodies, in order to treat intestinal colitis, immune dysfunction, disorders of metabolism and even cancer prevention (Tan et al 2019).

Apart from the benefits shown to the host, a bacterial strain must have unambiguous security status in order to be considered probiotic. In the case of Bacteroides, recently, a strain (DSM 23964) of B. xylanisolvens isolated from stools of healthy humans has been studied and it has been shown to have no virulence determinants which have been found in some opportunistic Bacteroides, such as the enterotoxin bft and enzymatic biodegradative activities of extracellular matrix and PSA. This strain does not have resistance to antibiotics – although it is resistant to some – and no plasmids have been detected, which makes the transfer of possible resistance very unlikely. Therefore, this strain seems very safe (Ulsemer et al 2012a). It has also been seen that it does not adhere to the walls of the intestine, but it resists the action of gastric enzymes and low pH. In addition, as indicated by the name of the species, it degrades xylan and other pectins. These heteropolysaccharides are prebiotics, compounds that are beneficial for the gut microbiota.

Other basic probiotic characteristics found in this strain of B. xylanisolvens are the production of SCFA and immunomodulatory properties. These properties and the safety and good tolerance of this strain have been verified by incorporating it in fermented milk, after inactivation by heat. This milk has been ingested in trials by healthy humans, with safe effects (Ulsemer et al 2012b). Its safety has also been confirmed in studies of toxicity in mice, where high doses of the strain have not produced toxic or mutagenic effects, neither haematological nor histopathological damage (Ulsemer et al 2012c).

On the basis of these studies, the European Food Safety Authority has given the approval as a new food of the use of fermented milks with B. xylanisolvens DSM 23964 pasteurized (EFSA 2015). However, there is no claim to consider it as a probiotic, especially because bacteria are not viable as the product has been pasteurized, and by definition, probiotics should be living microorganisms.


We have seen the relevance of Bacteroides as one of the main components of the human intestinal microbiota and mammals in general. In addition to its fundamental role in the intestine and the possibilities of its use as a probiotic, it is an ideal model for the study of gut bacteria, because it is relatively easy of cultivating and has the potential to be genetically manipulated (Wexler, Goodman 2017). Therefore, it is necessary to deepen the knowledge of Bacteroidales, and in particular to know how they metabolize the host’s nutrients or mucus, or how they respond to changes in the host’s diet, or how they interact with the other microorganisms of the digestive tract. A better understanding of all these mechanisms will favour the design of therapeutics aimed at modifying the microbiota of patients suffering from various diseases and metabolic disorders linked to the intestinal microbiota (Wexler, Goodman 2017).


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Plastic-eating bacteria

25th December 2018

Translated from the original article in Catalan.

Plastic ocean

We humans are destroying the planet Earth. Besides climate change (there are still ignorant people who do not believe it), the depletion of natural resources and the massive extinction of animal and plant species, one of the most visual effects is the coverage of the planet with rubbish. Since 71% of the surface is marine, most of the non-degrading waste finishes in the sea. In the oceans there are already large expansions covered by floating debris, especially plastics, called “plastic islands” (Figure 1). In the North Pacific area, where different sea currents come together, the “island” reaches 1500 km of radius, with plastics up to 200 meters deep, and continues to grow. There is more information of it, and also about the environmental consequences, in the Wikipedia article Great Pacific garbage patch.

F1 great-pacific-garbage-patch

Figure 1. Small portion of the Great Pacific Garbage Patch (From oceanandreserveconservationalliance.com)


PET plastics

Although there are many types of plastics, one of the most used and most abundant in waste and “plastic islands” is polyethylene terephthalate, known as PET or PETE (Figure 2). It is a type of thermoplastic polymer, vulgarly plastic, which belongs to the so-called polyesters, and is obtained by synthesis from petroleum. It is harmless, very resistant and lightweight and has multiple applications (Figure 3). Counting only bottles of PET for refreshing beverages, 1 million of them per minute are sold in the world. It is a recyclable material (see Pet bottle recycling in Wikipedia) but very resistant to biodegradation. In nature it can last some hundreds of years.

F2 PET molecular structure

Figure 2. PET, polyethylene terephthalate.


F3 pet uses www.technologystudent.com

Figure 3. Several applications of PET (From http://www.technologystudent.com).


PET is “eaten” by Ideonella sakaiensis

I. sakaiensis (Figure 4) are bacteria with rod shape, gram-negative, non esporulate aerobic heterotrophic, mobile with a flagellum, and catalase (+) and oxidase (+) (Tanasupawat et al 2016). They grow at neutral pH and are mesophilic, with optimum at 30-37°C. They belong to the phylogenetic group of betaproteobacteria, which include, besides many others, the known Neisseria (gonorrhoea and meningitis) and the nitrifying Nitrosomonas.

F4 Ideonella-sakaiensis falsecolorSEM Yoshida S

Figure 4. Scanning electron microscope images (false colour) of Ideonella sakaiensis cells grown on PET film for 60 h (From Yoshida et al 2016).


The 201-F6 strain, the first of the new species I. sakaiensis, was isolated from a landfill and identified in 2016 by a Japanese group of the Kyoto Institute of Technology that looked for bacteria using plastic as carbon source, from samples of remains of PET bottles (Yoshida et al 2016). They saw that these bacteria adhere to a low-grade PET film and can degrade it, by means of two enzymes characterized by these authors: a PETase and a MHETase, which produce terephthalic acid and ethylene glycol acid (Figure 5), which are benign environmental substances and that the bacteria can be metabolized. A colony of I. sakaiensis completely degraded a low-grade PET bottle in 6 weeks. High-grade PET products need to be heated to weaken them before the bacteria can degrade them. This is the first bacterium found as a PET degrader, and uses it as the only carbon source and energy source. Since PET has existed only for 70 years, these bacteria should have evolved in this short period until being able to degrade PET in a few weeks, instead of hundreds of years in nature (Sampedro 2016).

F5 Yoshida fig 3 right

Figure 5. Predicted metabolic pathway of PET degradation by I. sakaiensis: extracellular PETase hydrolyses PET giving monohydroxyethyl terephthalic (MHET) and terephthalic acid (TPA). MHETase hydrolyses MHET to TPA and ethylene glycol (EG). The TPA is incorporated through a specific transporter (TPATP) and is catabolized to cyclohexadiene and this to protocatechuic acid (PCA) by the DCDDH. Finally, the PCA ring is cut by a PCA 3.4 dioxygenase with oxygen, as known for degradation of phenolic compounds and other xenobiotics. The numbers in parentheses are the ORF of the corresponding genes (From Yoshida et al 2016).


Previously, only some tropical microfungi (Fusarium solani) were known to degrade PET, and they also excreted esterases. In this case, Fusarium would be used to modify the polyester fabric, to achieve more hydrophilic and easier to work (Nimchua et al 2008). It is important to remember the structural similarity of synthetic PET fabrics (Figure 3) to those of natural fibre such as cotton, since these contain cutin, which is a polyester, a waxy polymer from the external parts of the plants. Therefore, the enzymes of Fusarium or Ideonella must be relatively similar to those that were already in nature long before the plastics were invented.


Recent genetic improvement of the enzyme PETase of Ideonella sakaiensis

In order to better understand the function and specificity of the PETase, a group of American and British researchers have recently characterized the structure of this enzyme (Austin et al 2018), mainly by high resolution X-ray crystallography, comparing it with a homologous cutinase obtained from actinobacteria Thermobifida fusca. The main differences between the two have been a greater polarization in the surface of the PETase (pI 9.6) than in the cutinase (pI 6.3), and on the other hand (Figure 6), a greater width of the active-site cleft in the case of PETase of I. sakaiensis. The cleft widening would be related with an easy accommodation of aromatic polyesters such as PET.

F6 austin fig 2 modif

Figure 6. Compared structures (left) of the PETase of I. sakaiensis (above) and the cutinase of actinobacterium Thermobifida fusca (below), obtained by high resolution X-ray crystallography (0.92 Å). The active-site cleft is marked with a red dotted circle. Details (right) of the active site with different cleft widths in the PETase of I. sakaiensis (above) and the cutinase of T. fusca (below) are shown. (From Austin et al 2018).


Hypothesizing that the structure of the active site of the PETase would have resulted from a similar cutinase in an environment with PET, Austin et al (2018) proceeded to make mutations in the PETase active-site to make it more similar to cutinase and obtained a double mutant S238F/W159H which theoretically would make the entry of the active site closer (Figure 6). But their surprise was capital when they saw that the mutant degraded the PET better (an improvement of 20%), with an erosion of the PET film (Figure 7 C) even greater than the original PETase (Figure 7B). The explanation was that mutant changes in amino acid residues favoured PET intake in the active site, despite making a closest cleft (Austin et al 2018).

F7 austin fig 3 modif

Figure 7. Scanning electronic microscopy images of a piece of PET without microorganisms (A), after incubating 96 h with PETase of the I. sakaiensis 201-F6 (B), and with PETase of the double-mutant S238F/W159H (C) (From Austin et al 2018).


