Category Archives: Environment

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.

Bibliography

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

https://en.wikipedia.org/wiki/Great_Pacific_garbage_patch

https://en.wikipedia.org/wiki/PET_bottle_recycling

https://en.wikipedia.org/wiki/Polyethylene_terephthalate

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

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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.

 

Bibliography

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

Bacteria of vineyard and terroir, and presence of Oenococcus in Priorat (South Catalonia) grapes

2nd May 2015 

The vine growers believe that the land on which they grow vines gives the wines a unique quality, and that is called terroir. We can consider that the physiological response of the vines to the type of soil and climatic conditions, together with the characteristics of the variety and form of cultivation, result in a wine organoleptic properties that define their terroir (Zarraonaindia et al 2015 ). However, it is not known if there could be a very specific microbiota of each terroir, as this subject has been barely studied.

Wine microorganisms in the grapes? Saccharomyces is not there or it has not been found there

The main protagonists of wine fermentations, alcoholic one (yeast Saccharomyces cerevisiae) and malolactic one (lactic acid bacteria Oenococcus oeni) usually do not appear until the must grape is fermenting to wine, in the cellar. In normal healthy grapes, S. cerevisiae is hardly found.

Oenococcus oeni in the grapes ? We have found it !

Regarding O. oeni, so far very little has been published about its presence and isolation from the grapes. In some works, as Sieiro et al (1990), or more recently Bae et al (2006), some lactic acid bacteria (LAB) have been isolated from the surface of grapes, but not O. oeni. Only Garijo et al (2011) were able to isolate a colony (only one) of O. oeni from Rioja grapes. Moreover, DNA of O. oeni has been detected in a sample of grapes from Bordeaux (Renouf et al 2005, Renouf et al 2007) by PCR-DGGE of rpoB gene, although in these works no Oenococcus has been isolated.

I am pleased to mention that recently our team have managed to isolate O. oeni from grapes, and typify them, and we are now working on a publication about it (Franquès et al 2015). Indeed, our research team of lactic acid bacteria (BL-URV), together with colleagues working on yeasts from the same group “Oenological Biotechnology” (Faculty of Oenology at the Universitat Rovira i Virgili in Tarragona, Catalonia, Spain) is working on a European project, called “Wildwine “(FP7-SME-2012 -315065), which aims to analyse the autochthonous microorganisms of Priorat area (South Catalonia), and select strains with oenological potential. This project also involves the Priorat Appellation Council and the cellar Ferrer-Bobet, as well as research groups and associations wineries from Bordeaux, Piedmont and Greece. In the framework of this project we took samples of grapes (Grenache and Carignan) from several vineyards of Priorat (Figure 1), as well as samples of wines doing malolactic fermentation. From all them we got 1900 isolates of LAB. We optimized isolation from grapes from the pulp and juice with various methods of enrichment, and so we got 110 isolated bacteria from grapes, identified as O. oeni by specific molecular techniques. Once typified, we have found that the molecular profiles of these strains do not coincide with commercial strains and so they are autochthonous. In addition, some of these strains from grapes were also found in the corresponding wine cellars.

Fig 1 garna-cari Priorat

Figure 1. Taking samples of Grenache (left) and Carignan (right) in Priorat area to isolate lactic acid bacteria such as Oenococcus (Pictures Albert Bordons).


The microbiota of grapes

The grapes have a complex microbial ecology, including yeasts, mycelial fungi and bacteria. Some are found only in grapes, such as parasitic fungi and environmental bacteria, and others have the ability to survive and grow in wines: especially yeasts, lactic acid bacteria (LAB) and acetic acid bacteria. The proportion of all them depends on the maturation of the grapes and the availability of nutrients.

