I recently graduated from the University of Vermont with a Master of Science in Civil and Environmental Engineering. While there, I had the opportunity to study a full-scale anaerobic digester co-digesting cow manure and pre-treated food waste from start-up through stable operation over the course of a year with UVM’s Environmental Microbiome Engineering Research Group (EMERG). The study aimed to improve our understanding of the dynamics of the core microbial community in anaerobic digestion and assess gene abundance.

Anaerobic digestion is the conversion of organic matter to renewable energy by a complex community of naturally occurring microorganisms in an engineered environment. Anaerobic digestion and the production of biogas provide many benefits, such as diverting organic waste from landfills, promoting nutrient recovery, reducing greenhouse gas emissions, and producing renewable energy in the form of biogas. Biogas is a byproduct of anaerobic digestion and consists of approximately 60% methane, 40% carbon dioxide, and other trace gases. Once biogas is produced, it can be combusted to create electricity or upgraded into pipeline quality renewable natural gas.

The digester was studied through three stages. During the first stage, cow manure was the sole substrate fed to the anaerobic digester (AD). During the second stage, the digester was inoculated twice with sludge (SD) from an external full-scale digester. During the third stage, pre-treated food waste was added to the AD for co-digestion with the manure. Food waste was pre-treated by being fed to a hydrolysis tank (HT) prior to entering the AD.

Sample Collection

Once per week over the course of a year, I would collect samples from the digester and the hydrolysis tank. I was also able to collect samples of the sludge used to inoculate the digester before it was added to the AD. Following collection, samples were transported to the lab for processing and preservation. DNA was then extracted from the naturally occurring microorganisms in the samples and sent for metagenomic next-generation sequencing. The goal of sequencing was to: (i) analyze the microbial community, and (ii) analyze the abundance of genes of interest.

Anaerobic Digester Conditions

The first part of the study was to look at the monitored operational parameters. The red vertical line indicates the two days the digester was inoculated, and the green vertical line indicates the day co-digestion with pre-treated food waste began.

The digester’s pH was stable (around 7.5) throughout operation. The pH of the hydrolysis tank ranged from 3.5-4.5 and was significantly more acidic than the digester to promote the degradation and fermentation of complex food waste prior to entering the anaerobic digester. The pH in the hydrolysis tank did not seem to impact the pH in the anaerobic digester.

The organic loading rate (OLR) is the amount of organic waste fed to the digester on a daily basis. The OLR steadily increased as expected during start-up. The concentration of volatile fatty acids increased immediately after food waste addition, steadily decreasing over time even while the OLR increased. High volatile fatty acid concentrations can create inhibitory conditions in the digester which prevent methanogenic bacteria growth. The methane composition of biogas stabilized to 65% after co-digestion began, which is higher than other reported full-scale digesters co-digesting cow manure and organic wastes.

Microbial Community – Taxonomic Classification

After developing an understanding of the conditions inside the digester, metagenomic sequencing results were analyzed. Downstream analyses including sequence assembly, annotation, and mapping were performed with the SqueezeMeta pipeline. For taxonomic classification, the NCBI GenBank database was used.

The first abundance plot I created looked specifically at the methanogenic community, which consists solely of archaea. This is the community of microorganisms that are responsible for producing the biogas used for renewable energy. The total abundance of methanogenic archaea decreased significantly after the digester was inoculated on Day 27 and 29 and began to increase and stabilize only after co-digestion with food waste began on Day 104. The most abundant genera included Methanocorpusculum, Methanoculleus, and an unclassified Methanomicrobiales. These genera belong to the Methanomicrobiales order which are known to perform hydrogenotrophic methanogenesis, which is the production of methane from carbon dioxide and hydrogen gas. Also interesting, the most abundant archaea within the inoculum sludge were already abundant in the anaerobic digester prior to inoculation.

Gene Abundance

After assessing the methanogenic community, heat maps were created to get a closer look at the abundance of specific genes of interest. The KEGG database was used for gene annotation and all genes were identified by their KEGG identification numbers. The abundance value of each gene was calculated from the mapped reads using the transcript per million (TPM) approach, which normalizes the number of DNA reads mapped to a specific gene based on the total number of reads in a sample.

Sugar Degradation & Fermentation

The first heat map focused on genes that encode sugar degradation and fermentation in the different sampling locations, as seen in the Sugar Degradation and Fermentation Heat Map. First looking at sugar degradation, genes encoding glucokinase (glk) and xylulokinase (xylB) were highly abundant in the AD. This suggests that both five- and six- carbon sugars were being fermented. Sugar degradation genes in the HT were widely different than the AD. Specifically, genes encoding the phosphoketolase enzyme (xfp) were highly abundant in the HT.

Genes encoding simple fermentation, specifically lactate production (ldh), ethanol production (adh), and acetate kinase (ackA) were highly abundant in the HT compared to the AD. Lactate, ethanol, and acetate are all intermediate products in the anaerobic digestion pathway. Results from the sugar degradation and fermentation heatmap generally suggested that the main role of the HT was to promote the fermentation of complex organic waste.

Methanogenesis

Genes encoding methanogenesis, or methane production, were then investigated. Genes were grouped by acetoclastic methanogenesis and hydrogenotrophic methanogenesis. Acetoclastic methanogens use acetate to produce methane, while hydrogenotrophic methanogens use hydrogen gas and carbon dioxide to produce methane.

Results from the methanogenesis heat map indicate that all genes needed for hydrogenotrophic methanogenesis were present across the AD samples suggesting that hydrogenotrophic methanogenesis was the dominant route of methane production. This was further supported by the taxonomic classification results which showed the most abundant methanogenic archaea were known hydrogenotrophs.

Study Results & Findings

In total, it was found that a highly resilient methanogenic microbial community had already taken hold in the anaerobic digester and inoculation had little impact on the overall process. This suggests that so long as a stable pH is maintained and the organic loading rate is increased slowly, cow manure itself may be a suitable inoculum for the co-digestion of cow manure and pre-treated food waste. Differences in gene abundance across the HT and AD also suggest significant differences in fermentative pathways within these two reactors.

In total, this research could influence the start-up of other full-scale anaerobic digesters co-digesting cow manure and pre-treated food waste, allowing for more opportunities to reduce waste to landfills while enhancing renewable energy production.

Hoyle Tanner has a RISER (Resiliency, Innovation, Sustainability, Economics, and Renewables) committee that focuses on sustainable practices. Our environmental engineers would like to help your community. Connect with me to get started!

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