In addition, these authors have shown that this PETase degrades also other similar semi aromatic polyesters, such as polyethylene-2,5-furonicarboxylate (PEF), and therefore this enzyme can be considered an aromatic polyesterase, but it does not degrade aliphatic ones.

The conclusion of their work is that protein engineering is feasible in order to improve the performance of PETase and that we must continue to deepen in the knowledge of their relationships between structure and activity for the biodegradation of synthetic polyesters (Austin et al 2018).


Other plastic-eating microbes ?

The discovery of I. sakaiensis has been very important for the possibility of establishing a rapid recycling process for PET, but it is not the first organism that has been found as plastic consumer. By the way, we can see the formulas of the main plastics derived from petroleum in Figure 8.

F8 Shah 2008 Fig 1

Figure 8. Formulas of the most common petroleum plastics: polyethylene (PE), polyvinyl chloride (PVC), polypropylene (PP), polystyrene (PS), polyethylene terephthalate (PET or PETE) and polyurethane (PU) (From Shah et al 2008).


Reviewing the bibliography, we see that many cases of plastic degrading microorganisms have been described (Shah et al 2008), especially polyethylene, polyurethane and PVC: various PseudomonasRhodococcus and Comamonas among bacteria, and some Penicillium, Fusarium and Aspergillus between fungi.

Among the polyurethane consumers, mushrooms are highlighted (Howard 2002), and especially the plants endophyte Pestalotiopsis microspora, which can use polyurethane as the only source of carbon (Russell et al 2011).

On the other hand, the ability of the mealworms, the larval forma of the darkling beetle Tenebrio molitor, to chew and degrade the polystyrene foam is well known (Yang et al 2015). Fed only with the PS, these larvae degrade it completely in relatively short periods. As expected, the degradation of the PS is carried out by the intestinal bacteria of the animal (Figure 9). It has been demonstrated because degradation stops when administering antibiotics to the larva (Yang et al 2015). One of the isolated bacteria that has been shown to degrade PS is Exiguobacterium, from Bacillales group, but it is not the only one. In fact, when performing studies of metagenomics from gut of larvae eating PS, a large variety of bacteria have been found, and these vary depending on the kind of plastic, since the degradation of polyethylene has also been seen. Some of the bacteria with DNA found as predominant would be the enterobacteria Citrobacter and Kosakonia. It seems that the intestinal microbiota of Tenebrio is modified and adapted to the different ingested plastics (Brandon et al 2018).

F9 fig Abs Yang 2015 2

Figure 9. Biodegradation of polystyrene by the intestinal bacteria of Tenebrio, the mealworm (Yang et al 2015).


Finally, as we see the microbial biodegradation of non-biodegradable or recalcitrant plastics should not surprise us, since on the one hand, there are natural “plastics” such as polyhydroxybutyrate or polylactic acid that are easily degradable (Shah et in 2008), and on the other hand the adaptive capacity of the microorganisms to be able to break the most recalcitrant chemical bonds is very large. Microbes evolve rapidly, and acquire better strategies to break the plastics made by humans (Patel 2018). We have seen in this case the degradation of PET, which in less than 70 years some microbes have already found a way to take advantage of it.

The problem is that we are generating too much plastic waste in no time and the microorganisms have not had time yet to degrade them. It is clear that we will have to help our microbial partners, not generating more degrading polymers, and recycling and degrading them, by using these same degrading microbes, among other ways.


Austin HP et al (2018) Characterization and engineering of a plastic-degrading aromatic polyesterase. Proc Nat Acad Sci 115, 19, E4350-E4357

Brandon AM et al (2018) Biodegradation of Polyethylene and Plastic Mixtures in Mealworms (Larvae of Tenebrio molitor) and Effects on the Gut Microbiome. Environ Sci Technol 52, 6526-6533

Griggs MB (2017 april 24) These caterpillars chow down on plastic bags. Popular Science. http://www.popsci.com

Howard GT (2002) Biodegradation of polyurethane: a review. Int Biodeterior Biodegrad 42, 213-220




Patel NV (2018 april 17) Scientists stumbled upon a plastic-eating bacterium – then accidentally made it stronger. Popular Science. http://www.popsci.com

Russell JR et al (2011) Biodegradation of polyester polyurethane by endophytic fungi. Appl Environ Microbiol 77, 17, 6076-6084

Sampedro J (2016 marzo 10) Descubierta una bacteria capaz de comerse un plástico muy común. El País

Shah AA et al (2008) Biological degradation of plastics: a comprehensive review. Biotechnol Adv 26, 246-265

Tanasupawat et al (2016) Ideonella sakaiensissp. nov., isolated from a microbial consortium that degrades poly(ethylene terephtalate). Int J Syst Evol Microbiol 66, 2813-2818

Yang et al (2015) Biodegradation and mineralization of polystyrene by plastic-eating mealworms: Part 2. Role of gut microorganisms. Environ Sci Technol 49, 12087-12093

Yoshida et al (2016) A bacterium that degrades and assimilates poly(ethylene terephthalate). Science 351,1196–1199

Lactic acid bacteria of beers: the bad guys and the good ones

28th October 2018

It is not easy to “live” in the beer

In principle, lactic acid bacteria (LAB) and many other bacteria and generally most microorganisms, do not have it easy to survive in beer or other alcoholic beverages such as wine. This is one of the main reasons why wines and beers have been from ancient times the safest ways to drink hygienically something similar to water and that it was not contaminated, apart from boiled waters, such as tea and other herbal infusions.

The reasons for the difficult survival of microorganisms in beer are ethanol, the pH quite acidic (around 4), the lack of nutrients due to the fact that the yeasts have assimilated them, the little dissolved oxygen, the high concentration of carbon dioxide (0.5% by weight / volume) and the presence of humulone derived compounds (Figure 1) of hops: iso-alpha-acids, up to 50 ppm, which are microbiocides. All these obstacles make it very difficult for any microorganism to thrive. The most susceptible beers of unwanted microbial growth are those where some of the mentioned obstacles are dampened: beers with a higher pH of 4.5, or with little ethanol or little CO2, or with added sugars – which are nutrients -, or with little amount of compounds derived from hops (Vriesekoop et al 2012).

Fig 1 512px-S-Humulone_Isomerization.svg

Figure 1. Humulone (left) of the hop is degraded during beer elaboration to isohumulone (right) and other iso-alpha-acids, which are compounds bitter and microbiocides (Wikipedia; Sakamoto & Konings 2003)


The acid pH of the beer (slightly higher than the wine) inhibits many of the best-known pathogens (Figure 2). And the cases we see that could grow at this pH near 4 are inhibited by other factors such as ethanol.

Fig 2 Menz 2009 jib49-fig-0002-m

Figure 2. Range of acid pH for the growth of various bacteria, compared to the typical beer pH (Menz et al 2009).


The “bad” lactic acid bacteria of beer

Despite what we have just seen, some bacteria, particularly some LAB, have been able to adapt evolutionarily to the strict beer conditions, and they can survive and spoil them. In particular, the most frequent harmful species against the quality of beers are Lactobacillus brevis and Pediococcus damnosus (Figure 3). The first is the most frequent, and it can give tastes and undesired aromas, as well as turbidity to the final product. P. damnosus has the advantage of growing at low temperatures, and it can also produce undesired aromas, such as diacetyl (Vriesekoop et al 2012). Some Pediococcus and Lactobacillus may adhere to yeast, inducing them to sediment, which delays fermentation (Suzuki 2011).

Fig 3 brevis i pedio.png

Figure 3. Lactobacillus brevis (left) and Pediococcus damnosus (right) at the electronic scanning microscope.


Some Pediococcus may also be responsible for the appearance of biological amines in some beers, at risk for the consumer. Amines in a certain concentration are toxic, they may be present in some fermented foods such as cheese, cold meat and alcoholic beverages such as wines and beers, and are produced by decarboxylation of amino acids by LAB. The level of tyramine and other amines has been used as a measure of quality in some Belgian beers made with LAB (Loret et al 2005).

Apart from these LAB, other bacteria related to problems of beer contamination are acetic acid bacteria such as Acetobacter, typically associated with oxygen intake in packaging or distribution. Other harmful bacteria are some enterobacteria, such as Shimwellia pseudoproteus or Citrobacter freundii, which proliferate in the early stages of fermentation, and produce butanediol, acetaldehyde and other unwanted aromatic compounds (Vriesekoop et al 2012). Other harmful bacteria for beer, especially when bottled, are Pectinatus and Megasphaera, which are strict anaerobes, of the clostridial family, and can produce hydrogen sulphide and short chain fatty acids, all of them unpleasant (Suzuki 2011 ).


The “good” lactic acid bacteria of beer

LAB are well known for being some of the microbes that most benefits contribute to the food production, on the one hand as an economic means of preserving food, and on the other hand to improve their quality and organoleptic characteristics. That’s why they are the main agents of fermented foods, along with yeasts. We have seen some of the LAB’s food benefits in other posts in this blog: prehistoric cheeses, or breast milk microbiota, and even wine bacteria.