When the fruits are intact, the predominant microbiota are basidiomycetous yeasts as Cryptococcus and Rhodotorula, but when they are more mature, they begin to have micro fissures that facilitate the availability of nutrients and explain the predominance just before the harvest of slightly fermentative ascomycetes as Candida, Hanseniaspora, Metschnikowia and Pichia. When the skin is already damaged more damaging yeasts may appear, as Zygosaccharomyces and Torulaspora, and acetic acid bacteria. Among the filamentous fungi occasionally there may have some very harmful as Botrytis (bunch rot) or Aspergillus producing ochratoxin. Although they are active only in the vineyard, their products can affect wine quality.

On the other hand, environmentally ubiquitous bacteria have been isolated from the grapes skin, as various Enterobacteriaceae, Bacillus and Staphylococcus, but none of them can grow in wine (Barata et al 2012).

Coming back to the possible specific microbiota of terroir, it has been found that some volatile compounds contributing to the aroma of the wine, such as 2-methyl butanoic acid and 3-methyl butanol, are produced by microorganisms isolated in the vineyards, as Gram-positive bacterium Paenibacillus, or the basidiomycetous fungus Sporobolomyces or the ascomycetous Aureobasidium. Therefore, there could be a relationship between some of the microbial species found in grapes and some detected aromas in wine, coming from the must of course (Verginer et al 2010).

Metagenomics as analytical tool of microbiota from grapes

Since conventional methods of isolation and cultivation of microorganisms are slow, laborious and some microbes cannot be grown up in the usual isolation media, massive sequencing methods or metagenomics are currently used. These consist of analysing all the DNA of a sample, and deducing which are the present microorganisms by comparing the sequences found with those of the databases. For bacteria the amplified DNA of V4 fragment from 16S RNA gene is used (Caporaso et al 2012).

This technique has been used with samples of botrytized wines (Bokulich et al 2012) and various LAB have been found (but not Oenococcus), including some not normally associated with wine. It has also been used to see the resident microbiota in wineries and how it changes with the seasons, resulting that in the surfaces of tanks and machinery of the cellar there is a majority of microorganisms neither related with wine nor harmful (Bokulich et al 2013).

With this technique Bokulich et al (2014) have also analysed the grapes and they have seen clear differences between the proportions of bacterial groups (and fungi) from different places, different varieties, as well as environmental or bio geographical conditions. For example, when analysing 273 samples of grape musts from California, the 3 varieties (Cabernet, Chardonnay and Zinfandel) are quite discriminated in a principal components analysis with respect to the bacterial communities found in each sample (Figure 2).

Thus, the dominant bacterial taxa or groups in a variety or given environment could provide some specifics traits on those wines, and this could explain some regional or terroir patterns in the organoleptic properties of these wines (Bokulich et al 2014).

Fig 2 ACP Bokulich 2014

Figure 2. Principal component analysis of bacterial communities of grape musts samples of Sonoma (California) from 3 varieties (Cabernet in red, Chardonnay in green and Zinfandel in blue) (Bokulich et al 2014).


We have also carried out a massive sequencing study with the same grape samples from which we have obtained isolates of O. oeni, as said before (Franquès et al 2015), and in more than 600,000 analysed sequences of 16S rRNA, we have found mainly Proteobacteria and Firmicutes. Among these gram-positive, we have found sequences of lactic acid bacteria (15%) and from these we have successfully confirmed the presence of O. oeni in 5% of the sequences. Therefore, we have isolated O. oeni from grapes and we have detected their DNA in the samples.

The bacterial microbiota of the vineyards and soil

As we see, microbiota of grapes and wine has been studied a little, but the soil microbiota has not been characterized. This one can define more clearly the terroir, which is influenced by the local climate and characteristics of the vineyard.

In Figure 3 the main genera found in different parts of the vine and soil are summarized (Gilbert et al 2014).

Fig 3 Gilbert 2014

Figure 3. Main bacteria and fungi associated with organs and soil of Vitis vinifera (Gilbert et al 2014)


Recently an interesting scientific work (Zarraonaindia et al 2015) has been published on this subject, with the aim to see if the soil could be the main original source of bacteria that colonize the grapes. These authors took samples of soil, roots, leaves, flowers and grapes from Merlot vines, from different areas and years, of Suffolk, New York, and they analysed the bacterial DNA by 16S rRNA sequencing. They found that 40% of the species found were present in all samples of soil and roots, while there was more variability in leaves and fruits, and moreover, 40% of those found in leaves and fruits were also found in soils. All this suggests that many bacteria originate in the soil.

