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Winogradsky column and hydrogen sulfide

Winogradsky column and hydrogen sulfide


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For a science experiment for school, I have set up 15 Winogradsky columns, and will be looking at the microbial growth inside the columns over a period of 25 days.

I have read that, due to gas produced by the microbes, it is good to leave the cap loosely closed to prevent an explosion from gas buildup. However, I have also read that sulfate-reducing bacteria that grow in the column can produce toxic hydrogen sulfide gas. Wouldn't the hydrogen sulfide also escape in small amounts and pose a health risk? What would be the correct protocol to follow here?


Hydrogen sulfide can be toxic at high concentrations, however, the amount that your columns will make is likely very low, and will almost certainly degrade/oxidize into something safer before it hurts anyone (who isn't a small organism in the column!). It will noticeably smell bad at levels well below what is dangerous. So maybe just make sure that the columns are in a ventilated space.

Remember, what is happening in the column is basically just rotting organic matter, like leaving a bunch of food waste in a highly compacted, sealed environment. It is sort of intrinsically gross, but it isn't any more dangerous to your health than forgetting to take out the garbage for a month (actually, it's probably a lot less dangerous than that!).

For example, this guide says the following (after constructing columns with different inputs such as e.g. eggs):

You may have seen worms, snails, shrimp or other small organisms in the water, but probably not many (if any) in the bottle with the egg yolk, because hydrogen sulfide is toxic to most organisms!

So, depending on what you put in there, a very H2S-productive medium is likely to create enough H2S to kill tiny animals that are trapped inside the column. But apparently not enough to hurt large animals like you, as long as you don't huff the column.


Winogradsky Column

The Winogradsky column is a simple device for culturing a large diversity of microorganisms. Invented in the 1880s by Sergei Winogradsky, the device is a column of pond mud and water mixed with a carbon source such as newspaper (containing cellulose), blackened marshmallows or egg-shells (containing calcium carbonate), and a sulfur source such as gypsum (calcium sulfate) or egg yolk.

Incubating the column in sunlight for months results in an aerobic/anaerobic gradient as well as a sulfide gradient. These two gradients promote the growth of different microorganisms such as Clostridium, Desulfovibrio, Chlorobium, Chromatium, Rhodomicrobium, and Beggiato, as well as many other species of bacteria, cyanobacteria, and algae.

The column provides numerous gradients, depending on additive nutrients, from which the variety of aforementioned organisms can grow. The aerobic water phase and anaerobic mud or soil phase are one such distinction.

  • Because of oxygen’s low solubility in water, the water quickly becomes anoxic (a total depletion in the level of oxygen) towards the mud and water interface.
  • Anaerobic phototrophs are still present to a large extent in the mud phase, and there is still capacity for biofilm creation and colony expansion.
  • Algae and other aerobic phototrophs are present along the upper half of the columns’ surface and water. Green growth is often attributed to these organisms.

How to make a Winogradsky column?

Source here.

The column is a rough mixture of ingredients – exact measurements are not critical.

  • A tall glass or plastic tube (30 cm long, >5 cm wide) is filled one-third full of pond mud, omitting any sticks, debris, and air bubbles.
  • Supplementation of -0.25% w/w calcium carbonate and -0.50% w/w calcium sulfate or sodium sulfate is required (ground eggshell and egg yolk respectively are rich in these minerals), mixed in with some shredded newspaper or hay (for cellulose).
  • An additional anaerobic layer, this time of unsupplemented mud, brings the container to two-thirds full.
  • This is followed by water from the pond to saturate the mud and occupy half the remaining volume.
  • The column is sealed tightly to prevent evaporation of water and incubated for several months in strong natural light.

Colonies saw in Winogradsky column

After the column is sealed tightly,

  • The anaerobic bacteria will develop first, including Clostridium spp. These anaerobic bacteria will consume cellulose as an energy source. Once this commences they create CO2 that is used by other bacteria and thus the cycle begins.
  • Eventually, color layers of different bacteria will appear in the column.
  • At the bottom of the column will be green sulfur photosynthetic anaerobic bacteria .
  • The layer above will be purple which is sulfur anaerobic bacteria .
  • Followed by another column of purple anaerobic non-sulfur bacteria.
  • And at the top will be a layer of Cyanobacteria which is sulfur-oxidizing bacteria . This top layer of aerobic bacteria produces CO2, which feeds back into the column creating a further reaction.

Limitations of Winogradsky column

While the Winogradsky column is an excellent tool to see whole communities of bacteria, it does not allow one to see the densities or individual bacterial colonies. It also takes a long time to complete its cycle. However, its importance in environmental microbiology should not be overlooked and it is still an excellent tool to determine the major bacterial communities in a sample.

What biomolecule do the newspaper, eggshells and gypsum contribute to the Winogradsky column?