Therefore, LAB also have a good role in the production of beers: in particular, as we will see below, in the production of acidified malt, and in some peculiar styles of beer such as the Belgian Lambic and the Berliner Weissbier.

As you know, malt is the raw material for making beer. The cereal is subjected to the malting process, where cereal grains, mainly barley, are germinated, the enzymes hydrolyse the starch into sugars, and all of this is then heated obtaining the must, the substrate solution which will be fermented by the yeasts ferment, producing ethanol and carbon dioxide.

The acidification of the malt, that is, with a lower pH, has the advantages of activating many important enzymes in malting, giving a lower viscosity to the malt and therefore to the final beer. Although adding mineral acids or commercial lactic acid can achieve acidification, it is often recommended or legislated a biological acidification, which is achieved by adding LAB. The use of LAB starter cultures is a relatively new process and in addition to the commented benefits on the quality of the malt, it has been shown to also inhibit unwanted molds that are a real problem in malting and that can give mycotoxins. The compounds produced by LAB that can inhibit the fungi are the same lactic acid and the consequent pH drop, bacteriocins, hydrogen peroxide, and other compounds not well known as perhaps some peptides (Lowe & Arendt 2004).

The most commonly LAB strains used to acidify malt are Lactobacillus amylolyticus previously isolated from the same malt. These strains are moderately thermophile, resistant to compounds derived from humulone, and they have the advantage of being amylolytic in addition to producing lactic acid, which lowers the pH (Vriersekoop et al 2012).

Beers with LAB participating in the fermentation, such as Lambic and Berliner Weissbier styles, belong to the type of spontaneous fermentation beers. The other types of controlled fermentation beers are the best-known Ale and Lager, both inoculated with specific yeasts. Ale beers are those of high fermentation, where Saccharomyces cerevisiae yeast used tends to remain on the surface and the fermentation temperature is above 15-20ºC. Lager ones are those of low fermentation, originally from Bavaria, where yeast S. pastorianus (S. carlsbergensis) tends to settle at the bottom of the fermenter and the temperature is between 7 and 13ºC.


Belgian Lambic beer

Traditional Belgian beers (in Dutch lambiek or lambik) are known for their sensorial characteristics due to LAB activity. They are traditional in Brussels itself and in the neighbouring region of Pajottenland, in the Zenne river valley, in the Flemish Brabant on the SW of the Belgian capital. One of the villages in this valley is Lembeek, which could be the origin of the name of this beer.

These beers of spontaneous fermentation represent the oldest style of making beer in the developed world, for some centuries. For a few years now (since around 2008), similar beers are made in the USA, called “American coolship ales” (Ray 2014).

Lambic beer is made with barley malt and a minimum of 30% of non-malted wheat. The cones of a special hops, completely dried and aged for 3 years, are added to the must. They are added not for their aroma or bitterness, but rather as antimicrobial, to prevent above all, the growth of gram-positive pathogenic bacteria in the fermentation broth.

Also to avoid these contaminants and to promote the microbiota typical of the Lambic fermentation, these beers are brewed only between October and May, since in summer there are too many harmful microorganisms in the air that could spoil the beer, and it is necessary to lower the temperature after boiling. Boiling of the must is done intensively, with an evaporation of 30%.

After boiling, the broth is left in open deposits, and in this way the microorganisms of the air present in the fermentation rooms of the brewery (usually at the top of the building) are acquired, and of the outside air, since the tradition says that the windows must be left open. It is assumed that the captured microbes are specific to the Zenne Valley. These open deposits are the koelschip in Dutch (coolship in English), like swimming pools (Figure 4). Being well open, with a lot of surface (about 6 x 6 m) and shallow depth (about 50 cm), they favour the collection of microbes from the room and from the outside. Another purpose of this form is the fastest cooling of boiled broth to start fermentation. They can be made of wood, copper, or stainless steel more recently.


Figure 4. Koelschip (in Dutch) or coolship in English, the open deposits, as swimming-pools, where the Lambic beer process begins (Brasserie Cantillon, Brussels).


The “inoculated” broth in this spontaneous way is left only one night in the coolship, and on the following day this must is pumped into fermentation tanks where there will stay a year, during which the sugar content will go down, up to about 30 g/L. Then it is transferred to oak barrels, previously used for sherry or port, and there it can be left for another two years, at temperatures of 15-25ºC. Some barrels are the same used since 100 years ago. The final product is a cloudy beer, with a pale yellow, very little CO2, dry, acidic, with about 6-8º of ethanol. It reminds a bit like the sherry and especially the cider, and with a slightly bitter taste (Jackson 1999).

In this long process of fermentation, up to 3 years, of course there is a diversity in the composition of the microbial population. In a first phase there is a certain predominance of Kloeckera yeasts and especially enterobacteria during the first month. After 2 months, Pediococcus damnosus and Saccharomyces spp. predominate, and alcoholic fermentation begins. After 6 months of fermentation the predominant yeast is Dekkera bruxellensis (Spitaels et al 2014), or what is the same, Brettanomyces (Kumara & Verachtert 1991), of which Dekkera is the sexual form.

Fig 5 Spitaels fig3

Figure 5. Species of isolates in MRS and VRBG agar media, for lactic acid bacteria and enterobacteria respectively, during the process of making a Lambic beer. The number of isolates is given between brackets (Spitaels et al 2014).


We see (Figure 5) as in particular after 2 months the predominant bacterium is the LAB P. damnosus. It was appointed in the first studies as “P. cerevisiae“, but this name was finally not admitted because it included other species. The count of these in MRS is 104UFC per mL until the end of fermentation. Acidification seems to be rapidly taking place in the transition from the first stage to that of maturation, coinciding with the growth of P. damnosus, which produces lactic acid, although Dekkera/Brettanomyces and acetic acid bacteria also contribute to the acidification (Spitaels et to 2014).

In other trials with the American coolship ales (ACA) of Lambic style, Lactobacillus spp. have also been found, and in a metagenomic study (Bukolich et al 2012) of these ACA, DNA of several Lactobacillales has been detected. At the end of the process, a predominance of Pediococcus (Figure 6, panel C) was also observed. In the same figure in panel A we observe how the predominant unicellular fungus is also Dekkera/Brettanomyces.

Fig 6 Bukolich fig 2

Figure 6. TRFLP analysis (polymorphisms of lengths of PCR-amplified terminal restriction fragments) of total DNA extracted from the fermentation samples of ACA beers (similar to Lambic) during 3 years, using primers for: ITS1/ITS4 of 26S rDNA for yeasts (panel A), 16S rDNA for bacteria (panel B), and specific ones for LAB (panel C). Samples marked with * did not give amplification (Bukolich et al 2012).


Lambic derived beers: Gueuze, Faro, fruity and others

The basic Lambic, which is difficult to purchase, is only found in a few Brussels cafes and the production area. In fact, Lambic is the basis for elaborating the others, much more common to consume:

The Faro is a Lambic sweetened with brown sugar and sometimes with spices.

The fruity Lambic are those that have been added whole fruits or fruit syrup. They can be with bitter cherry (kriek), which are the most traditional, or with raspberry, peach, grapes, strawberry, and sometimes also apple or pineapple or apricot or other.

And finally, the Gueuze, which are sparkling and easy to find. They are made by mixing young Lambics (from 6 months to 1 year) with other more mature ones (2-3 years) in thick glass bottles similar to those of champagne or cava and left for a second fermentation with the remaining sugars from the young Lambic. This would have been begun by a mayor of Lembeek in 1870 that owned a brewery and applied the fermentation techniques in the bottle that had been successful in the Champagne some years before (Cervesa en català 2012). The word Gueuze can have the same etymological origin as gist(yeast in Flemish) and it could also refer to the fact that it produces bubbles of CO2, that is, gas (Jackson 1999). However, another historical version would be that this beer was called “Lambic de chez le gueux” (Welsh from poor people) because the mentioned mayor of Lembeek had similar socialist ideas to those of the “Parti des Gueus” founded by the Calvinists from Flanders in the 16th century to fight against the Spanish empire. And since beer is feminine in French, the gueuxfeminine is gueuze, here it is.

In this refermentation in the bottle the populations of Dekkera/Brettanomycesand LAB are maintained, although other unicellular fungi such as CandidaHansenula, Pichia or Cryptococcus (Verachtert & Debourg 1999) appear in limited numbers.

Fig 7 lambics3 swanbournecellars

Figure 7. Several beer Gueuze and fruity Lambic, mostly Belgian (from www.swanbournecellars.com.au/).


The Berliner Weissbier (Figure 8) is another beer relatively similar to Lambic ones. It is also brewed with an important part of wheat must, it is cloudy, acidic and with 3% ethanol. It is traditional in Berlin and the north of Germany, made from the s. XVI and the most popular alcoholic beverage in Berlin until the end of the s. XIX. It was called the “northern champagne” by the Napoleon’s soldiers. Spontaneous fermentation of must involves a mixture of Dekkera/Brettanomyces, Saccharomycesand hetero-fermentative Lactobacillus.

