Biochar Puts Microbes on a DIET – Electron Transfer in the Soil Microbiome

By: Caroline Bresnan

A microbe’s life in the dirt is far from uneventful. Hundreds of species of microorganisms both compete and cooperate for survival in the rich, dark world of the soil microbiome. Recently, studies have shown that certain microbes can share electrons with each other, which allows them to perform feats (or, should I say, feasts) of metabolism that would otherwise be impossible. This phenomenon is called direct interspecies electron transfer, or DIET.

A colony of Geobacter metallireducens (green). Source

Professor Lovley discovered that G. metallireducens’ conductive pili make it a potential source of bioenergy. If you give a colony of G. metallireducens an electrode to grab with their pili, they can make enough electricity to power a calculator! Some day, Professor Lovley hopes to make a bacterial battery that can power larger appliances, perhaps even a car! (Click here to learn more about Professor Lovley’s microbial fuel cells.)

So how does all this relate to direct interspecies electron transfer (DIET)? Well, even though G. metallireducens prefers to send its electrons to a metal, sometimes there just isn’t any metal around. In that case, another place to put those electrons is a neighboring cell. A bacterial species called Geobacter sulfurreducens, which is closely related to G. metallireducens, has very similar conductive pili. When grown together, these two bacteria can form clusters, interlocking their pili to make their own power grid (Summers et al., 2010).

First, G. metallireducens extracts high-energy electrons from a food source like ethanol. After using the electrons for its own energy needs, it sends them to G. sulfurreducens, which also uses them for energy. G. metallireducens is like a power plant making electricity, while G. sulfurreducens is like your house which uses that electricity for important things like heat, light, and charging your computer. Though DIET between the two Geobacters is extremely effective, it can take over thirty days for both species to adapt to the new method of metabolism (Summers et al., 2010).

G. metallireducens (green) and G. sulfurreducens (red) can exchange electrons
by intertwining their pili. Like a power plant, G. metallireducens generates energy
that G. sulfurreducens can use. The pili act like tiny power lines between the two.
Source

Being able to perform DIET means bacteria can be more flexible in their metabolic strategies. When it comes to surviving in the soil, this ability gives them an edge. In the world of agriculture, a product called biochar may give DIET-performing species an even bigger advantage. Biochar is a charcoal-like substance made by burning organic material under low-oxygen conditions (Marris, 2006). Agricultural waste such as peanut shells and wood chips are perfect for the purpose. Many studies have shown that biochar substantially increases plant growth in addition to preserving moisture and nutrients in the soil. These properties are especially important for improving crop yields from depleted soils in developing countries.

Biochar may not look like much, but as a
soil additive, it can have a big impact.
Source

Biochar is also touted for helping to remove carbon dioxide from the atmosphere, which may reduce the effects of global climate change. The carbon in biochar is locked in an inert form that will not decompose into CO2, unlike the carbon in regular plant waste (Lehmann, 2007). There is evidence that biochar could trap carbon in the soil for thousands of years. Though biochar has many admirable qualities, its effects on the soil microbiome are not well understood. In particular, some recent findings highlight concerns about biochar’s potential to increase methane emissions (Chen et al., 2014).

Many experiments, like the one depicted above, have demonstrated

how biochar increases plant growth. (The biochar soil is on the left.)

Source

Last year, a team of researchers led by Shanshan Chen wanted to investigate how biochar affects DIET. They hypothesized that species like G. metallireducens and G. sulfurreducens could use biochar instead of their pili for DIET because biochar is electrically conductive. G. metallireducens would deposit electrons onto the biochar, while G. sulfurreducens would remove them. This would create a constant flow of electricity. Previous studies found that even mutant bacteria lacking pili could perform DIET when grown on materials similar to biochar.

The team of Chen et al. made co-cultures of G. metallireducens and G. sulfurreducens, some of which contained biochar and some of which did not. The cultures were set up in such a way that the bacteria would have to cooperate in order to grow. Each day, the researchers looked for evidence that the two species were performing DIET. This involved monitoring the concentration of ethanol, which is a food source for G. metallireducens, and succinate, which is a waste product of G. sulfurreducens. If the amount of ethanol decreased and the amount of succinate increased, Chen et al. could then conclude that the bacteria were cooperating through DIET.

This graph is busy, so focus on the black lines
first. They represent the co-cultures grown with
biochar, which began consuming ethanol and
making succinate within two days. The flatter
grey line with triangles represents the co-cultures
without biochar, which had very little metabolic
activity. (Chen et al., 2014)

For co-cultures without biochar, the concentrations of ethanol and succinate hardly changed. However, the co-cultures growing with biochar began consuming ethanol and producing succinate in less than two days. Previous studies showed that it can take thirty days for the microbes to adapt to DIET using just their pili. These results strongly suggested that the bacteria were relying on biochar as a conduit to transfer electrons. To support this conclusion, the researchers took pictures of the biochar at the microscopic level, and saw bacteria living on it. DNA analysis of the bacteria growing on the biochar confirmed that both species were attached to the surface.