Regarding the type of bacteria, they found that Proteobacteria (especially Pseudomonas and Methylobacterium) predominated (Figure 4), mainly in the aerial parts of the plant. There were also Firmicutes as expected, and Acidobacteria and Bacteroides.

Fig 4 microbiota vineyard

Figure 4. Composition of the bacterial community, at Phylum level, in samples from different organs of the vine and its soil (Zarraonaindia et al 2015).


Although variations were observed in all samples depending on the year (there may be different climatic conditions) and according to different edaphic factors (pH, C: N, humidity), the principal-components analysis (Figure 5) showed that the main types of samples (soil, roots, leaves, grapes) differ quite well, and bacterial taxon composition in samples of grape juice before fermentation is similar to that of grapes.

Fig 5 distribució grups mostres OTUs

Figure 5. Principal-components analysis showing the similarities in terms of the composition of bacterial taxonomic groups, among sample types, including musts (Zarraonaindia et al 2015).


This suggests that the bacterial community found in grapes remains relatively stable until the processing to musts, and that it is more stable than the differences between organs. At the same time, a large number of representatives of bacterial phyla of the grapes come from the soil. This can be explained because when grapes are harvested by hand, they are often placed in boxes that are left on the ground, or for mechanical harvest, the machinery used removes the soil and generates dust, which can colonize the grapes.

Therefore, the soil microbiota is a source of bacteria associated with vines and may play a role in the must and therefore in the wine, and potentially in the formation of the terroir characteristics. Some of these bacteria may have some roles not yet known in productivity or disease resistance of the plant, or contribute to the organoleptic characteristics of wine (Zarraonaindia et al 2015).

In addition, and thinking in wine microorganisms responsible for fermentations, as said, in our laboratory we have confirmed that there are some O. oeni strains in grapes and we have confirmed this by detecting their DNA in the same grapes.

References

Bae S, Fleet GH, Heard GM (2006) Lactic acid bacteria associated with wine grapes from several Australian vineyards. J Appl Microbiol 100, 712-727

Barata A, Malfeito-Ferreira M, Loureiro V (2012) The microbial ecology of wine grapes (Review). Int J Food Microbiol 153, 243-259

Bokulich NA, Joseph CML, Allen G, Benson AK, Mills DA (2012) Next-generation sequencing reveals significant bacterial diversity of botrytized wine. Plos One 7, e36357

Bokulich NA, Ohta M, Richardson PM, Mills DA (2013) Monitoring seasonal changes in winery-resident microbiota. Plos One 8, e66437

Bokulich NA, Thorngate JH, Richardson PM, Mills DA (2014) Microbial biogeography of wine grapes is conditioned by cultivar, vintage, and climate. PNAS nov 25, E139-E148

Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Huntley J, Fierer N, Owens SM, Betley J, Fraser L, Bauer M, Gormley N, Gilbert JA, Smith G, Knight R (2012) Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J 6, 1621–1624

Franquès J, Araque I, Portillo C, Reguant C, Bordons A (2015) Presence of autochthonous Oenococcus oeni in grapes and wines of Priorat in South Catalonia. Article in elaboration.

Garijo P, López R, Santamaría P, Ocón E, Olarte C, Sanz S, Gutiérrez AR (2011) Eur Food Res Technol 233, 359-365

Gilbert JA, van der Lelie D, Zarraonaindia I (2014) Microbial terroir for wine grapes. PNAS 111, 5-6

Renouf V, Claisse O, Lonvaud-Funel A (2005) Understanding the microbial ecosystem on the grape berry surface through numeration and identification of yeast and bacteria. Aust J Grape Wine Res 11, 316-327

Renouf V, Claisse O, Lonvaud-Funel A (2007) Inventory and monitoring of wine microbial consortia. Appl Microbiol Biotechnol 75, 149-164

