Tag Archives: Microorganisms

Nitrogen continues to confound convention

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Ah nitrogen…  It is the most abundant molecule in the air that we breathe (close to 80%), yet plants always seem to be starving for it.  Annually, nitrogen fertilizers are a $75 billion dollar industry. The problem is that the nitrogen gas that we breathe (N2) is very nonreactive, because the two nitrogen atoms are held together by a massively powerful triple bond.  So N2 must be broken down to some other more usable form (such as ammonia) – a process we call nitrogen fixation.  Most nitrogen fixers are microorganisms that live in soils or symbiotically within plants.  Unfortunately, N-fixation is energetically very costly, so even organisms that can fix nitrogen will generally happily use nitrogen compounds from the soil or leaf litter (the layer of fallen leaves above the soil) if they are available, rather than expending enormous energy to fix it for themselves. The general formula for nitrogen fixation (ignoring protons, electrons and energy transfers) is…

DynarskiEquation

A few years ago Scott Morford, Benjamin Houlton and Randy Dahlgren (the first two are co-authors of the present study) stunned the ecological world by identifying a previously unsuspected source of nitrogen – weathering of bedrock such as the mica schist pictured below. This bedrock was formed from seabeds which were rich in organic matter and had a high concentration of nitrogen compounds When the rock breaks down, both carbon and nitrogen compounds leach into the soil. Katherine Dynarski became interested in nitrogen fixation as an undergrad at Villanova University, so it was natural for her to move to the University of California at Davis to begin her graduate work with Morford and Houlton on how nitrogen cycles through ecosystems.

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Nitrogen-rich mica schist bedrock. Credit: Katherine Dynarski.

Dynarski got involved in this specific project essentially by accident. She was helping a fellow graduate student collect rocks at adjacent forests on contrasting bedrock (one high-N mica schist, and one low-N basalt), and figured that while she was out there, she might as well measure some N-fixation rates. In leaf litter and the soil below, most N-fixation is done by free-living soil bacteria. Dynarski expected higher N-fixation rates in the litter collected above the N-poor bedrock, reasoning that the microorganisms would need to fix nitrogen from the air, because there was little present in the litter.  In contrast, she expected to find lower N-fixation rates in litter collected above the N-rich bedrock, reasoning that the micro-organisms could save considerable energy by using existing nitrogen that had leached into the soil and leaf litter layer. She was shocked when she ran the samples and found exactly the opposite of her expectation, which led her to develop a more substantial project looking at the relationship between bedrock and N fixing microbes.

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Katherine Dynarski conducting gas incubations to measure N-fixation rates in the field. Credit: Scott Mitchell.

Working in northern California and Western Oregon, Dynarski and her colleagues identified sites whose bedrock was low in nitrogen (below 500 parts per million N) or high in nitrogen (above 500 ppm N). The researchers used soil and leaf litter samples from 14 paired sites – high N bedrock with nearby low N bedrock. They analyzed soil and leaf litter samples from each plot for concentration of nitrogen, carbon (C), phosphorus (P) and molybdenum (Mo) – the latter two elements have been shown in other systems to limit the rate of N-fixation.  The researchers also collected samples of underlying bedrock and analyzed N and Mo content of these rocks.

Recall that the conventional paradigm is that microorganisms should have lower N-fixation rates in N-rich environments.  There was negligible N-fixation occurring in the soil, but considerable N-fixation in the leaf litter above.  Thus the conventional prediction was that N-fixation rates would be higher in leaf litter above low-N bedrock. As I mentioned previously, Dynarski found the exact opposite to be true in one site; would this unconventional finding be confirmed by the 14 sites explored in this study?

The answer is yes!  Considerably more N-fixation was detected in leaf litter above high N bedrock than in leaf litter above low N bedrock.

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Mean leaf litter N-fixation rates and low-N and High_N bedrock sites.  Error bars are one standard deviation. P = 0.014.

You will notice the large error bars above the graph.  As it turns out, N-fixation rates vary dramatically – even on a very small spatial scale, which is why the researchers took multiple samples from each site. Some sample sites (hotspots) have unusually high rates of N-fixation.  These hotspots are also strongly correlated with high carbon concentration, with greater C in the leaf litter associated with much higher rates of N-fixation.

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Litter N-fixation rates in relation to % soil carbon at N-fixation hotspots. Hotspots are defined as having fixation rates greater than 1 kg N per hectare per year.

Dynarski and her colleagues also discovered that, in general, leaf litter above high-N bedrock tended to have more C and P than did leaf litter above low-N bedrock.  Given this finding (along with the hotspot finding) we are now ready to explore the question of why microbes are expending more energy to fix nitrogen in regions where more nitrogen is naturally available.

