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Performance problems with dairy farm anaerobic digester linked to overly dominant Fungi

Biogas production from anaerobic digestion of organic waste is a biotechnological process involving complex bacterial, archaeal and likely fungal communities. The presence and function of active fungi in anaerobic digesters have been barely investigated (1). In this project, Aspergillus fungi were observed at a surprisingly dominant abundance in a Jersey cow dairy farm anaerobic digestion process that had been experiencing persistent, unresolvable foaming challenges. This raised suspicions by the customer’s fermentation engineers about the potential for Aspergillus to be contributing to the foaming problem, as well as negatively impacting biogas yields.

Background

A medium size dairy farm had been experiencing persistant, unresolvable foaming problems with their anaerobic digester for more than a year. They called on Microbe Detectives to see if the root cause could be microbiological. The selected diagnostic strategy had three parts.

Part 1

One sample of biosolids was collected with a specialized DNA analysis sample collection kit, from the influent, acidogenesis phase, and methanogenesis phase of the anaerobic digestion process. Each sample was analyzed with DNA amplicon sequencing of the 16S and 18S rRNA genes, identifying nearly all Bacteria, Archaea, and Eukarya in each sample at the genus level or above and their relative percentage abundance.

Part 2

A second set of samples was collected from a Holstein cow dairy farm anaerobic digestion process that was owned and operated by the same farmer. One sample of biosolids was collected from the acidogenesis phase, and methanogenesis phase. Each sample was collected and analyzed using the same DNA methods applied in Part 1. The Holstein dairy farm anaerobic digester was reported by the farmer to be performing well, and did not have a foaming issue.

Part 3

The data collected from Part 1 and Part 2 where then compared to see what we could discover, and potentially link to the foaming problem, warranting further investigation.

Top 10 Most Abundant Microbes

Farm A Anaerobic Digester, Bacteria and Archaea

Lachnospiraceae were the most abundant bacteria and archaea observed in the influent, acidogenesis phase, and methanogenesis phase. Lachnospiraceae are a family of obligately anaerobic, spore-forming bacteria that ferment diverse plant polysaccharides to short-chain fatty acids (butyrate, acetate) and alcohols (ethanol) (2). These bacteria are in the order Eubacteriales and are known to be the most abundant microbe group in the rumen, or second stomach, of dairy cows (3). Lachnoclostridum are genera members of the Lachnospiraceae family. Together, the % rel. abundance observed was 52% in the influent biosolids, 51% in the acidogenesis phase, and 9% in the methanogenesis phase.

Farm A Anaerobic Digester, Eukarya

Aspergillus was observed to be the most abundant eukaryotic microbe in the influent (47%), acidogenesis phase (49%), and methanogenesis phase (58%). It was notable that Aspergillus was observed to increase in abundance in methanogenesis to 58% rel. abundance, as shown below. This clearly demonstrated its dominance among eukaryotic microbes.

Farm B Anaerobic Digester

Our analysis of the top 10 most abundant microbes observed in the anaerobic digester at Farm B mainly focused on searching for notable differences in the microbiota, as compared to the anaerobic digester at Farm A. As shown below, a notable differentiating characteristic observed was a more diverse ecology and lack of a dominating eukaryotic microbe group, as compared to the anaerobic digester at Farm A.

Comparison of Eukarya % Abundance

The differentiating characteristics of the microbiota observed in the anaerobic digesters were also revealed by comparing the % rel. abundance of eukarya kingdoms observed. Specifically, in the methanogenesis phase of the anaerobic digester at Farm A, Fungi % rel. abundance was a dominant 74%. In the methanogenesis phase of the anaerobic digester at Farm B, a much more even distribution of the kingdoms was observed, where Fungi represented only 38% rel. abundance.

Comparison of Archaea % Abundance

Further evidence of key differences between the microbiota observed in the anaerobic digester at Farm A compared to Farm B was revealed by comparing % rel. abundance of archaea. All methanogens are members of the Archaea domain. These microbe groups play a key role in decomposing organics and producing renewable energy in the form of methane biogas.

As shown below, the % relative abundance of Archaea observed were significantly greater in the anaerobic digester at Farm B, 5.3% versus 0.7% in the acidogenesis phase, and 12.3% versus 2.6% in the methanogenesis phase. These are significant differences that indicate superior biogas production yields at Farm B.

Comparison of Methanogen % Abundance

A comparison of methanogen % relative abundance confirmed the differences with Archaea abundance. Methanogen % rel. abundance observed in the methanogenesis phase at Farm B was nearly five times greater than in the methanogenesis phase at Farm A, as illustrated below.

The Takeaway

In reviewing this data with Malcolm Fabiyi, PhD, an expert in anaerobic digestion and bioaugmentation, we were able to shed further light as follows:

  • The DNA data showed that Farm A had been extensively overrun by Aspergillus.
  • In the affected farm, the population of methanogens was depressed by the Fungi. The implication is that a route for methanogen seeding was significantly reduced, and as a result, archaea mediated processes, especially methanogenesis were compromised. 
  • Aspergillus sp. are known to generate biosurfactants which can affect the surface tension of digestate and enhance foaming tendency.
  • Foaming can occur as a result of an accumulation of fermentation products (VFAs, CO2, H2, etc.) due to reduced capacity for conversion of these intermediates to methane by hydrogenotrophic (CO2 + H2) and acetotrophic methanogens (convert VFAs to CH44 and CO2).
  • As proteinaceous matter gets degraded during fermentation, digester alkalinity increases and the combination of alkali and VFA can lead to foam formation.
  • Other mechanisms that could have contributed to foaming includes poor mixing, and excessive gas accumulation (e.g., CO2)

References

  1. Langer SG, Gabris C, Einfalt D, Wemheuer B, Kazda M, Bengelsdorf FR. Different response of bacteria, archaea and fungi to process parameters in nine full-scale anaerobic digesters. Microb Biotechnol. 2019 Nov;12(6):1210-1225. doi: 10.1111/1751-7915.13409. Epub 2019 Apr 17. PMID: 30995692; PMCID: PMC6801161.
  2. Boutard, M; Cerisy, T (13 November 2014). “Functional Diversity of Carbohydrate-Active Enzymes Enabling a Bacterium to Ferment Plant Biomass”. PLOS Genetics. 10 (11): e1004773. doi:10.1371/journal.pgen.1004773. PMC 4230839. PMID 25393313
  3. Seshadri, R; Leahy, SC (19 March 2018). “Cultivation and sequencing of rumen microbiome members from the Hungate1000 Collection”. Nature Biotechnology. 36 (4): 359–367. doi:10.1038/nbt.4110. PMC 6118326. PMID 29553575

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