  • News papers: Source of carbon (Cellulose).
  • Egg shell: Source of Calcium carbonate
  • Gypsum: Source of Sulfur (Calcium sulfate)

Importance of Winogradsky column

One can discover several interesting things in the columns.

  • Winogradsky columns are model microbial ecosystems.
  • The microbes create two gradients, one of oxygen, the second of hydrogen sulfide. The oxygen is highest at the top and lowest at the bottom, the hydrogen sulfide gas is highest at the bottom and lowest at the top.
  • When grown in the light, different bacteria have different pigments growing in many different layers, giving the different layers different colors.

(i) Green photosynthetic bacteria and algae are on top,

(ii) Upper decomposers (using aerobic respiration) follow,

(iii) There is usually both a red and a rust-colored layer of mud including photosynthetic bacteria that do not produce oxygen. (Except at the top, the photosynthetic bacteria are only under the glass or plastic, not deep inside the mud).

(iv) Lower decomposers (using anaerobic respiration) and chemosynthetic bacteria using sulfur are in the lowest layers.

(i) The number of bacteria species in the ecosystem is also reduced, there are mainly upper decomposers using aerobic respiration and lower decomposers using anaerobic respiration, and lower chemosynthetic organisms using sulfur.


Winogradsky Column

"pond" in a controlled environment containing a variety of prokaryotes
which coexist in nature.The column will enable students to study some
of the many organisms that might be obtained from a sample of a natural
pond sediment and water (or salt or brakish water environment).

Starting with a soil andwater sample from the environment, you should be able to procure a

large assortment of prokaryotes which will florish in the wide range of
environments which could be supplied by placing the column in various
external environments. Internally, if the column is set up
correctly you should get an aenerobic layer in the mud on the bottom, then
a microaerophillic zone, a aerobic zone in the water, and a layer of
surface air at the top. The oxygen concentration will
decrease as you descend the column and the hydrogen sulfide concentration will
decrease as you rise in the column. If gases are kept within the
container, the hy drogen sulfide should remain in the gas layer and not
escape.

Oxygen will be exchanged with the top gas layer in the
column, and will be obtained from external enviroment if the lid is
removed (or if the column is not airproof!). Other gases such as
CO2, methane, and hydrogen sulfide will rise to the top air layer also,
with the potential of being recycled back into the liquid/solid
sections of the column if there are microbes that can utlilize these
compounds.

Manufactored potting soil might supply some prokaryotes but probablly not the assortment that can
be gotten from nature. Some soil mixes are pasturized almost
all do not contain any "soil" at all but other products of
decomposition (com posted tree bark, composted animal waste).
The process of composting produces significant heating.
Although his heat is not enough to sterilze the potting soil,
it is generally enough heat to seriously reduce the numbers and variety
of microorganisms present. If the experiement was performed
with potting soil, the column would contain microorganisms but would
probably take longer to show distinct layers. Potting soil
would probably not contain as much anerobic type of microbes as natural
soil, but there would probably end up enough in the column to start the
anerobic layer.

Some source of carbon is added to the soil to provide food for the
microbes, although my source of soil certainly did have carbon already
added in the form of leaves which were not totally removed.
Given that it is possible to generate satisfactory compost starting only with shredded newspaper, water, some soil for microbial source, heat, and a lot of time a high celluslose additive like newspaper or sawdust should work as a food source if you are patient.


Most other sourc es of carbon contain substances besides carbon which would have the potential to feed microbes that prefer some of these substances such as sugars, starches and proteins. The advantage to a high celluslose additive is that it contains a lot of carbon, albeit in a slowly digestable form.

External enviroments can supply sunlight or not, termperature variations, and in theory reduced oxygen although most non-lab situations would have difficulty in supplying the last environment.

    Some sort of sediment and water from the same environment. Winter mud is fine.


Monday, February 16, 2009

Winogrsky Column

Winogradsky columns are a classic ecology/microbiology project. The idea is to create an model

"pond" in a controlled environment containing a variety of prokaryotes
which coexist in nature.The column will enable students to study some
of the many organisms that might be obtained from a sample of a natural
pond sediment and water (or salt or brakish water environment).

Starting with a soil andwater sample from the environment, you should be able to procure a

large assortment of prokaryotes which will florish in the wide range of
environments which could be supplied by placing the column in various
external environments. Internally, if the column is set up
correctly you should get an aenerobic layer in the mud on the bottom, then
a microaerophillic zone, a aerobic zone in the water, and a layer of
surface air at the top. The oxygen concentration will
decrease as you descend the column and the hydrogen sulfide concentration will
decrease as you rise in the column. If gases are kept within the
container, the hy drogen sulfide should remain in the gas layer and not
escape.