The researchers considered two hypotheses.  First, it takes N to make N.  N-fixation is catalyzed by N-rich enzymes. It may be that leaf litter above low-N bedrock is too N-poor to provide microbes with enough nitrogen make these enzymes. So the additional nitrogen from high-N bedrock is just enough to allow microbes to produce the N-fixation enzymes.

The second hypothesis is that the litter above low-N bedrock is also low in C, P and Mo, all of which are required for N-fixation. Thus the positive effect of these nutrients overwhelms the negative effect of additional nitrogen on the rate of nitrogen fixation.  According to this hypothesis, the conventional paradigm of high nitrogen availability reducing the rate of N-fixation is correct, but other factors may be equally or more important in natural ecosystems.

Fortunately, this conundrum is easily resolved.  Dynarski and her colleagues took some leaf litter samples and added a small amount of nitrogen to them.  These N-additions significantly reduced N-fixation rates at both low and high bedrock N sites.  Thus environmental N does reduce biological N-fixation, but other factors, such as the availability of other essential nutrients, can overwhelm the inhibitory effect of environmental nitrogen in natural ecosystems

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A Douglas fir forest in the Oregon Coast Range, where some of this research was conducted.  Credit: Katherine Dynarski.

The researchers conclude that nitrogen input from bedrock weathering leads to increased C storage and P retention, ultimately enhancing rates of N-fixation. About 75% of Earth’s surface is underlain by rocks with substantial N reservoirs, so we need to continue exploring the effects of weathering bedrock on ecosystem processes and functioning.

note: the paper that describes this research is from the journal Ecology. The reference is Dynarski, K. A., S. L. Morford, S. A. Mitchell, and B. Z. Houlton. 2019. Bedrock nitrogen weathering stimulates biological nitrogen fixation. Ecology 100(8):e02741. 10.1002/ ecy.2741. Thanks to the Ecological Society of America for allowing me to use figures from the paper. Copyright © 2019 by the Ecological Society of America. All rights reserved.

A saltier Great Salt Lake supports a shifting ecosystem

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In science, like many other fields, “who you know” can be critical to success. Eric Boyd from Montana State University was introduced to the Great Salt Lake (GSL) ecosystem by his colleague Bonnie Baxter, a professor at Westminster College and the Great Salt Lake Institute in Salt Lake City, Utah.  Baxter was fascinated by microbialites- deposits of carbonate mud of diverse shape and structure, that harbor an impressive diversity and abundance of microorganisms.  Some of these microorganisms are photosynthetic, using dissolved organic carbon from the water to build carbohydrates; as such they are the primary producers which feed the rest of the ecosystem. Baxter impressed upon Boyd the need to understand the ecosystem, which feeds huge populations of two consumer species, the brine fly Ephydra gracilis and the brine shrimp Artemia franciscana. Up to 10 million birds, representing about 250 species, feed on these two species over the course of a year.

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Eric Boyd collects samples from the north arm of GSL. Credit: Bonnie Baxter.

In 1959 a railroad causeway was built that divided GSL into a south and north arm, which differ from each other in one critical way.  The south arm receives freshwater input from three rivers, while the north arm’s only freshwater input is rain and snowmelt.  Both arms are hypersaline; the south arm is 4-5 times saltier than typical ocean water, while the north arm is about twice as salty as the south arm. Boyd and Baxter recognized that these salinity differences were probably impacting the microbial communities in the two arms; in fact preliminary observations indicated that microbialite communities were no longer forming in the north arm.  So when Melody Lindsay began her doctoral research with Boyd, she elected to investigate how salinity was influencing the microbialite communities in the lake.

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Melody Lindsay (right) and Bonnie Baxter (left) planning to sample in the south arm of GSL.  Credit: Jaimi Butler.

Lindsay and her colleagues collected samples of microbialite mats from the south arm of the lake where the salinity of the water measured 15.6% (as a comparison, typical ocean water is about 3.5%).  At each of six salinity levels (8, 10, 15, 20, 25 and 30%), the researchers set up three microcosms of 150 ml of lakewater, which they then inoculated with 10 grams of homogenized microbial mat. They then sampled microbial diversity and abundance four and seven weeks after beginning the experiment.

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Exposed microbialites along the south arm’s shoreline.  Credit: Eric Boyd.

This experiment was conceptually simple, but technically a bit of a challenge.  Microorganisms are difficult to identify and count; and in fact it is likely that some of the species were new to science. Fortunately, researchers can use molecular approaches (quantitative PCR) to measure the quantity of each type of 16S rRNA gene in each microcosm. Each species of microorganism has distinct rRNA genes, so different base sequences indicate different microorganisms.  This allows researchers to estimate how much of each species is present. One restriction is that closely related species will have almost identical rRNA genes, so they may be difficult to distinguish from each other.