In a second set of experiments, Chen et al. introduced a new microorganism, Methanosarcina barkeri, which belongs to the domain of archaea. Like bacteria, archaea are single-celled organisms that don’t have nuclei, but DNA analysis has shown that archaea and bacteria are very distinct. Some archaea are famous for living in extreme environments like deep-sea geothermal vents. However, M. barkeri prefers a less dramatic home in the soil. As the name Methanosarcina  implies, this archaea makes methane as a waste product of its metabolism. Through DIET, M. barkeri can use electrons from another microbe to make even more methane from CO2.

As before, the researchers made several co-cultures of G. metallireducens and M. barkeri both with and without biochar. This time, they measured the concentrations of ethanol and methane. Similar to the previous experiment, the cultures without biochar showed very little activity. Co-cultures with biochar, however, quickly began to consume ethanol and generate methane. To support these results, the researchers took pictures of the microbes growing on the biochar, and confirmed that both species were present with DNA analysis.

A scanning electron micrograph, which shows both G. metallireducens and
G. sulfurreducens attached to biochar. (Chen et al., 2014).

Chen et al.’s results show that biochar promotes cooperative metabolism between microorganisms. Just as a power plant needs customers to use its electricity, G. metallireducens must have a place to send its electrons. G. sulfurreducens and M. barkeri are the customers, who could not thrive without the energy from their microbial power plant. Biochar’s conductive properties allow it to act as the power lines between the two. This arrangement helps both species prosper.

In the soil microbiome, biochar provides a big advantage to microbes that can perform DIET, possibly helping them to outcompete other organisms. But is this necessarily a good thing? The archaea M. barkeri can produce extra methane when growing on biochar. As a greenhouse gas, methane is 20 times more potent than carbon dioxide (EPA, 2014)! Therefore, even though biochar might reduce the amount of CO2 in the atmosphere, by promoting M. barkeri’s growth through DIET, it might stimulate even more methane production. Furthermore, M. barkeri is only one of many methane-producing species that could be affected.

Many biochar enthusiasts are calling for its widespread use since it improves crop growth. Even so, what would happen to methane levels if we spread biochar on every field in America? What about every field in the world? Comparisons can be made to the pesticide DDT, which helped eliminate malaria in the United States, but also devastated the natural ecosystem and nearly wiped out the bald eagle. For now we can only speculate about biochar’s effects; there is still a lot of testing to be done. This study only looked at three microorganisms growing in the lab, and there are hundreds more living in the soil, which may have different reactions to biochar. Future studies will have to take a bigger look at the complex interactions of plants, microbes, and biochar if we hope to understand the full implications of adding this material to the soil.

Caroline is a senior and biology major at Mount Holyoke College.

References

Chen S, A Rotaru, PM Shrestha, NS Malvankar, F Liu, W Fan, KP Nevin, and DR Lovely. 2014. Promoting direct interspecies electron transfer with biochar. Nature Scientific Reports. 4(5019): 1-7.

EPA. 2014. Overview of greenhouse gases: methane. United States Government. Web.

Geobacter Project. 2014. Basic Science with an Applied Product. University of Massachusetts, Amherst: Department of Microbiology. Web.

Lehmann, J. 2007. A handful of carbon. Nature. 447: 143-144.

Liu F, A Rotaru, PM Shrestha, NS Malvankar, KP Nevin, and DR Lovely. 2012. Promoting direct interspecies electron transfer with activated carbon. Energy Environ. Sci. 5: 8982-8989.

Lovely DR, SJ Giovannoni, DC White, JE Champine, EJP Phillips, YA Gorby, and S Goodwin. 1993. Geobacter metallireducens gen. nov. sp. nov., a microorganism capable of coupling the complete oxidation of organic compounds to the reduction of iron and other metals. Archives of Microbiology. 159: 336-344.

Marris E. 2006. Black is the new green. Nature. 442: 624-626.

Summers ZM, HE Fogarty, C Leang, AE Granks, NS Mavankar, and DR Lovely. Direct exchange of electrons within aggregates of an evolved syntrophic coculture of anaerobic bacteria. Science. 330: 1413-1415.

Zhang T, P Tremblay, AK Chaurasia, JA Smith, TS Bain, and DR Lovely. 2013. Anaerobic benzene oxidation via phenol in Geobacter. Applied and Environmental Microbiology. 79(24): 7800-7806.