Oxygen will be exchanged with the top gas layer in the
column, and will be obtained from external enviroment if the lid is
removed (or if the column is not airproof!). Other gases such as
CO2, methane, and hydrogen sulfide will rise to the top air layer also,
with the potential of being recycled back into the liquid/solid
sections of the column if there are microbes that can utlilize these
compounds.

Manufactored potting soil might supply some prokaryotes but probablly not the assortment that can
be gotten from nature. Some soil mixes are pasturized almost
all do not contain any "soil" at all but other products of
decomposition (com posted tree bark, composted animal waste).
The process of composting produces significant heating.
Although his heat is not enough to sterilze the potting soil,
it is generally enough heat to seriously reduce the numbers and variety
of microorganisms present. If the experiement was performed
with potting soil, the column would contain microorganisms but would
probably take longer to show distinct layers. Potting soil
would probably not contain as much anerobic type of microbes as natural
soil, but there would probably end up enough in the column to start the
anerobic layer.

Some source of carbon is added to the soil to provide food for the
microbes, although my source of soil certainly did have carbon already
added in the form of leaves which were not totally removed.
Given that it is possible to generate satisfactory compost starting only with shredded newspaper, water, some soil for microbial source, heat, and a lot of time a high celluslose additive like newspaper or sawdust should work as a food source if you are patient.


Most other sourc es of carbon contain substances besides carbon which would have the potential to feed microbes that prefer some of these substances such as sugars, starches and proteins. The advantage to a high celluslose additive is that it contains a lot of carbon, albeit in a slowly digestable form.

External enviroments can supply sunlight or not, termperature variations, and in theory reduced oxygen although most non-lab situations would have difficulty in supplying the last environment.

    Some sort of sediment and water from the same environment. Winter mud is fine.


Sergey Nikolayevich Winogradsky

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Sergey Nikolayevich Winogradsky, Winogradsky also spelled Vinogradsky, (born Sept. 1, 1856, Kiev, Russian Empire [now in Ukraine]—died Feb. 25, 1953, Brie-Comte-Robert, France), Russian microbiologist whose discoveries concerning the physiology of the processes of nitrification and nitrogen fixation by soil bacteria helped to establish bacteriology as a major biological science.

After studying natural sciences at the University of St. Petersburg in 1881, Winogradsky went (1885) to Strassburg, Ger. In 1887 he established the specific physiology of sulfur bacteria, demonstrating that the colourless form of these bacteria can obtain energy by oxidizing hydrogen sulfide to sulfur and then to sulfuric acid in the absence of light.

In 1888 Winogradsky went to the University of Zürich, where he discovered (1889–90) the microbial agents responsible for nitrification (the oxidation of ammonium salts to nitrites and nitrites to nitrates). He established two new genera—Nitrosomonas (nitrite formers) and Nitrosococcus ([Nitrobacter] nitrate formers)—for the two new types of microorganisms concerned in the process. He returned to St. Petersburg and worked for the Imperial Institute of Experimental Medicine until his first retirement in 1905. Forced out of Russia by the October Revolution of 1917, he resumed his career in 1922 at the Pasteur Institute in Paris, where he remained until he again retired in 1940.


Winogradsky was born in Kiev (then in the Russian Empire). In this early stage of his life, Winogradsky was "strictly devoted to the orthodox faith", though he later became irreligious.

He entered the Imperial Conservatoire of Music in St Petersburg in 1875 to study piano. However, after two years of music training, he entered the University of Saint Petersburg in 1877 to study chemistry under Nikolai Menshchutkin and botany under Andrei Sergeevich Famintzin.

He received a diploma in 1881 and stayed at the St. Petersburg University for a degree of master of science in botany in 1884. In 1885, he began work at the University of Strasbourg under the renowned botanist Anton de Bary Winogradsky became renowned for his work on sulfur bacteria.

In 1888, he relocated to Zurich, where he began investigation into the process of nitrification, identifying the genera Nitrosomonas and Nitrosococcus, which oxidizes ammonium to nitrite, and Nitrobacter, which oxidizes nitrite to nitrate.

He returned to St. Petersburg for the period 1891-1905, and headed the division of general microbiology of the Institute of Experimental Medicine during this period, he identified the obligate anaerobe Clostridium pasteurianum, which is capable of fixing atmospheric nitrogen.

In 1901, he was elected honorary member of the Moscow Society of Naturalists and, in 1902, corresponding member of the French Academy of Sciences.

He retired from active scientific work in 1905, dividing his time between his private estate and Switzerland. In 1922, he accepted an invitation to head the division of agricultural bacteriology at the Pasteur Institute at an experimental station at Brie-Comte-Robert, France, about 30 km from Paris. During this period, he worked on a number of topics, among them iron bacteria, nitrifying bacteria, nitrogen fixation by Azotobacter, cellulose-decomposing bacteria, and culture methods for soil microorganisms. Winogradsky retired from active life in 1940 and died in Brie-Comte-Robert in 1953.