Overall, microorganism abundance was 152% greater after four weeks and 128% greater after seven weeks at the 15% salinity. Recall that these samples came from microbialites associated with 15.6% salinity, so this finding indicates good growth at the salinity which the microorganisms have recently experienced.  Interestingly, microorganisms thrived even better at 10% salinity.  But higher salinity levels, particularly  25% and 30%, were very detrimental to microbial growth.

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Change in abundance of 16S rRNA gene from microcosms incubated for four and seven seeks in comparison to abundance at week 0 for each salinity.  Significant differences are comparisons with abundance at week 0. NS = no significant difference, * P<0.1, ** P<0.01, *** P<0.001, **** P<0.0001. Error bars = 1SE.

The researchers broke down their results into taxonomic Orders, based on the 16S rRNA sequence of each gene. The two most common Orders were Sphingobacteriales and Spirochaetales, which both grew best at low salinity. The next most common Orders were a cyanobacterium from the Order Croococcales, and an alga from the Order Naviculales.  Species from these two taxonomic Orders are foundational to the ecosystem, because they are photosynthetic and relatively large. These dominant producers either directly, or indirectly, feed the rest of the ecosystem. Croococcales grew best at intermediate salinities (10-20%), while Naviculales did best at 8-15%, but also reasonably well at 20% salinity (see the figure below for a summary of the most common Orders).

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Abundance of taxonomic Orders of Microorganisms incubated at different salinities at 4 and 7 weeks, in comparison to initial abundance (week 0 = yellow square). Darker green squares indicate a greater increase, and darker brown squares indicate a greater decrease in abundance.  The most common Orders are on top, least common are on the bottom. Het = heterotroph, PP = primary producers, PhH = photoheterotroph.

Overall, primary productivity, as measured by how much dissolved organic carbon was taken up by the photosynthesizers, was greatest at 10 and 15%, and declined sharply above 20% salinity.  In addition, brine shrimp, one of the two important animal consumers of microorganisms, hatched and survived best at the lowest salinities.

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Two mating brine shrimp under the watchful eyes of an observer. Credit: Hans Hillewaert.

Lindsay and her colleagues conclude that conditions in the south arm are conducive to microbialite communities and the consumers they support.  However, the north arm has much lower productivity, with salinity levels so high that salt is spontaneously crystalizing out of solution in some areas. Given that climate change models predict increased drought severity over the next century in the GSL region, it is very likely that salinity levels will rise throughout the lake.  Over the same time period, humans are expected to increase water usage from the rivers that flow into the lake, which will further drop water levels in the lake, increase salinity in GSL, and dry out many of the microbial mats. This loss of ecosystem production is expected to cascade up the ecosystem, reduce brine shrimp abundance and ultimately the abundance and diversity of migratory birds that feed on them.

note: the paper that describes this research is from the journal Ecology. The reference is Lindsay, M. R.,  Johnston, R. E.,  Baxter, B. K., and  Boyd, E. S.  2019.  Effects of salinity on microbialite‐associated production in Great Salt Lake, Utah. Ecology  100( 3):e02611. 10.1002/ecy.2611. Thanks to the Ecological Society of America for allowing me to use figures from the paper. Copyright © 2019 by the Ecological Society of America. All rights reserved.

Recovering soils suffer carbon loss

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When dinosaurs roamed the Earth, and I was in high school, acid rain became big news.  Even my dad, who as an industrial chemist believed that industry seldom sinned, acknowledged that he could see how coal plants could release sulfur (and other) compounds, which would be converted to strong acids, borne by prevailing winds to distant destinations, and deposited by rain and snow into soils. Forest ecosystems in North America and Europe are happily, albeit slowly, recovering from the adverse effects of acid deposition, but there are some causes for concern.  At the Hubbard Brook Experimental Forest in New Hampshire, USA, researchers experimentally remediated some of the impacts of acid deposition by adding calcium silicate to a watershed (via helicopter!). A decade later, this treatment had caused a 35% decline in the total carbon stored in the soil. This result was very unexpected and alarming, as this could mean that acid-impacted temperate forests may become major sources of CO2, with more carbon running off into streams, and some becoming atmospheric CO2, as the effects of acid rain wane. Richard Marinos and Emily Bernhardt wanted to determine exactly what caused this carbon loss to better understand how forests will behave in the future as they recover from acidification.

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The forest at Hubbard Brook in the Autumn. Credit: Hubbard Brook Ecosystem Study at hubbardbrook.org

The problem is that calcium and acidity (lower pH is more acid: higher pH is more alkaline) have different and complex effects on plants, soil microorganisms and the soils in which they live. Several previous studies demonstrated that higher soil pH (becoming more alkaline) caused an increase in carbon solubility, while higher calcium levels caused carbon to become less soluble. Soluble organic carbon forms a tiny fraction of total soil carbon, but is very important because it can be used by microorganisms for cellular respiration, and also can be leached from ecosystems as runoff. In general, soil microorganisms benefit as acidic soils recover because heavy metal toxicity is reduced, enzymes work better, and mycorrhizal associations are more robust.  Complicating the picture even more, both elevated calcium and increased pH have been associated with increased plant growth, but increased calcium is also associated with reduced fine root growth.