Fig 8 Berliner Weissbier boozedancing_Fotor

Figure 8. Berliner Weisse beer (from G-LO, @boozedancing wordpress).


Beers similar to Lambic brewed in Spain

In the same way that the commented American Coolship Ales, Lambic style beers are also made in many other countries and, in the case of Spain, coinciding with the boom of artisanal beers, they are also elaborated, especially the fruity Lambic ones. According to the Birrapedia website, 6 of these are currently being processed, all of which are cherries. Two of them are made in Lleida, one in Barcelona, one in Alicante, one in the Jerte valley, and another in Asturias.


Resistance of lactic acid bacteria from beer to hop compounds

Lactobacillus and Pediococcus, both bad and good we have seen, and other contaminating bacteria of beers, have the ability to withstand hop compounds, which, as we have seen, are natural microbiocides. This resistance can be due to various defence systems, both active and passive (Sakamoto & Konings 2003). The active systems include efflux pumps, such as HorA and HorC, which carry the iso-alpha-acids (Figure 1) out the cell. HorA does it with ATP consumption, and HorC using the proton driving force (Figure 9). The corresponding genes horA and horC were originally found in L. brevis, but later they were also found in L. lindneri, L. paracollinoides and in the best known P. damnosus(Suzuki et al., 2006).

Curiously, HorA shows a resemblance of 54% to OmrA, a membrane transporter of Oenococcus oeni, related to the tolerance of this bacterium from wine to ethanol and other stressors (Bourdineaud et al 2004) (See some more about O. oeni in my post on the bacteria of the vine and the wine). Therefore, it is probable that HorA also has functions of exclusion of other compounds aside from those of the hops. It has been seen that these horAand horC resistance genes and their flanking regions are well preserved and have sequences almost identical to the different species that have them. Therefore, it is very likely that some have been acquired from others by means of horizontal gene transfer, by plasmids or transposons, as is usual in many other bacteria (Suzuki 2011).

Fig 9 brevis Suzuki fig 8

Figure 9. Mechanisms of resistance to hop compounds in Lactobacillus brevis (Suzuki 2011).


As we see in Figure 9, protons are pumped out by an ATPase, and the consumption of ATPs is compensated by forming it thanks to the consumption of substrates such as citrate, malate, pyruvate or arginine. Another mechanism of resistance, passive in this case, is the modification of the composition of membrane fatty acids, with the addition of more saturated ones, such as C16:0, which reduces the membrane fluidity and makes it difficult the entrance of the hop compounds. This also reminds us of the changes in membrane of O. oeni related to the resistance to ethanol (Margalef-Català et al 2016). The cell wall also changes its composition in the presence of the hop alpha-iso-acids, increasing the amount of high molecular weight lipoteichoic acid, which would also be a barrier. We also see (Figure 9) how hop compounds can lower the intracellular levels of Mn2+, and then a greater synthesis of Mn-dependent proteins is observed, and a greater capture of Mn2+ from outside. Finally, cells of L. brevis reduce their size when they are in beer (Figure 10), probably in order to decrease the extracellular surface, thus minimizing the effect of external toxic compounds (Suzuki 2011).

Fig 10 brevis mida Suzuki

Figure 10. Effects of beer adaptation (left) in the size of Lactobacillus brevis cells compared to well grown cells in rich media MRS (right). The bars are 5 mm (Suzuki 2011).


All these mechanisms have been studied in L. brevis strains harmful to beer, but it is assumed that the resistance of beneficial bacteria from Lambic and others would be due to the same mechanisms, since they are of the same bacterial species.

As a conclusion to all said, we see that LAB have outstanding roles as beneficial in various aspects of brewery and malting, despite their most known role of harmful in the processing of the most common beers.



Birrapedia (seen 18 august 2018) Cervezas de tipo Fruit Lambic elaboradas en España. https://birrapedia.com/cervezas/del-tipo-fruit-lambic-elaboradas-en-espana

Bokulich NA et al (2012) Brewhouse resident microbiota are responsible for multi-stage fermentation of American Coolship Ale. PLoS One, 7, e35507

Bourdineaud J et al (2004) A bacterial gene homologous to ABC transporters protect Oenococcus oeni from ethanol and other stress factors in wine. Int J Food Microbiol 92, 1-14.

Cervesa en català (2012) Fitxes de degustació – Timmermans Gueuze Tradition http://cervesaencatala.blogspot.com.es/2012/06/fitxes-de-degustacio-timmermans-gueuze.html

Jackson, Michael (1999) Belgium’s great beers. Beer Hunter Online, July 30, 1999

Kumara HMCS & Verachtert H (1991) Identification of Lambic super attenuating micro-organisms by the use of selective antibiotics. J Inst Brew 97, 181-185

Loret S et al (2005) Levels of biogenic amines as a measure of the quality of the beer fermentation process: data from Belgian samples. Food Chem 89, 519-525

Lowe DP & Arendt EK (2004) The use and effects of lactic acid bacteria in malting and brewing with their relationships to antifungal activity, mycotoxins and gushing: a review. J Inst Brew 110, 163-180

Margalef-Català et al (2016) Protective role of glutathione addition against wine-related stress in Oenococcus oeni. Food Res Int 90, 8-15

Menz G et al (2009) Pathogens in beer, in Beer in Health and Disease Prevention, (Preedy, V. R. Ed.), 403–413, Academic Press, Amsterdam

Ray AL (2014) Coolships rising: the next frontier of sour beers in the U.S.  First we feast 27 feb 2014

Sakamoto K & Konings WN (2003) Beer spoilage bacteria and hop resistance. Int J Food Microbiol 89, 105-124

Spitaels F et al (2014) The microbial diversity of traditional spontaneously fermented lambic beer. PLOS One 9, 4, e95384

Suzuki K et al (2006) A review of hop resistance in beer spoilage lactic acid bacteria. J Inst Brew 112, 173-191

Suzuki K (2011) 125th Anniversary Review: microbiological instability of beer caused by spoilage bacteria. J Inst Brew 117, 131-155

The Beer Wench (2008) My obsession with wild beers. Nov. 20, 2008 https://thecolumbuswench.wordpress.com/tag/lambic/

Verachtert H & Debourg A (1999) The production of gueuze and related refreshing acid beers. Cerevisia, 20, 37–41

Vriesekoop F et al (2012) 125th Anniversary review: Bacteria in brewing: the good, the bad and the ugly. J Inst Brew 118, 335-345


Bacillus as probiotics

12th August 2017

The probiotics

Probiotics are living microorganisms that, when ingested in adequate amounts, can have a positive effect on the health of guests (FAO / WHO 2006; World Gastroenterology Organization 2011, Fontana et al., 2013). Guests can be humans but also other animals. Lactic acid bacteria, especially the genus Lactobacillus and Bifidobacterium, both considered as GRAS (Generally recognized as safe), are the microbes most commonly used as probiotics, but other bacteria and some yeasts can also be useful. Apart from being able to be administered as medications, probiotics are commonly consumed for millennia as part of fermented foods, such as yoghurt and other dairy products (see my article “European cheese from 7400 years ago..” “December 26th, 2012). As medications, probiotics are generally sold without prescription, over-the-counter (OTC) in pharmacies.

I have already commented on the other posts of this blog the relevance of probiotics (“A new probiotic modulates microbiota against hepatocellular carcinoma” August 24th, 2016), as well as the microbiota that coexists with our body (“Bacteria in the gut controlling what we eat” October 12th, 2014; “The good bacteria of breast milk” February 3rd, 2013) and other animals (“Human skin microbiota … and our dog” December 25th, 2015; “The herbivore giant panda …. and its carnivore microbiota” September 30th, 2015).

Besides lactic acid bacteria and bifidobacteria, other microorganisms that are also used to a certain extent as probiotics are the yeast Saccharomyces cerevisiae, some strains of Escherichia coli, and some Bacillus, as we will see. Some clostridia are also used, related to what I commented in a previous post of this blog by March 21st, 2015 (“We have good clostridia in the gut ...”).


The Bacillus

In fact, Bacillus and clostridia have in common the ability to form endospores. And both groups are gram-positive bacteria, within the taxonomic phylum Firmicutes (Figure 1), which also includes lactic acid bacteria. However, bacilli (Bacillus and similar ones, but also Staphylococcus and Listeria) are more evolutionarily closer to lactobacillalles (lactic acid bacteria) than to clostridia ones. The main physiological difference between Clostridium and Bacillus is that the first are strict anaerobes while Bacillus are aerobic or facultative anaerobic.

Fig 1 tree gram+ eng

Figure 1. Phylogenetic tree diagram of Gram-positive bacteria (Firmicutes and Actinobacteria). Own elaboration.