Abstract

Plastic pollution in the current scenario requires a sustainable and eco-friendly treatment process. Single-use plastics accumulate more than recyclable plastic wastes. Low-Density Polyethylene (LDPE) is one among the plastic family with inert characteristics. The traditional method, such as landfilling, develops pollution resistant micro-organisms. It is involved in the exploitation of the native microbes to the fullest. The soil of the Kodungaiyur, agriculture site, and Otteri dumpyard were used, which resulted in nearly 22.97 ± 2.7115%, 15.91667 ± 2.73775%, and 10.74 ± 0.502925% of LDPE degradation in 30 days without nutrient supplements. The enrichment of the column by organic nutrients increased the degradation of LDPE. The column enrichment was confirmed by the sulfur oxidizing bacteria (SOB) Escherichia coli and Pseudomonas stutzeri, which produced 195 mg/mL of sulfate ions. The FTIR of the LDPE degradation showed the polymer's oxygenation, while the electron microscopic images revealed cracks. In addition, an attempt was made to fit the experimental time-series data into suitable mathematical models to look at prediction and elementary forecasting. Three mathematical models, namely the customized moving averages model (CMAM), simple liinear regression model (SLRM), and a modified linear regression model (MLRM) with a lag, were able to represent the real experimental data complementarily.


5 CONCLUSION

This study reveals that two environmental change factors (i.e., temperature and nutrient availability) caused large and non-additive variation in the composition of aquatic microbial communities and the abiotic conditions of the ecosystem. The advantages of this system include high parallelization and replication, easy and non-destructive sampling, the versatility of testable conditions and manipulations, and are of additional interest besides in situ ecosystem studies. We believe that these insights are only the tip of the iceberg of what can be learned from such a model micro-ecosystems with strong and even stratified spatial environmental gradients. Even in the described study, we could have included many more elements, such as characterization of the conditions at the sampling site, measurements of organic compounds composition in the water column, and control without cellulose. Further research with this new experimental system could take many paths, including studying the stability of the communities to press and pulse perturbations, and how this stability may depend on aspects of community composition, such as functional composition and intraspecific diversity the extent and significance of evolutionary processes such as mutation and selection for mediating effects of environmental change and observation of community composition via metagenomic methods, to capture not just the bacterial component of the micro-ecosystems, but also to research the functional significance of other likely inhabitants, such as viruses. Finally, one could research why these micro-ecosystems did not become entirely oxic or entirely anoxic, and why there was little evidence of the discontinuous responses to environmental change that are predicted for systems with strong positive feedbacks, such as this one.


Phototrophic Bacteria

The phototrophic bacteria are a large and diverse category of bacteria that do not represent a taxon but, rather, a group of bacteria that use sunlight as their primary source of energy. This group contains both Proteobacteria and nonproteobacteria. They use solar energy to synthesize ATP through photosynthesis. When they produce oxygen, they perform oxygenic photosynthesis. When they do not produce oxygen, they perform anoxygenic photosynthesis. With the exception of some cyanobacteria, the majority of phototrophic bacteria perform anoxygenic photosynthesis.

One large group of phototrophic bacteria includes the purple or green bacteria that perform photosynthesis with the help of bacteriochlorophylls, which are green, purple, or blue pigments similar to chlorophyll in plants. Some of these bacteria have a varying amount of red or orange pigments called carotenoids. Their color varies from orange to red to purple to green (Figure (PageIndex<2>)), and they are able to absorb light of various wavelengths. Some green sulfur bacteria are able to photosynthesize at the bottom of the ocean using the light wavelengths emitted from geothermally heated rocks around hydrothermal vents! 1 Traditionally, photosynthetic bacteria are classified into sulfur and nonsulfur bacteria they are further differentiated by color (e.g. purple sulfur bacteria).

Figure (PageIndex<2>): The purple and green sulfur bacteria shown in this Winogradsky column use bacteriochlorophylls to perform photosynthesis.

The sulfur bacteria perform anoxygenic photosynthesis, using sulfites as electron donors and releasing free elemental sulfur. Nonsulfur bacteria use organic substrates, such as succinate and malate, as donors of electrons.

The purple sulfur bacteria oxidize hydrogen sulfide into elemental sulfur and sulfuric acid and get their purple color from the pigments bacteriochlorophylls and carotenoids. Bacteria of the genus Chromatium are purple sulfur Gammaproteobacteria. These microorganisms are strict anaerobes and live in water. They use carbon dioxide as their only source of carbon, but their survival and growth are possible only in the presence of sulfites, which they use as electron donors. Chromatium has been used as a model for studies of bacterial photosynthesis since the 1950s.