Sieiro C, Cansado J, Agrelo D, Velázquez JB, Villa TG (1990) Isolation and enological characterization of malolactic bacteria from the vineyards of North-western Spain. Appl Environ Microbiol 56, 2936-2938

Verginer M, Leitner E, Berg G (2010) Production pf volatile metabolites by grape-associated microorganisms. J Agric Food Chem 58, 8344-8350

Zarraonaindia I, Owens SM, Weisenhorn P, West K, Hampton-Marcell J, Lax S, Bokulich NA, Mills DA, Martin G, Taghavi S, Van der Lelie D, Gilbert JA (2015) The soil microbiome influences grapevine-associated microbiota. mBio 6, e02527-14

The tropospheric bacteria of rain and snow

Nowadays there is more evidence that the bacteria found in the high troposphere (8-15 km) could influence the density of clouds and rain.

Firstly, we must remind that the troposphere is the lowest part of the atmosphere, and the 8-15 km layer is the high troposphere, near the tropopause that borders the stratosphere, above the Mount Everest. Here there are some of the highest clouds.

 atmosfera capesThe layers of Earth’s atmosphere (www.theozonehole.com/atmosphere)

So, in a recent study (DeLeón-Rodríguez et al, 2013) it has been shown that the viable bacteria (by epifluorescence microscopy and quantitative PCR) at a 10 km altitude (samples taken above the Caribbean Sea and the Atlantic West) represent 20% of the particles with size between 0.25 and 1 mm, and bacteria are at least 10 times more abundant than fungi, with numbers of 105 per m3, with a 60% of viable cells. This suggests that bacteria are an important and underestimated fraction of microparticles of atmospheric aerosols, even at higher concentrations than lower altitudes.

The authors have analyzed the bacteria by pyrosequencing (Roche 454) the rRNA genes. They have seen that the tropospheric microbiome has a good variety of bacterial taxa that vary dynamically according to the atmospheric turbulence and in the presence of hurricanes. Some of the most abundant bacteria found are those using compounds C1-C4 (e.g., oxalic acid) present in the atmosphere, so these bacteria are metabolically active at these altitudes. This reinforces the idea of the active role of bacteria in the troposphere, and that there are not only inert spores (fungal) floating through the air.

In this sense, this metagenomic analysis also confirms the presence of bacteria that are able to catalyze the formation of ice crystals and hence the cloud condensation. This process of nucleation (ice nucleation, IN) occurs when the water molecules coalesce around a seed particle, for example dust. Depending on the temperature, these complexes can grow to become water droplets or ice, leading to the formation of rain or snow. Given that the high troposphere dust particles are scarce, it is evident the role of bacteria in this phenomenon.

One of the key roles in the nucleation of ice (IN) by bacteria is that they catalyze ice formation at temperatures close to 0°C, unlike the formation of ice nuclei by the inorganic particles, which is done at temperatures lower, below -10°C, and without any core particle the ultra-pure water freezes at -40°C.

Ice nucleation by bacteria has been reproduced in the laboratory (Christner et al, 2008) with samples of rain and snow from around the world (Canada, USA, Pyrenees, Alps and Antarctica), showing that in the samples treated with lysozyme (which hydrolyzes bacterial cell wall) or treated with heat, the IN activity was reduced almost 100% at a temperature of -5°C. Therefore, bacteria are responsible of the IN at these relatively high temperatures.

The bacteria most commonly associated with the IN activity are species associated with plants, such as Pseudomonas syringae or Xanthomonas campestris, which also often have been detected in atmospheric aerosols and clouds. P. syringae has also been found in the hail stones.

Pseudo syringae www.forestry.gov.ukPseudomonas syringae (www.forestry.gov.uk)

The phenomenon of IN by P. syringae was already observed in 1974 (Maki et al.) and after it has been shown (Gurian-Sherman & Lindow 1993) that IN strains of this species and others have in the outer membrane of the cell wall, as a active IN, a protein of 180 kDa, composed of repeats of a consensus octapeptide. This protein forms a planar arrangement that traps water molecules producing a mold for ice formation.