Bacterial endospores (Figure 2) are the most resistant biological structures, as they survive extreme harsh environments, such as UV and gamma radiation, dryness, lysozyme, high temperatures (they are the reference for thermal sterilization calculations), lack of nutrients and chemical disinfectants. They are found in the soil and in the water, where they can survive for very long periods of time.

Fig 2 bacillus Simon Cutting

Figure 2. Endospores (white parts) of Bacillus subtilis in formation (Image of Simon Cutting).


Bacillus in fermented foods, especially Asian

Several Bacillus are classically involved in food fermentation processes, especially due to their protease production capacity. During fermentation, this contributes to nutritional enrichment with amino acids resulting from enzymatic proteolysis.

Some of these foods are fermented rice flour noodles, typical of Thailand and Burma (nowadays officially Myanmar). It has been seen that a variety of microorganisms (lactic acid bacteria, yeasts and other fungi) are involved in this fermentation, but also aerobic bacteria such as B. subtilis. It has been found that their proteolytic activity digests and eliminates protein rice substrates that are allergenic, such as azocasein, and therefore they have a beneficial activity for the health of consumers (Phromraksa et al. 2009).

However, the best-known fermented foods with Bacillus are the alkaline fermented soybeans. As you know, soy (Glycine max) or soya beans are one of the most historically consumed nourishing vegetables, especially in Asian countries. From they are obtained “soy milk”, soybean meal, soybean oil, soybean concentrate, soy yogurt, tofu (soaked milk), and fermented products such as soy sauce, tempeh, miso and other ones. Most of them are made with the mushroom Rhizopus, whose growth is favoured by acidification or by direct inoculation of this fungus. On the other hand, if soy beans are left to ferment only with water, the predominant natural microbes fermenting soy are Bacillus, and in this way, among other things, the Korean “chongkukjang” is obtained, “Kinema” in India, the “thua nao” in northern Taiwan, the Chinese “douchi”, the “chine pepoke” from Burma, and the best known, the Japanese “natto” (Figure 3). Spontaneous fermentation with Bacillus gives ammonium as a by-product, and therefore is alkaline, which gives a smell not very good to many of these products. Nevertheless, natto is made with a selected strain of B. subtilis that gives a smoother and more pleasant smell (Chukeatirote 2015).

These foods are good from the nutritional point of view as they contain proteins, fibre, vitamins, and they are of vegetable or microbial origin. In addition, the advertising of the commercial natto emphasizes, besides being handmade and sold fresh (not frozen), its probiotic qualities, saying that B. subtilis (Figure 4) promotes health in gastrointestinal, immunologic, cardiovascular and osseous systems (www.nyrture.com). They say the taste and texture of natto are exquisite. It is eaten with rice or other ingredients and sauces, and also in the maki sushi. We must try it !


Figure 3. “Natto”, soybeans fermented with B. subtilis, in a typical Japanese breakfast with rice (Pinterest.com).

Fig 4 Bs nyrture-com micrograf electro colorejada

Figure 4. Coloured electronic micrograph of Bacillus subtilis (Nyrture.com).


Bacillus as probiotics

The endospores are the main advantage of Bacillus being used as probiotics, thanks to their thermal stability and to survive in the gastric conditions (Cutting 2011). Although Clostridium has also this advantage, its strict anaerobic condition makes its manipulation more complex, and moreover, for the “bad reputation” of this genus due to some well-known toxic species.

Unlike other probiotics such as Lactobacillus or Bifidobacterium, Bacillus endospores can be stored indefinitely without water. The commercial products are administered in doses of 10^9 spores per gram or per ml.

There are more and more commercial products of probiotics containing Bacillus, both for human consumption (Table 1) and for veterinary use (Table 2). In addition, there are also five specific products for aquaculture with several Bacillus, and also shrimp farms are often using products of human consumption (Cutting 2011).

For use in aquaculture, probiotic products of mixtures of Bacillus (B. thuringiensis, B. megaterium, B. polymixa, B. licheniformis and B. subtilis) have been obtained by isolating them from the bowel of the prawn Penaeus monodon infected with vibriosis. They have been selected based on nutrient biodegradation and the inhibitory capacity against the pathogen Vibrio harveyi (Vaseeharan & Ramasamy 2003). They are prepared freeze-dried or microencapsulated in sodium alginate, and it has been shown to significantly improve the growth and survival of shrimp (Nimrat et al., 2012).

As we see for human consumption products, almost half of the brands (10 of 25) are made in Vietnam. The use of probiotic Bacillus in this country is more developed than in any other, but the reasons are not clear. Curiously, as in other countries in Southeast Asia, there is no concept of dietary supplements and probiotics such as Bacillus are only sold as medications approved by the Ministry of Health. They are prescribed for rotavirus infection (childhood diarrhoea) or immune stimulation against poisoning, or are very commonly used as a therapy against enteric infections. However, it is not clear that clinical trials have been carried out, and they are easy-to-buy products (Cutting 2011).


Table 1. Commercial products of probiotics with Bacillus, for human consumption (modified from Cutting 2011).

Product Country where it is made Species of Bacillus
Bactisubtil ® France B. cereus
Bibactyl ® Vietnam B. subtilis
Bidisubtilis ® Vietnam B. cereus
Bio-Acimin ® Vietnam B. cereus and 2 other
Biobaby ® Vietnam B. subtilis and 2 other
Bio-Kult ® United Kingdom B. subtilis and 13 other
Biosporin ® Ukraine B. subtilis + B. licheniformis
Biosubtyl ® Vietnam B. cereus
Biosubtyl DL ® Vietnam B. subtilis and 1 other
Biosubtyl I and II ® Vietnam B. pumilus
Biovicerin ® Brazil B. cereus
Bispan ® South Korea B. polyfermenticus
Domuvar ® Italy B. clausii
Enterogermina ® Italy B. clausii
Flora-Balance ® United States B. laterosporus *
Ildong Biovita ® Vietnam B. subtilis and 2 other
Lactipan Plus ® Italy B. subtilis *
Lactospore ® United States B. coagulans *
Medilac-Vita ® China B. subtilis
Nature’s First Food ® United States 42 strains, including 4 B.
Neolactoflorene ® Italy B. coagulans * and 2 other
Pastylbio ® Vietnam B. subtilis
Primal Defense ® United States B. subtilis
Subtyl ® Vietnam B. cereus
Sustenex ® United States B. coagulans

* Some labelled as Lactobacillus or other bacteria are really Bacillus


Table 2. Commercial products of probiotics with Bacillus, for veterinary use (modified from Cutting 2011).

Product Animal Country where it is made Species of Bacillus
AlCare ® Swine Australia B. licheniformis
BioGrow ® Poultry, calves and swine United Kingdom B. licheniformis and B. subtilis
BioPlus 2B ® Piglets, chickens, turkeys Denmark B. licheniformis and B. subtilis
Esporafeed Plus ® Swine Spain B. cereus
Lactopure ® Poultry, calves and swine India B. coagulans *
Neoferm BS 10 ® Poultry, calves and swine France B. clausii
Toyocerin ® Poultry, calves, rabbits and swine Japan B. cereus


The Bacillus species that we see in these Tables are those that really are found, once the identification is made, since many of these products are poorly labelled as Bacillus subtilis or even as Lactobacillus (Green et al. 1999; Hoa et al. 2000). These labelling errors can be troubling for the consumer, and especially for security issues, since some of the strains found are Bacillus cereus, which has been shown to be related with gastrointestinal infections, since some of them produce enterotoxins (Granum & Lund 1997; Hong et al. 2005)

The probiotic Bacillus have been isolated from various origins. For example, some B. subtilis have been isolated from the aforementioned Korean chongkukjang, which have good characteristics of resistance to the gastrointestinal tract (GI) conditions and they have antimicrobial activity against Listeria, Staphylococcus, Escherichia and even against B. cereus (Lee et al. 2017).

One of the more known probiotics pharmaceuticals is Enterogermina ® (Figure 5), with B. subtilis spores, which is recommended for the treatment of intestinal disorders associated with microbial alterations (Mazza 1994).

Figuresv1 copy.ppt

Figure 5. Enterogermina ® with spores of Bacillus subtilis (Cutting 2011)


Bacillus in the gastrointestinal tract: can they survive there ?

It has been discussed whether administered spores can germinate in the GI tract. Working with mice, Casula & Cutting (2002) have used modified B. subtilis, with a chimeric gene ftsH-lacZ, which is expressed only in vegetative cells, which can be detected by RT-PCR up to only 100 bacteria. In this way they have seen that the spores germinate in significant numbers in the jejunum and in the ileum. That is, spores could colonize the small intestine, albeit temporarily.

Similarly, Duc et al. (2004) have concluded that B. subtilis spores can germinate in the gut because after the oral treatment of mice, in the faeces are excreted more spores that the swallowed ones, a sign that they have been able to proliferate. They have also detected, through RT-PCR, mRNA of vegetative bacilli after spore administration, and in addition, it has been observed that the mouse generates an IgG response against bacterial vegetative cells. That is, spores would not be only temporary stagers, but they would germinate into vegetative cells, which would have an active interaction with the host cells or the microbiota, increasing the probiotic effect.