The green sulfur bacteria use sulfide for oxidation and produce large amounts of green bacteriochlorophyll. The genus Chlorobium is a green sulfur bacterium that is implicated in climate change because it produces methane, a greenhouse gas. These bacteria use at least four types of chlorophyll for photosynthesis. The most prevalent of these, bacteriochlorophyll, is stored in special vesicle-like organelles called chlorosomes.

Purple nonsulfur bacteria are similar to purple sulfur bacteria, except that they use hydrogen rather than hydrogen sulfide for oxidation. Among the purple nonsulfur bacteria is the genus Rhodospirillum. These microorganisms are facultative anaerobes, which are actually pink rather than purple, and can metabolize (&ldquofix&rdquo) nitrogen. They may be valuable in the field of biotechnology because of their potential ability to produce biological plastic and hydrogen fuel.

The green nonsulfur bacteria are similar to green sulfur bacteria but they use substrates other than sulfides for oxidation. Chloroflexus is an example of a green nonsulfur bacterium. It often has an orange color when it grows in the dark, but it becomes green when it grows in sunlight. It stores bacteriochlorophyll in chlorosomes, similar to Chlorobium, and performs anoxygenic photosynthesis, using organic sulfites (low concentrations) or molecular hydrogen as electron donors, so it can survive in the dark if oxygen is available. Chloroflexus does not have flagella but can glide, like Cytophaga. It grows at a wide range of temperatures, from 35 °C to 70 °C, thus can be thermophilic.

Another large, diverse group of phototrophic bacteria compose the phylum Cyanobacteria they get their blue-green color from the chlorophyll contained in their cells (Figure (PageIndex<3>)). Species of this group perform oxygenic photosynthesis, producing megatons of gaseous oxygen. Scientists hypothesize that cyanobacteria played a critical role in the change of our planet&rsquos anoxic atmosphere 1&ndash2 billion years ago to the oxygen-rich environment we have today. This group is discussed further in 3.1.3.1.

Figure (PageIndex<3>): (a) Microcystis aeruginosa is a type of cyanobacteria commonly found in freshwater environments. (b) In warm temperatures, M. aeruginosa and other cyanobacteria can multiply rapidly and produce neurotoxins, resulting in blooms that are harmful to fish and other aquatic animals. (credit a: modification of work by Dr. Barry H. Rosen/U.S. Geological Survey credit b: modification of work by NOAA)

Table (PageIndex<2>) summarizes the characteristics of some important groups of phototrophic bacteria.

Table (PageIndex<2>): Characteristics of Phototrophic Bacteria.
Phylum Class Example Genus or Species Common Name Oxygenic or Anoxygenic Sulfur Deposition
Cyanobacteria Cyanophyceae Microcystisaeruginosa Blue-green bacteria Oxygenic None
Chlorobi Chlorobia Chlorobium Green sulfur bacteria Anoxygenic Outside the cell
Chloroflexi (Division) Chloroflexi Chloroflexus Green nonsulfur bacteria Anoxygenic None
Proteobacteria Alphaproteobacteria Rhodospirillum Purple nonsulfur bacteria Anoxygenic None
Betaproteobacteria Rhodocyclus Purple nonsulfur bacteria Anoxygenic None
Gammaproteobacteria Chromatium Purple sulfur bacteria Anoxygenic Inside the cell


Discussion

Winogradsky column community structure

Winogradsky column microbial populations can be exceptionally diverse. As many as 30 phyla and 323 genera were present in an individual column, and among all columns 31 phyla and 414 genera were identified. Similarly high diversity in taxonomic groups has been observed in past studies of sediments, including over 40 phyla identified in a study of salt marsh sediments, and 18 phyla identified in a suboxic freshwater pond [29], [30]. The Shannon index also indicated an exceptionally diverse community, falling between 6 and 7 for SWI and deeper layers. This level of diversity is not unprecedented but is notable a prior study of salt marsh sediment microbial communities indicated Shannon index values of over 7 [31]. Furthermore, because the rarefaction curves reach a plateau, our sequencing effort was sufficient to effectively capture the diversity of the samples even at low sampling depth.

