Category Archives: Biotechnology
25th December 2018
Translated from the original article in Catalan.
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.
Figure 1. Small portion of the Great Pacific Garbage Patch (From oceanandreserveconservationalliance.com)
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.
Figure 2. PET, polyethylene terephthalate.
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.
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).
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.
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).
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.
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 Pseudomonas, Rhodococcus 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).
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
Howard GT (2002) Biodegradation of polyurethane: a review. Int Biodeterior Biodegrad 42, 213-220
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
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).
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.
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).
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.
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.
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 Candida, Hansenula, Pichia or Cryptococcus (Verachtert & Debourg 1999) appear in limited numbers.
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.
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).
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).
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
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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
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12th August 2017
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 ...”).
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.
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.
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).
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).
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.
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:
- The microorganism should be well characterized at the species level, using phenotypic and genotypic methods (e.g. 16S rRNA).
- The strain in question should be deposited in an internationally recognized culture collection.
- 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.
- Determination of its antimicrobial activity, and the resistance profile, including the absence of resistance genes and the inability to transfer resistance factors.
- Preclinical evaluation of its safety in animal models.
- Confirmation in animals demonstrating its effectiveness.
- Human evaluation (Phase I) of its safety.
- Human evaluation (Phase II) of its effectiveness (if it does the expected effect) and efficiency (with minimal resources and minimum time).
- 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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All we that studied “Bios” probably remember two known aspects of the symbiotic relationships of plant roots with microorganisms:
1) The bacterial Rhizobium nodules on the roots of legumes (Figure 1). These bacteria, with the nitrogenase complex, are among the few organisms capable of fixing atmospheric N2 transforming it into organic nitrogen, which is used by the plant, and symbiotically, the plant provides organic compounds to the bacteria. Thanks to these bacteria, plants such as legumes do not require nitrogen fertilizers.
Figure 1. Rhizobium nodules
2) The mycorrhizae, that is, the symbiotic relationships between fungi and plant roots. The most commonly known are the mushrooms always associated with some trees (Figure 2), such as the Lactarius sanguifluus associated with pines. In fact, mycorrhizae are present in most plants. Through this symbiosis, the fungi receive organic nutrients of the plant, and this can capture more easily water and mineral nutrients (especially P, Zn and Cu) by means of the fungus. In addition, mycorrhizae increase the resistance of plants to diseases coming from the soil and facilitate them inhabiting badlands.
Figure 2. Mycorrhizae of mushrooms with trees. Image from Shannon Wright
But these are only the best known of the symbiotic relationships between microorganisms and plant roots. Indeed, as the soil is full of microorganisms, many of these, including bacteria, fungi, algae, protozoa or viruses, are beneficial, symbiotic or otherwise, for the plants. And what is biotechnologically more interesting, more potential applications of these microorganisms to benefit crop plants are being found, which can be a good alternative to the use of fertilizers and pesticides.
Different microorganisms can have direct positive effects on plant nutrition as nitrogen fixation, mineralization of organic compounds, and solubilisation of elements not available to the plant (such as phosphates, K, Fe), but also indirectly positive effects, such as the production of hormones and growth factors, or protection against pathogens (García 2013).
Thus, there is a growing interest in the biological control of plant pathogens. It has been proven that some of these pathogens are inhibited by antibiotics produced by microorganisms in the rhizosphere (Raaijmakers et al 2002). Bacteria are being used (bacterization) for some years in soil or with seeds or other plant parts, with the aim of improving the growth and health of the plant.
Some of the best known and used bacteria in this sense have been Bacillus and Paenibacillus. Several species of these genera of aerobic spore bacteria are abundant in agricultural soils and can promote plant health in different ways, suppressing pathogens with antibiotic metabolites, stimulating plant defence, facilitating nutrient uptake by the plant, or promoting symbiosis with Rhizobium or with mycorrhizae (McSpadder 2004).
The genus Paenibacillus was reclassified from Bacillus in 1993, and includes P. polymyxa, a species N2 – fixing, which is used in agriculture and horticulture. This and other Paenibacillus species give complex and regular colonial forms in agar, even surprising (Figure 3), which vary according to environmental conditions. For this, a self-organizing and cooperative behaviour between individual bacterial cells is needed, using a system of chemical communication. This bacterial social behaviour would be an evolutive precursor of multicellular organisms.
Figure 3. Colonies of Paenibacillus dendritiformis, 6 cm diameter each, branched (left) and chiral (right) morphotypes. From Wikipedia Creative Commons.
The colonization of plant roots by these bacteria has been demonstrated, and also that they do it by forming biofilms (Figure 4). The inoculation of these bacteria to the roots promotes the growth, as shown in peppers (Figure 5). This appears to be due to the nitrogen fixing bacteria, which increases the formation of plant proteins and chlorophyll, thus increasing photosynthesis and physiological activities. And on the other hand, it has been shown that these bacteria produce siderophores, which facilitate Fe uptake by the plant (Lamsal et al 2012).
Figure 4. Colonization of Paenibacillus polymyxa and biofilm formation on roots of Arabidopsis thaliana. Adapted from Timmusk et al 2005.
Figure 5. Promoting growth effect of peppers (Capsicum annuum) by inoculation with Bacillus subtilis (AB17) and Paenibacillus polymyxa (AB15), respect the non-inoculated control. From Lamsal et al. 2012.
Moreover, bacteria such as Paenibacillus can be effective against plant pathogens. For example, it has been shown that a strain of P. lentimorbus (B-30488r) reduces the incidence of disease done by the fungus Alternaria solani in tomato. It has been tested (Figure 6) that after inoculating with Paenibacillus a plant infected with Alternaria, resistance to the fungus was induced in the plant. The bacteria degraded the cell walls of the fungus and also inhibited it by competition of nutrients. In addition, it was found that Paenibacillus has no negative effect on the microbial population in the rhizosphere of tomato (Khan et al 2012). These treatments are a good alternative to the use of fungicides, avoiding the environmental and health problems of these compounds.
Figure 6. Schema of the influence of Paenibacillus lentimorbus B-30488r in the interactions of tomato plant with Alternaria solani, a fungus pathogen (Khan et al 2012).
Finally, these Paenibacillus can also be useful to avoid the transmission of human pathogens such as Salmonella through the crop plants. Indeed, on the east coast of the USA a few years ago were detected outbreaks of Salmonella on tomatoes due to contamination of water. When they analyzed the microbiome present in the roots of tomatoes and these were compared with those of other places where there were no Salmonella contamination occurred, it was found that these tomatoes of the East Coast had no Paenibacillus, which were present in tomatoes of other places. With this, they decided to inoculate tomatoes with several Paenibacillus and found that Salmonella disappeared. Among the inoculated strains, one was selected as more effective, P. alvei TS -15 , for which a patent was obtained as a biocontrol agent of foodborne human pathogens (Brown et al. 2012) .
Thus, knowledge of the soil microbiota and the many forms of relationships between microorganisms and plants lead to find new strategies for using “good” microbes to prevent food safety problems of transmission of pathogens, while at the same time it can be a good ecological alternative to the massive use of pesticides.
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