With all this, perhaps it would be necessary to consider many Bacillus as not allochthonous of the GI tract, but as bacteria with a bimodal growth and sporulation life cycle, both in the environment and in the GI tract of many animals (Hong et al. 2005).

Regarding the normal presence of Bacillus in the intestine, when the different microorganisms inhabiting the human GI tract are studied for metagenomic DNA analysis of the microbiota, the genus Bacillus does not appear (Xiao et al., 2015). As we can see (Figure 6), the most common are Bacteroides and Clostridium, followed by various enterobacteria and others, including bifidobacteria.

Fig 6 Xiao nbt.3353-F2

Figure 6. The 20 bacterial genera more abundant in the mice (left) and human (right) GI tract (Xiao et al. 2015).


In spite of this, several species of Bacillus have been isolated from the GI tract of chickens, treating faecal samples with heat and ethanol to select only the spores, followed by aerobic incubation (Barbosa et al. 2005). More specifically, the presence of B. subtilis in the human microbiota has been confirmed by selective isolation from biopsies of ileum and also from faecal samples (Hong et al. 2009). These strains of B. subtilis exhibited great diversity and had the ability to form biofilms, to sporulate in anaerobiosis and to secrete antimicrobials, thereby confirming the adaptation of these bacteria to the intestine. In this way, these bacteria can be considered intestinal commensals, and not only soil bacteria.


Security of Bacillus as probiotics

The oral consumption of important amounts of viable microorganisms that are not very usual in the GI treatment raises additional doubts about safety. Even more in the use of species that do not have a history of safe use in foods, as is the case of sporulated bacteria. Even normal bowel residents may sometimes act as opportunistic pathogens (Sanders et al. 2003).

With the exception of B. anthracis and B. cereus, the various species of Bacillus are generally not considered pathogenic. Of course, Bacillus spores are commonly consumed inadvertently with foods and in some fermented ones. Although Bacillus are recognized as GRAS for the production of enzymes, so far the FDA has not guaranteed the status of GRAS for any sporulated bacteria with application as a probiotic, neither Bacillus nor Clostridium. While Lactobacillus and Bifidobacterium have been the subject of numerous and rigorous tests of chronic and acute non-toxicity, and a lot of experts have reviewed data and have concluded that they are safe as probiotics, there is no toxicity data published on Bacillus in relation to their use as probiotics. When reviewing articles on Medline with the term “probiotic” and limited to clinical studies, 123 references appear, but Bacillus does not appear in any of them (Sanders et al. 2003).

Instead, there are some clinical studies where Bacillus strains have been detected as toxigenic. All this explains that some probiotic Bacillus producers refer to them with the misleading name of Lactobacillus sporogenes, a non-existent species, as can be seen from NCBI (https://www.ncbi.nlm.nih.gov/taxonomy/?term = Lactobacillus + sporogenes).

Finally, we should remember the joint report on probiotics of FAO (United Nations Food and Agriculture Organization) and WHO (World Health Organization) (FAO / WHO 2006), which suggests a set of Guidelines for a product to be used as a probiotic, alone or in the form of a new food supplement. These recommendations are:

  1. The microorganism should be well characterized at the species level, using phenotypic and genotypic methods (e.g. 16S rRNA).
  2. The strain in question should be deposited in an internationally recognized culture collection.
  3. To evaluate the strain in vitro to determine the absence of virulence factors: it should not be cytotoxic neither invades epithelial cells, and not produce enterotoxins or haemolysins or lecithinases.
  4. Determination of its antimicrobial activity, and the resistance profile, including the absence of resistance genes and the inability to transfer resistance factors.
  5. Preclinical evaluation of its safety in animal models.
  6. Confirmation in animals demonstrating its effectiveness.
  7. Human evaluation (Phase I) of its safety.
  8. Human evaluation (Phase II) of its effectiveness (if it does the expected effect) and efficiency (with minimal resources and minimum time).
  9. Correct labelling of the product, including genus and species, precise dosage and conservation conditions.



The use of Bacillus as probiotics, especially in the form of dietary supplements, is increasing very rapidly. More and more scientific studies show their benefits, such as immune stimulation, antimicrobial activities and exclusive competition. Their main advantage is that they can be produced easily and that the final product, the spores, is very stable, which can easily be incorporated into daily food. In addition, there are studies that suggest that these bacteria may multiply in GI treatment and may be considered as temporary stagers (Cutting 2011).

On the other hand, it is necessary to ask for greater rigor in the selection and control of the Bacillus used, since some, if not well identified, could be cause of intestinal disorders. In any case, since the number of products sold as probiotics that contain the sporulated Bacillus is increasing a lot, one must not assume that all are safe and they must be evaluated on a case-by-case basis (Hong et al. 2005).



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Casula G, Cutting SM (2002) Bacillus probiotics: Spore germination in the gastrointestinal tract. Appl Environ Microbiol 68, 2344-2352.

Chukeatirote E (2015) Thua nao: Thai fermented soybean. J Ethnic Foods 2, 115-118.

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Duc LH, Hong HA, Barbosa TM, Henriques AO, Cutting SM (2004) Characterization of Bacillus probiotics available for human use. Appl Environ Microbiol 70, 2161-2171.

FAO/WHO (2006) Probiotics in food. Health and nutritional properties and guidelines for evaluation. Fao Food and Nutrition Paper 85. Reports of Joint FAO/WHO expert consultations.

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Hoa, N. T., L. Baccigalupi, A. Huxham, A. Smertenko, P. H. Van, S. Ammendola, E. Ricca, A. S. Cutting (2000) Characterization of Bacillus species used for oral bacteriotherapy and bacterioprophylaxis of gastrointestinal disorders. Appl Environ Microbiol 66, 5241–5247.

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Nimrat S, Suksawat S, Boonthai T, Vuthiphandchai V (2012) Potential Bacillus probiotics enhance bacterial numbers, water quality and growth during early development of white shrimp (Litopenaeus vannamei). Veterinary Microbiol 159, 443-450.

Phromraksa P, Nagano H, Kanamaru Y, Izumi H, Yamada C, Khamboonruang C (2009) Characterization of Bacillus subtilis isolated from Asian fermented foods. Food Sci Technol Res 15, 659-666.

Sanders ME, Morelli L, Tompkins TA (2003) Sporeformers as human probiotics: Bacillus, Sporolactobacillus, and Brevibacillus. Compr Rev Food Sci Food Safety 2, 101-110

Vaseeharan, B., P. Ramasamy (2003) Control of pathogenic Vibrio spp. by Bacillus subtilis BT23, a possible probiotic treatment for black tiger shrimp Penaeus monodon. Lett Appl Microbiol 36, 83–87

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Xiao et al. (2015) A catalogue of the mouse gut metagenome. Nature Biotechnol 33, 1103-1108.

Fig 0 pinterest-com cool bacillus-subtilis-science-comics


A new probiotic modulates gut microbiota against hepatocellular carcinoma

24th August 2016

Over the last years the beneficial effects of the human intestinal microbiota on various health markers have been displayed, such as inflammation, immune response, metabolic function and weight. The importance of these symbiotic bacteria of ours has been proved. You can see these other posts related with our microbiota: “The good clostridia avoid us from allergies“, “Gut bacteria controlling what we eat” or “Good bacteria of breast milk

At the same time it has been seen that probiotics can be a good solution for many diseases with affected gut microbiota. Indeed, the beneficial role of probiotics to reduce gastrointestinal inflammation and prevent colorectal cancer has been proven.

However, recently it has been found that probiotics may have beneficial effects in other parts of the body beyond the gastrointestinal tract, particularly with immunomodulatory effects on an hepatocellular carcinoma (HCC). In this way, researchers at the University of Hong Kong, along with other from University of Eastern Finland, have published a study (Li et al, PNAS, 2016), where they have seen reductions of 40% in weight and size of HCC liver tumours in mice which were administered with a new mixture of probiotics, “Prohep.”

Hepatocellular carcinoma (HCC) is the most common type of liver cancer is the 2nd most deadly cancers, and it is quite abundant in areas with high rates of hepatitis. In addition, sorafenib, the drug most widely used to reduce the proliferation of tumour, is very expensive. The cost of this multikinase inhibitor is €3400 for 112 tablets of 200 mg, the recommended treatment of four pills a day for a month. Instead, any treatment with probiotics that would proved to be effective and could replace this drug would be much cheaper.

The new probiotics mix Prohep consists of several bacteria: Lactobacillus rhamnosus GG (LGG), Escherichia coli Nissle 1917 (ECN) and the whole inactivated by heat VSL#3 (1: 1: 1) containing Streptococcus thermophilus, Bifidobacterium breve, Bf. longum, Bf. infantis, Lb. acidophilus, Lb. plantarum, Lb. paracasei and Lb. delbrueckii.