Our results show that these Winogradsky column communities contain few highly abundant taxa and a large number of more rare microbes. Most genera identified in the Winogradsky columns were present at less than 1% relative abundance, while between 19 and 30 genera were found at greater than 1% relative abundance. The Berger-Parker index demonstrated that community dominance was similar among all subsurface samples, and rarefaction curves of OTU richness continued to rise with additional sequences per sample (Figure 3). This distribution of few abundant and numerous rare microbes is typical of soil and other microbial communities studied using high throughput techniques [32]–[36]. In some cases, rare microbes have been cultured or shown to be functionally important in the ecosystem [37], [38]. However, the concept that microbial communities contain a “rare biosphere” is controversial, as several studies have suggested that measurements of the rare biosphere, and therefore diversity, are inflated by sequencing errors [39]–[43]. Indeed, with increasing sampling depth and constant frequency of sequencing error, one would expect richness and singleton OTUs to continue to increase indefinitely due to error alone. The diversity of very low abundance OTUs therefore could be explained equally well by either rare microbes, sequencing artifacts, or a combination of both. We attempted to minimize the impact of sequencing errors on our data by clustering OTUs at 97% identity (rather than 100%), and quality filtering our sequences using both chimera detection and removal of extremely rare sequences. The remaining OTUs occurred in at least 2 samples and were represented by at least 50 sequences, which makes it less likely that these OTUs were the products of individual sequence errors or sequencing artifacts.

Effect of environmental variables on Winogradsky microbial community

Our results suggest that the composition of the community in a Winogradsky column is shaped by a founder effect followed by diversification in stratified niches. When sediment is collected from the pond site, a founding population is captured and poured into the column. The high diversity of soil and sediment communities ensures that there are microbes that can thrive in the variety of niches that result in the column, including gradients of oxygen and hydrogen sulfide along the depth of the column. It is likely that the chemical and physical properties of the sediment contribute to the establishment of these niches as well. Colonization of the human microbiome is also subject to a similar founder effect followed by diversification. Delivery mode (vaginal vs. Cesarean section) influences the structure of the founding population that colonizes the infant [44], and over time site specific communities develop on the skin, gut, and other niches on or in the human body [45].

In our study, we found evidence of the founder effect on Winogradsky communities, as sediment source was an important driver of the composition of the resulting community. Beta diversity measures showed that regardless of depth or organic carbon source, columns made from the same sediment source were more similar to each other than columns made from different sediment sources (Figures 3 and 4). Sediment source biomarkers indicated that the same niches in Winogradsky columns may be filled by phylogenetically distant microbes according to a founder effect. Overall, there were more biomarkers for Eph's Pond than for Buxton Pond columns (Figure 5B), consistent with our alpha diversity results, which showed that Eph's Pond columns contained more diverse and rich microbial communities. Anaerobic members of the Proteobacteria, including sulfur cycle anaerobes within the Deltaproteobacteria, were higher in abundance in Eph's Pond columns at all layers than in the same layers of Buxton Pond columns. By contrast, the anaerobic phylum Firmicutes (including sulfate reducers within this phylum) was higher in abundance in Buxton Pond columns than Eph's Pond columns. These patterns suggest that both columns contained anaerobic and sulfur cycle niches dominated by different microbes according to a founder effect.

Once that founding population is added to the column, depth, and to a lesser extent, organic carbon source, allow the growth of specific bacteria in the different niches that are created. At the very top of the columns, the community, dominated by Cyanobacteria, was the least diverse and least rich (Figure 2). Samples collected by drilling into the SWI were the most variable but were no more diverse or rich than samples from greater depth. This indicates that, despite depth-based shifts in environmental conditions in Winogradsky columns, microbial diversity at all subsurface points remains high. Because we don't have information on the structure of the pond microbial community or the organic matter content of the sediments, we are unable to explain why supplementation caused only a minor shift in the population. The added leaf litter or vegetable scraps may have provided too little supplemental carbon to make a sufficient difference, or both sources may have provided similar supplements, changing the population from the founding pond sediments but not from eachother.

Using beta diversity analyses, we found separation of the phylogenetic and taxonomic composition of the communities by depth (upper layers: top surfaces and SWI, and lower layers: 4 cm, 8 cm, 12 cm). The separation of surface and SWI samples from deeper samples was more pronounced in weighted UNIFRAC, where relative abundance is taken into account. Weighted UNIFRAC has been suggested to be more appropriate in highlighting community differences based on shifts in abundance, such as those associated with differences in metabolite concentration [24]. We therefore propose that the differences seen between upper-level and lower-level samples reflect the major shifts in microbial community composition and structure that occur as conditions shift from oxic to anoxic. Similar patterns have seen in ponds receiving abundant organic matter and in flooded paddy soils, in which anoxic conditions rapidly develop close below the surface and anaerobic microbes produce methane and H2S [3], [29]. It will be interesting to determine if similarly high diversity as well as depth and sediment source effects are seen in Winogradsky columns prepared with materials from more distinct environments.

Composition of Winogradsky column communities

Proteobacteria, Firmicutes and Bacteroidetes made up more than 75% of the community of each sample. Proteobacteria were highest in abundance at the tops of columns and decreased in abundance with increasing depth. Proteobacteria are frequently identified as an abundant member of sediment microbial populations, and Alphaproteobacteria and Betaproteobacteria have been linked to oxic zones of vertical oxygen gradients in previous studies [3], [29], [46]–[48]. Our work shows that this association of members of the Proteobacteria with oxic zones is duplicated in Winogradsky columns. Interestingly, in both Eph's and Buxton Pond columns, in lower-depth samples, the communities were either abundant in Proteobacteria or Firmicutes, but not both, suggesting that local conditions favor growth of one or the other.