This feature makes that these bacteria are responsible for most of frost damage in plants, besides than P. syringae is pathogen of many plants at room temperature by the production of a compound (coronatin) who keeps the stomata open, causing the bacterial invasion of plant tissues (Nigel Chaffey, 2012).

fulla tomaquet Alan Collmer, Cornell University

Tomato leaf infected with Pseudomonas syringae (Alan Collmer, Cornell University/Wikimedia Commons)

Coming back to the frost damage, most plants can withstand up to -5°C without much damage if these bacteria are absent, but the presence of the IN protein-forming bacteria such as P. syringae in numbers of only 1000 cells by g of plant increases dramatically the damage by freezing. These damages also facilitate the penetration of bacteria and infection.

ice twigs 2Frozen plant (MO Plants& Maureen Gilmer)

This feature of ice nucleation by P. syringae is also utilized for the production of artificial snow. Although this can be made usually by the forced expansion of a pressurized mixture of water and air under appropriate conditions of temperature and humidity (e.g. ≤ 2°C at 20% humidity, or ≤ -2°C at 60%), snow production is favoured by the addition of nucleation agents, which can be inorganic, organic or the mentioned bacterial protein.

 Siemens - We take you to the topSnow cannons (www.siemens.com)

Coming back to the clouds, we must remind that bacteria are far less the sole agents of nucleation forming condensation droplets resulting in rain or snow. The cloud condensation nuclei, CCN, also called cloud seeds, can be very different types of microparticles of sizes around 0.1 – 1 mm. When this aerosol of microdroplets is condensed, it forms drops of 0.02 mm in the clouds, which give falling raindrops of 2 mm.

The microparticles are mostly of natural origin such as dust, sea salt, volcanic sulphates or organic microparticles result of the oxidation of volatile compounds. Some of these may be of industrial origin, as well as soot and other particles resulting from combustion. Another important biological source of CCN is the aerosols of sulphate and methanosulphate produced from dimethyl sulphur, which is made by phytoplankton in the oceans.

Anyway, despite atmospheric microbiology is still in its infancy, as we have seen there are more and more data on the importance of bacteria and other microorganisms on bioprecipitation of rain and snow. To find out more about their role, research must go beyond the description of the abundance of microorganisms in the atmosphere, and to understand the biological, physical and chemical properties of the transport processes involved. This will require interdisciplinary approach seemingly different disciplines such as oceanography, bacterial genetics and physics of the atmosphere, for example.

nuvol-bacterisLet us imagine the bacteria (Pseudomonas syringae, photo: microbewiki.kenyon.edu) in the middle of the threatening clouds (photo: lanroca.wordpress.com)

References

Chaffey N. (2012) COR, nice one, Mr Microbe !. AoB Blog.

Christner B. et al. (2008) Geographic, seasonal, and precipitation chemistry influence on the abundance and activity of biological ice nucleators in rain and snow. PNAS 105, 48, 18854-18859.

DeLeón-Rodríguez N. et al. (2013) Microbiome of the upper troposphere: species composition and prevalence, effects of tropical storms, and atmospheric implications. PNAS 110, 7, 2575-2580.

Gurian-Sherman D. & S.E. Lindow (1993) Bacterial ice nucleation: significance and molecular basis. FASEB J. 7, 14, 1338-1343.

Hardy J. (2008) The rain-making bacteria. Micro-Bytes.

http://microbialmodus.wordpress.com/tag/ice-nucleating-bacteria/

https://en.wikipedia.org/wiki/Cloud_condensation_nuclei

https://en.wikipedia.org/wiki/Snowmaking

Maki L.R. et al.(1974) Ice nucleation induced by Pseudomonas syringae. App!. Microbiol. 28, 456-460.

Morris C.E. et al. (2011) Microbiology and atmospheric processes: research challenges concerning the impact of airborne micro-organisms on the atmosphere and climate, Biogeosciences 8, 17-25

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