In the mentioned work, Li et al. (2016) fed mice with Prohep for a week before inoculating them with a liver tumour, and observed a 40% reduction in tumour weight and size in comparison to control animals. As shown in Figure 1, the effect was significant at 35 days, and also for those who were given the Prohep the same day of tumour inoculation. Obviously, the effect of tumour reduction was much more evident when the antitumour compound Cisplatin was administered.

These researchers saw that tumour reduction was due to the inhibition of angiogenesis. This is the process that generates new blood vessels from existing ones, something essential for tumour growth. In relation to the tumour reduction, high levels of GLUT-1 + hypoxic were found. That meant that there was hypoxia caused by the lower blood flow to the tumour, since this was 54% lower in comparison to controls.


Fig 1 Li-Fig1B tumor size - days tumor

Figure 1. Change in tumour size. ProPre: administration of Prohep one week before tumour inoculation; ProTreat: administration of Prohep the same day of tumour inoculation; Cisplatin: administration of this antitumoral. (Fig 1B from Li et al, 2016).


These authors also determined that there was a smaller amount of pro-inflammatory angiogenic factor IL-17 and of Th17 cells of the immune system, cells also associated with cancer. The lower inflammation and angiogenesis could limit the tumour growth.

Moreover, these researchers established that the beneficial effects of probiotics administration were associated with the abundance of beneficial bacteria in the mice gut microbiota, analysed by metagenomics. So, probiotics modulate microbiota, favouring some gut bacteria, which produce anti-inflammatory metabolites such as cytokine IL-10 and which suppress the Th17 cell differentiation.


Fig 2 gut microbiota Eye of Science

Figure 2. Bacteria of the human intestinal microbiota seen by scanning electron microscope (SEM) (coloured image of Eye of Science / Science Source)


Some of the bacteria identified by metagenomics in the microbiota of mice that were administered with Prohep were Prevotella and Oscillibacter. The first is a bacteroidal, gram-negative bacterium, which is abundant in the microbiota of rural African child with diets rich in carbohydrates. Oscillibacter is a gram-positive clostridial, known in humans as a producer of the neurotransmitter GABA. Both are an example of the importance of some clostridial and bacteroidals in the gut microbiota. In fact, they are majority there, and although they are not used as probiotics, are found increasingly more positive functions, such as avoiding allergies (see “The good clostridia avoid us from allergies“).

It is known that these bacteria produce anti-inflammatory metabolites and therefore they would be the main involved in regulating the activity of immune cells that cause tumour growth. The observed reduction of tumour in these experiments with mice would be the result of combined effect of these administered probiotic bacteria together with the microbiota itself favoured by them. We see a potential outline of these actions in Figure 3.

Fig 3 Sung fig 2

Figure 3. Simplified diagram of the possible mechanisms of gut bacteria influencing on the polarization of Th17 cells in the lamina propria of the intestinal mucosa. The microbiota bacteria activate dendritic cells, which secrete cytokines (IL-22, IL-23, IL-27). The bacteria can promote Th17 immunity inducing IL-23, which can be involved by means of TLR ligands signal or extracellular ATP or serum amyloid A (SAA). Meanwhile, some probiotic strains could inhibit the development of Th17 by means of the production of IL-12 and IL-27, in addition to promoting the growth and colonization of the bacteria that induce Th17 (Sung et al 2012, Fig. 2).


Although we know that the cancer progression is a very complex process and that in the tumour microenvironments there is an infiltration of many different types of immune system cells, such as T cells, neutrophils, killer cells, macrophages etc, the Th17 helper cell subpopulation appears to be prevailing in the tumour progression, and therefore these effects of probiotics and microbiota open good prospects.

It is still early to say whether these findings will contribute to the treatment of human liver cancer, and therefore research in humans is needed, in order to see if these probiotics could be used as such or in tandem with some drug, depending on the tumour stage and size. In any case, all this opens a new range of possibilities for research of the molecular mechanisms of the beneficial effects of probiotics beyond the intestinal tract.



El-Nezami H (2016 april 27) HKU develops novel probiotic mixture “Prohep” that may offer potential therapeutic effects on liver cancer. The University of Hong Kong (HKU) 27 Apr 2016

El-Nezamy H, Lee PY, Huang J, Sung YJ (2015) Method and compositions for treating cancer using probiotics. Patent WO 2015021936 A1

Li J, Sung CYJ, Lee N, Ni Y, Pihlajamäki J, Panagiotou G, El-Nezami H (2016) Probiotics modulated gut microbiota suppresses hepatocellular carcinoma growth in mice. PNAS E1306-E1315

Oelschlaeger TA (2010) Mechanisms of probiotic actions – A review. Int J Med Microbiol 300, 57-62

Packham C (2016) Probiotics dramatically modulate liver cancer growth in mice. Medical Press, Med Research 23 Feb 2016

Silgailis M (2016) Treating some cancers with probiotics in the future ? Probiotic Prohep. Lacto Bacto: Health, Microbes and More 23 Feb 2016

Sung CYJ, Lee NP, El-Nezami H (2012) Regulation of T helper by bacteria: an approach for the treatment of hepatocellular carcinoma. Int J Hepatology ID439024, doi:10.1155/2012/439024

UEF News and Events (2016) A novel probiotic mixture may offer potential therapeutic effects on hepatocellular carcinoma. University of Eastern Finland 1 Mar 2016


The effect of rapid climate change in the Arctic ecosystem

March 20th, 2016

The Arctic Ocean

Interestingly and coincidentally, “Arctic” comes from the Greek word αρκτος -arctos-, which means “bear” and is a reference to the constellation Ursa Minor, where is the North Star, which indicates the geographic North Pole .

The Arctic constitutes a unique ecosystem of the Earth, consisting of a large ice field, or ice-covered ocean, sometimes regarded as the northern part of the Atlantic Ocean, and it is surrounded by land, which is permafrost, with complete absence of trees. Life in the Arctic consists of organisms adapted to ice, including zooplankton and phytoplankton, fish, marine mammals, birds, land animals, plants and human societies fully adapted to the extreme conditions of the environment.

Due to global warming, isotherms are moving northward at a rate exceeding 50 km per decade over the past 30 years, so if we define the Arctic from a defined temperature or the tree line, its size is diminishing, being the reduction of sea ice the most visible effect.


Anthropogenic climate change: global warming, especially in the Arctic


Yes: climate change is here and it is generated by human activities, that is, it is anthropogenic. Previously there have been on Earth fluctuations in global temperature caused by natural phenomena, usually long-term and cyclical variations. For example, glaciations since about 2 million years are repeated every 100,000 years, and last ice age ended 15,000 years ago. So we are living now in an interglacial period and the next ice age could become not before 50,000 years. The cause of this cycle of glaciations seems to be orbital variations of the Earth, resulting in a lower insolation in high latitudes of the northern hemisphere during glacial periods.

Solar activity, like other stars, has cycles and roughly every 600 years there are periods of little activity (absence of very few solar spots and auroras), with lower energy output, which corresponds to cold periods in the Earth’s climate. The last minimal was in the period 1645-1715, and therefore from the middle of the eighteenth century we enjoy a maximum solar activity, with small cycles of minimum and maximum every 11 years.

Discounting these natural variations, it is clear that throughout the 20th century and especially since the 1960s there has been a steady increase in global average temperature (Figure 1), reaching almost 1ºC more than the beginning of the 20th century. In the early years of the current century the trend is worsening. The last 10 years have been the warmest since there are records, and the forecast is to continue increasing. Most experts agree that humans exert a direct impact on the heating process known as the greenhouse effect. The causes of this effect are some of gaseous components of the atmosphere, especially CO2, which has grown in parallel with rising temperatures, from about 300 ppm at the beginning of 20th century to nearly 400 ppm today. This CO2 and other gases as water vapour, methane and other exclusively anthropogenic absorb radiation and the result is that the atmosphere warms further.

Fig 1 gistemp_preI_2015 reg Temp

Figure 1. Increase in average global temperature compared to the beginning of 20th century (from GISTEMP).


This global warming is particularly evident in the Arctic. The temperature increases are higher in northern latitudes, especially 60-70º N, where this past December 2015 (Figure 2) have raised to 9ºC above average in large areas of North America and Eurasia. This is called Polar Warming Amplification (PWA). The cause of this overheating in the Arctic respect of the rest of Earth is partly due to the loss of snow and ice (retroactive effect) because the largest area of land and water absorbs more solar energy than white ice (albedo effect), but also the PWA is partly due to the dynamic atmospheric transport, which transports heat energy from the clouds and subtropical regions to the north (Taylor et al 2013).

Fig 2 GISTEMP planisferi

Figure 2. Thermal anomaly registered in December 2015 with respect to the average 1951-1980 (from GISTEMP).


Besides the consequences of this warming on the Arctic ice that we will comment below, another serious problem is the melting of permafrost, since then methane gas trapped under the frozen ground is released. This way, vast quantities of methane are released, and this greenhouse gas is contributing further to accelerate the global warming.