Firmicutes and Bacteroidetes were low in abundance at the tops of columns and increased in abundance with depth. Firmicutes, mainly Clostridium Cluster one, has previously been associated with anoxic zones in vertical oxygen gradients [3], [46] and the genus Clostridium has long been described as an abundant member of the bottom layers of Winogradsky columns. The high abundance of the class Clostridia at lower layers supports past culture-based associations between Clostridia and anoxic zones of sediment.

Bacteroidetes is another anaerobic phylum and its increasing abundance with depth is likely a reflection of decreasing oxygen concentration with depth. Certain members of the Phylum Bacteroidetes are capable of degrading complex organic compounds like cellulose [49] and chitin and are likely key to the carbon cycle in the Winogradsky column. The simpler carbon compounds produced by these decomposers can be utilized by other fermenting organisms. Overall the distribution of aerobic and anaerobic groups at different depths reinforces the implications of beta diversity analyses and strongly suggests a major impact of oxygen concentration on microbial communities in Winogradsky columns.

The sulfur cycle is a key biogeochemical cycle that is driven primarily by microbial processes. The Winogradsky column has been used to enrich for sulfur cycle organisms, and used as a teaching tool for demonstrating the sulfur cycle. High-throughput sequencing of Winogradksy columns allows for detailed analysis of the ecology and spatial distribution of sulfur cycle microbes and provides an example of taxonomically distant microbes filling the same niches in microbial communities. Dissimilative reduction of sulfur compounds is carried out by sulfur reducing bacteria for energy conservation. Anaerobic respiration of sulfate (SO4 2− ) generates hydrogen sulfide (H2S), which has a distinctive odor and spontaneously forms ferrous sulfide with iron, visible as a black coloration of the soil. The sulfur reducing bacteria are not a monophyletic group, but rather are defined physiologically. Many sulfur reducers are in the class Deltaproteobacteria, but some Firmicutes and Archaea are also capable of sulfate reduction [50]. In these columns, the particular sulfur/sulfate reducers found differed by sediment source used. The sulfate reducing Firmicutes in the family Peptococcaceae were identified as a biomarker for Buxton Pond columns while other sulfur/sulfate reducers were rare or absent (Figure 7). In Eph's Pond columns, sulfur/sulfate reducing organisms were more diverse and abundant and were almost exclusively Deltaproteobacteria, most of which were biomarkers for Eph's Pond columns (Figure 7). Deltaproteobacteria are commonly found in sediment communities and have been identified as an abundant member of black layer communities in wetlands [29], [51], [52]. The family Desulfobacteraceae was the most abundant sulfate reducer and was distributed throughout Eph's Pond columns. In the family Syntrophaceae about one third of the sequences classifiable to a genus belonged to Syntrophus, which cannot reduce sulfate however other sequences may belong to sulfate reducing members of this family. The Syntrophobacteraceae family is also comprised of a mix of sulfur reducers and non-sulfur reducers. The differences in sulfur/sulfate reducing communities between the two sediment sources demonstrate the functional overlap between highly divergent microbial groups.

Sulfur oxidizers complete the cycle by oxidizing H2S to elemental sulfur (S 0 ) and SO4 2− through phototrophy or chemolithotrophy. The Purple Sulfur Bacteria (PSB) use H2S, and sometimes other reduced sulfur compounds, as an electron donor in photosynthesis. They have red, orange, blue and yellow pigments, resulting in the red-violet zone of the Winogradsky column, in the upper-middle portion of the column. The process generates elemental sulfur that is stored in intracellular globules and can be later oxidized to SO4 2− . The PSB are all members of the class Gammaproteobacteria. Like the PSB, the Purple Non-sulfur Bacteria (PNSB) use H2S in photosynthesis, but generally have lower H2S concentration optima than the PSB. These are members of the Alphaproteobacteria and Betaproteobacteria. The Green Sulfur Bacteria (GSB) use H2S as an electron donor in photosynthesis, generating S 0 and SO4 2− . They are strict anaerobes, and are typically found in the “green zone” located in the lower-middle of the Winogradsky column. Neither the PSB nor the PNSB were found to be abundant in these columns (Figure 7). Although the GSB were more abundant, they were distributed unevenly in samples throughout the column rather than in the expected lower-middle zone. This is not surprising given that distinct green and red-violet zones were not apparent in these columns, although patches of color were present throughout at different intensities. Further, our sampling technique (drilling into the side of the column) may have excluded or destroyed surface-attached members of the microbial community.