To help unravel this complexity, Marinos and Bernhardt experimentally tested the effects of increasing pH and increased calcium on soil organic carbon (SOC) solubility, microbial activity and plant growth.  They collected acidic soil from Hubbard Brook Experimental Forest, which formed three distinct layers: leaf litter on top, organic horizon below the leaf litter, and mineral soil below the organic horizon.

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Soil excavation site at Hubbard Brook. Credit: Richard Marinos.

The researchers then filled 100 2.5-liter pots with these three soil layers (in correct sequence) and planted 50 pots with sugar maple saplings, leaving 50 pots unplanted.  Pots were moved to a greenhouse, and that November given one of five treatments: calcium chloride addition (Ca treatment), potassium hydroxide addition (alkalinity treatment), Ca + alkalinity treatment combined, a deionized water control, and a potassium chloride control. The potassium chloride control had no effect, so we won’t discuss it further.

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Potted sugar maple saplings used for the experiments. Credit Richard Marinos.

The following July, Marinos and Bernhardt harvested all of the pots, carefully separating plant roots from the soil, and analyzing the organic horizon and mineral soil levels separately (there wasn’t enough leaf litter remaining for analysis). The researchers measured SOC by mixing soil from each pot with deionized water, centrifuging at high speed to extract the water-soluble material, combusting the material at high temperature and measuring how much CO2 was generated. The result is termed water extractable organic carbon (WEOC).

Remember that previous studies had shown that higher calcium levels decreased carbon solubility, while higher alkalinity increased carbon solubility. Surprisingly, Marinos and Bernhardt found that in unplanted pots, the Ca treatment reduced WEOC in both soil layers, while the alkalinity treatment decreased WEOC in the organic horizon, but not in mineral soil. In pots planted with maple saplings, the Ca treatment had no effect on WEOC, while the alkalinity treatment, and the Ca + alkalinity treatment, increased WEOC markedly.

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Water-Extractable Organic Carbon in soil without plants (left column) and with plants (right column). Top graphs are organic horizon soils and bottom graphs are mineral horizon soils. Error bars are 1 standard error.

The next question was how might soil microorganisms fit into the plant-soil dynamics?

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Soil respirations rates (top) over the short term (days 1-7 post-harvest) and (bottom) the long term (days 8-75 post-harvest). Error bars are 1 standard error.

Soil microorganisms use carbon products for cellular respiration, so the researchers expected that soils with more SOC would have higher respiration rates.  They measured soil respiration 1, 2, 4, 8, 16, 35 and 72 days after the harvest, so they could evaluate both short-term and long-term effects. In unplanted pots, soil respiration rates were unaffected by treatment.  But in planted pots, the alkalinity treatment increased soil respiration rates considerably in the short term (top graphs), but much less so in the long-term (bottom graphs). Putting the WEOC data from the figure above together with the respiration data from the two figures to your left, you can see that in pots with plants, increased alkalinity was associated with more SOC and higher respiration rates.

The researchers weighed the saplings after harvest and discovered that the sugar maples grew best in soils treated with calcium. Two previous studies had treated fields with calcium silicate and found better sugar maple growth in the treated fields.  Marinos and Bernhardt argue that their study provides evidence that it is the Ca enrichment, and not the increased pH, that caused increased growth for both of those studies.

Perhaps the most surprising finding is that higher alkalinity increased soil microbial activity only in pots with plants, and had no effect on soil microbial activity in pots without plants. Somehow, the plants in an alkaline environment are increasing the rate of microbial respiration, perhaps by releasing carbohydrates produced by photosynthesis into the soil, which could then stimulate decomposition of SOC by the microorganisms. Finding that this effect largely disappeared a few days after harvest (bottom graph above), supports the idea that the plants are releasing a substance that helps microorganisms carry on cellular respiration. But this idea awaits further study. In the meantime, we have a better understanding of how forest recovery from acid rain affects one aspect of the carbon cycle, though many other human inputs may interact with this recovery process.

note: the paper that describes this research is from the journal Ecology. The reference is Marinos, R. E. and Bernhardt, E. S. (2018), Soil carbon losses due to higher pH offset vegetation gains due to calcium enrichment in an acid mitigation experiment. Ecology, 99: 2363-2373. doi:10.1002/ecy.2478. Thanks to the Ecological Society of America for allowing me to use figures from the paper. Copyright © 2018 by the Ecological Society of America. All rights reserved.