Less and less ice in the Arctic

Linear trends of sea ice extent and sea ice in the Arctic from 1979 to date are negative year after year, for any month is considered, but it is more clear by comparing Septembers, at the end of the summer when the ice is melting (Figure 3). Of the approximately 7 million km2 minimum in September (the maximum in March is about 16 million), about 100,000 km2 are melt per year, almost 9% every 10 years (Serreze et al 2007), so that there is now almost half ice than in 1979 (Figure 4).

Fig 3 seaice1979vs2012 The Cryosphere Today

Figure 3. Comparison of the extent of sea ice (in red): September 1979 and 2012 (from The Cryosphere Today).

Fig 4 fig 7 Reeves mod

Figure 4. Average monthly extension of Arctic sea ice since 1979 (Reeves et al 2013).


In addition to the reduction in surface ice, keep in mind the reduction in volume, representing now a third of what it was in September 1979.

There is a big difference between the different models for predicting the disappearance of Arctic sea ice. Half of them expect the total disappearance by September 2100. Predictions move since September 2040 the less optimistic until well past 2100 for the other (Serreze et al 2007).

Other problems resulting from the disappearance of sea ice are the ship traffic, which could shorten distances trips between the ports of northern countries, and on the other hand the exploitation of oilfields and other fossil fuels and minerals, since there is a large part of global reserves in the Arctic (Figure 5).

Fig 5 reeves figs 4 i 5

Figure 5. Left: forecast paths for open sea ships (blue) and for icebreakers (red) for 2040-2059. Right: Distribution of the potential major reserves of oil and gas (yellow) and licenses (red) and wells in operation or to operate (black). The dashed line indicates the limit of Conservation of Arctic Flora and Fauna (CAFF) declared by the Working Group of the Arctic Council (www.arctic-council.org). Figures from Reeves et al (2013).


Ecological consequences of the disappearance of the Arctic ice pack

There are many living beings linked to the ice. The polar bears roam on the Arctic ice, so we are feared for his fate. Many fish, seals and crustaceans (krill) form a food chain that starts from the algae that grow under the ice in a very consistent environment, rich in nutrients, especially favourable for marine life (Figure 6 A). Moreover, floating sea ice in summer is a good corridor for dispersion of terrestrial vertebrates (for instance arctic foxes) and plants.

The gradual disappearance of sea ice and warming in the Arctic coast involves a series of ecological imbalances (Figure 6 B). We see for example how walruses forced to remain grouped on the ground are more predisposed to disease transmission. The loss of sea ice diminishes dispersion by ice corridors and then the land populations are most isolated, thus gene flow is restricted. Polar bears and other predators that hunt on the sea ice have it much harder and their populations are at risk. Phytoplankton productivity decreases significantly, thereby reducing zooplankton, and then the whole food chain (fish, seals, etc.) is affected (Post et al 2013).

Fig 6 Post F1.large

Figure 6. Ecological interactions influenced by sea ice. A: The distribution and seasonality of sea ice affects the abundance, distribution and interactions of the entire ecosystem in balance. B: The longest period without ice and less sea ice extent have disastrous consequences on the balance of the ecosystem (Post et al 2013).

The polar bear tries to survive

The polar bear (Ursus maritimus) is considered an endangered animal. There are only about 25,000 worldwide. The impact of climate change affects the exclusive habitat of polar regions and forecasts suggest that in a few years from now the ice of the Arctic will melt permanently and polar bears may become extinct because of warming area.

The polar bear is basically carnivorous, unlike others such as brown bears, and remains above the ice hunting seals. With the gradual disappearance of the ice it has more trouble finding preys, and some have begun to learn how to catch salmon rivers, as we see in the images (Figure 7).

Fig 7a maxresdefault

Fig 7b Videos-de-Animales-oso-polar-cazando-salmon

Figure 7. White Bear dedicated to fishing salmons in order to survive (www.youtube.com/watch?v=9m_Q9Ojbcmw).


We have also seen groups of polar bears at sea fishing (see video) and dive emerging alternately as if they were dolphins or porpoises. Despite these small adaptations, the food is very low and it is clear that their populations are declining rapidly.


Orcas thrive north

The disappearance of the northern ice is a dramatic ecological change that is causing the disappearance of some species like the polar bear, but interestingly these imbalances benefit some other emerging species. This is the case of the killer whale (Orcinus orca), which is thriving more and more to the north (Figure 8).

Fig 8a killer-whale-mother-calf-antarctica-820x473

Fig 8b Young 2011 Polar Res Fig1

Figure 8. Places (marked with numbers) of the Canadian Arctic where groups of orcas were repeatedly photographed between 2004 and 2009 (Young et al 2011).


Eskimo Inuit people live around the American Arctic (from Quebec to Alaska including Hudson Bay and adjacent islands) and the west coast of Greenland, and they are the first witnesses since the mid-twentieth century observing whales in their waters, unknown before. Moreover, in recent years scientists have made numerous orca’ sightings, they have been photographed individually (Young et al 2011), and their travels have been followed through bioacoustics (Ferguson et al 2010) and other techniques.

Fig 9a Narwhals_breach-1024x651

Fig 9b narwhal_hunt_top_image-e1415394076242

Figure 9. (Top): Narwhals with the characteristic great tusk, which gave rise to the myth of the unicorn. (Low): Group of orcas attacking narwhals cornered on the beach. Watch the video of PBS Nature.


For some years attacks by orcas on narwhals (as in Figure 9) have been observed repeatedly by Inuit Eskimos and studied in detail by several scientists. Laidre et al (2006) observed that before approaching whales, the narwhals tend to group, are more quiet and swim closer to the beach in shallow waters. During the attack, the narwhals disperse significantly but nevertheless mortality is very high. After predation, which can last several hours, oily stains are observed in sea surface, which come from fat of depredated narwhals (Figure 10).

Fig 10 orques greix

Figure 10. Group of orcas surrounded by patches of oil on the sea surface from the fat of attacked narwhals (Laidre et al 2006).


Orcas’ attacks on narwhals are so common and effective that are beginning to affect the population. The effects are even worse in other cetaceans with smaller population such as whales of Greenland or bowhead (Balena mysticetus), which are now virtually extinct (Figure 11).

Fig 11 orques prey Ferguson 2010

Figure 11. Scheme of preys’ proportions by a group of orcas from Hudson Bay (Ferguson et al 2010).


In conclusion, anthropogenic climate change is affecting the Arctic ecosystem severely (and all the other ecosystems), and although this problem is becoming known, effective policy measures to reduce emissions of CO2 and other greenhouse gases are so scarce that hardly will arrive in time. We are leading the planet Earth to a massive extinction of species and ecological changes ever seen in the history of humans.

Fig 0 polar-bear

The picture says it all: polar bear habitat is running out.



Arctic Council: http://www.arctic-council.org

Ferguson S.H., Higdon J.W. & Chmelnitsky E.G. (2010) The rise of killer whales as a major Arctic predator. In S.H. Ferguson, et al. (eds.): A little less Arctic: top predators in the world’s largest northern inland sea, Hudson Bay. Pp. 117–136. New York: Springer

GISTEMP, Goddard Institute for Space Studies Surface Temperature Analysis (NASA-GISS): http://data.giss.nasa.gov/gistemp/

Hawkings E (2014) nov 28: http://www.climate-lab-book.ac.uk/2014/hiatuses-in-the-rise-of-temperature/

Laidre KL, Heide-Jørgensen MP, Orr J (2006) Reactions of narwhals, Monodon monoceros, to killer whale, Orcinus orca, attacks in the Eastern Canadian Arctic. Can. Field Nat., 120, 457–465

Morell V (2012) Killer whale menu finally revealed. http://www.sciencemag.org/news/2012/01/killer-whale-menu-finally-revealed

PBS Nature: http://www.pbs.org/wnet/nature/invasion-killer-whales-killer-whales-attack-pod-narwhals/11165/

Post et al. (2013) Ecological Consequences of Sea-Ice Decline. Science. DOI: 10.1126/science.1235225: http://www.carbonbrief.org/knock-on-effects-for-wildlife-as-the-arctic-loses-ice

Reeves RR et al (2014) Distribution of endemic cetaceans in relation to hydrocarbon development and commercial shipping in a warming arctic. Marine Policy 44, 375-389

Serreze MC, Holland MM, Stroeve J (2007) Perspectives on the Arctic’s shrinking sea-ice cover. Science 315, 5818, 1533–6.

Taylor PC, Cai M, Hu A, Meehl J, Washington W, Zhang GJ (2013) Decomposition of feedback contributions to Polar Warming Amplification. J Climate 26, 7023-43

The Cryosphere Today: http://arctic.atmos.uiuc.edu/cryosphere/

Wikipedia: https://en.wikipedia.org/wiki/Climate_change

Wikipedia: https://en.wikipedia.org/wiki/Arctic

Young BG, Jeff W. Higdon JW, Steven H. Ferguson SH (2011) Killer whale (Orcinus orca) photo-identification in the eastern Canadian Arctic. Polar Research Vol 30

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