Sulfur oxidation can also occur through chemolithotrophy, the use of a reduced sulfur compound as an electron donor in aerobic or anaerobic respiration. The chemolithotrophic sulfur oxidizer Thiobacillus was fairly abundant in the column, but especially in the upper-middle zones. They are shown in Figure 8 as Hydrogenophilaceae the vast majority of the sequences identified in this family belong to the genus Thiobacillus. Another important chemolithotrophic sulfur oxidizer is the filamentous non-photosynthetic Beggiatoa, [53]. This species is strongly associated with Sergei Winogradsky, as its isolation and characterization by Winogradsky led to the concept of lithoautotrophy [54]. No Beggiatoa sequences were found in these columns.

We also identified a collection of microorganisms in the 4 cm and 8 cm layers involved in methane cycling. The phylum Euryarchaeota, one of the two Archaeal phyla detected in the columns, was a biomarker for samples at 8 cm. A prior study of microbial community composition in suboxic freshwater ponds identified Euryarchaeota as the only archaeal phylum in the sediment [29]. Euryarchaeota includes methanogenic microbes. Interestingly, the methanotrophic Methylococcales (a Gammaproteobacteria) was a biomarker for 4 cm samples, suggesting that methane produced in deeper layers is being used by methanotrophs above. The detection of Methylococcales in this layer is interesting, given that the black coloration of the sediment (indicative of metal sulfide precipitation) suggests an anoxic environment.

Natural history of Eph's and Buxton Ponds

Both Buxton Pond and Eph's Ponds are located near Williams College, Willamstown, MA. Buxton Pond is entirely shaded and receives a substantial amount of leaf litter, while Eph's pond receives more direct sunlight. Buxton Pond is also closer to small roads. Both ponds have in the past experienced exposure to human waste. A sewer rupture in 1994 allowed raw sewage to flow into Eph's Pond for several days no remediation was performed. Until the mid-1990s Buxton Pond was the receiving water body for one of the Buxton boarding prep school's cesspools. Several fecal microbiome microbes were biomarkers for Buxton Pond (Bacteroides, Enterobacteriales, and Ruminococcus), perhaps reflecting past fecal contamination. However, these are also cellulose degraders, and may be present in greater abundance than in Eph's Pond due to the differences in the amount of leaf litter that falls on the ponds. Columns from both ponds have microbes associated with contamination with aromatic organic compounds (Dehalobacter, Desulfomonile, and Dechloromonas), consistent with their history and location in a lightly developed municipality.

Winogradsky columns as model communities

Microbial communities contribute critically to biogeochemical cycles, bioremediation, alternate fuel production, primary productivity, and numerous other processes critical in supporting ecosystems. They are also extraordinarily diverse and complex, containing numerous rare microbes, and are shaped by an enormous variety of factors including the presence or absence of other microbes, metabolic conditions, temperature, and pH. Studies of microbial communities in natural sediments are difficult to control for the impact of such variables. Here, we demonstrate the potential of Winogradsky columns for applying 16 s rRNA sequencing to the study of complex microbial communities. Winogradsky columns are easy to create, replicate, and manipulate, which allows for a degree of control not possible in field studies. We have demonstrated that Winogradsky column communities can be exceptionally diverse and display consistent patterns in microbial abundance based on depth, similar to natural ecosystems. Columns could also be manipulated to mimic the effects of changing temperatures, pollution, drought, or other effects relevant to current environmental challenges. Supplementing columns with different metabolites would offer a simple method for elucidating the effects of individual metabolites on stratified microbial communities. Time-course studies would offer insights in to the dynamics of communities over time as well as questions of succession and competition in sediment microbial communities. Winogradsky columns are self-contained and manipulatible ecosystems of diverse microbes and in combination with high-throughput sequencing could become a powerful tool for studies of microbial ecology.

Winogradsky columns in undergraduate education

Winogradsky columns are commonly used in undergraduate microbiology courses, for example as tools to teach principles of nutrient cycling [55]. Metagenomics is an increasingly important part of microbiology, and together, the two present a unique opportunity to introduce students to ground-breaking technology, laboratory techniques such as extraction of DNA and PCR, and analysis of large, complex datasets. Several successful programs have already integrated education and genomics research [56]. Some of the work presented in this paper was performed within introductory microbiology courses at Vassar College and Williams College, and we envision that short or more extensive units on Winogradsky metagenomics could be an effective component of undergraduate microbiology courses, or could be the focus of an advanced undergraduate course on microbial ecology or metagenomics. We have developed teaching materials to guide teachers on using wet-lab and bioinformatic techniques for Winogradsky metagenomics in their courses [57], and emphasize that the data generated in these studies are a publicly available resource that can be used to supplement laboratory instruction.


Watch the video: Winogradsky anabolism f17 (October 2022).