Preservation of Methanogenic Cultures to Enhance Anaerobic Digestion

The use of anaerobic biotechnology is increasing as a sustainable process to treat various organic wastes. Methanogens convert organic COD into CH4 and play the key role to drive thermodynamically unfavorable biochemical fermentation reactions and keep the digestion process steady and efficient. Progressive understanding of anaerobic microbiology with digester functionality may help to develop efficient, customized methanogenic cultures to enhance anaerobic bioprocesses. Preservation of methanogenic cultures via drying would be a cost-effective option for research and practical applications. However, preservation of methanogenic cultures is challenging due to methanogen sensitivity to O2 toxicity and drying, and very limited work is reported on their preservation. The work described herein involves preservation and subsequent storage of various methanogenic cultures in oxic conditions as well as applications to improve performance of anaerobic digesters and standardize laboratory testing. Five methanogenic cultures were customized under different growth conditions. The cultures were preserved using freeze- and heat-drying, and subsequently stored for short and long periods in the presence of air. Their activity was then assayed by measuring specific methanogenic activity. The influences of growth conditions and protective agent addition were investigated to improve methanogenic activity after preservation. Clone library and qPCR techniques were used to identify and quantify methanogenic communities before and after drying. The usefulness of preserved cultures was examined to bioaugment transiently upset anaerobic digesters and as seed inocula for a standard laboratory test, the biochemical methane potential (BMP) assay. The effect of bioaugmentation was correlated with methanogenic community structure using the DGGE molecular fingerprinting technique. All customized methanogenic cultures were significantly active even after handling, drying and subsequent storage in the presence of air, suggesting methanogenic culture preservation and storage in air is feasible. Freeze-dried cultures maintained higher methanogenic activity than heat-dried cultures. The culture developed in the presence of limited O2 exhibited higher methanogenic activity than cultures developed in strict anaerobic conditions regardless of the drying method employed. Glucose as a protective agent resulted in higher methanogenic activity, more so in freeze drying than heat drying. Some methanogenic community members were found to be more tolerant to drying stress than others. Dried methanogenic cultures were found to be viable options to use as a bioaugment to improve treatment efficiency of anaerobic digesters after toxic upset and for the BMP assay

Methyl Coenzyme M Reductase (mcrA) Gene Abundance Correlates with Activity Measurements of Methanogenic H2/CO2-Enriched Anaerobic Biomass

Biologically produced methane (CH4) from anaerobic digesters is a renewable alternative to fossil fuels, but digester failure can be a serious problem. Monitoring the microbial community within the digester could provide valuable information about process stability because this technology is dependent upon the metabolic processes of microorganisms. A healthy methanogenic community is critical for digester function and CH4 production. Methanogens can be surveyed and monitored using genes and transcripts of mcrA, which encodes the α subunit of methyl coenzyme M reductase – the enzyme that catalyses the final step in methanogenesis. Using clone libraries and quantitative polymerase chain reaction, we compared the diversity and abundance of mcrA genes and transcripts in four different methanogenic hydrogen/CO2 enrichment cultures to function, as measured by specific methanogenic activity (SMA) assays using H2/CO2. The mcrA gene copy number significantly correlated with CH4 production rates using H2/CO2, while correlations between mcrA transcript number and SMA were not significant. The DNA and cDNA clone libraries from all enrichments were distinctive but community diversity also did not correlate with SMA. Although hydrogenotrophic methanogens dominated these enrichments, the results indicate that this methodology should be applicable to monitoring other methanogenic communities in anaerobic digesters. Ultimately, this could lead to the engineering of digester microbial communities to produce more CH4 for use as renewable fuel

Biochemical Methane Potential Assays and Anaerobic Digester Bioaugmentation Using Freeze Dried Biomass

In this study, freeze dried methanogenic biomass (FDMB) was used as inoculum in place of conventional, non-dried biomass for biochemical methane potential (BMP) assays and as a bioaugment to improve upset digester recovery. Methanogenic biomass was freeze dried and stored in an air atmosphere. Significant methanogenic activity was preserved in FDMB even with drying and storage in air; specific methanogenic activity (SMA) values were 65 ± 4.5% and 42 ± 10.4% for hydrogen:carbon dioxide (H2:CO2) and acetate, respectively, compared to non-dried biomass. There was no significant difference in BMP results for the four substrates tested (glucose, non-fat dry milk, thin stillage and dog food) when using FDMB and non-dried biomass as inocula. However, BMP assays using FDMB inocula took longer to complete. Methane (CH4) production from digesters exposed to a model toxicant (oxygen [O2]) recovered faster when bioaugmented with FDMB compared to digesters that received autoclaved biomass or no bioaugmentation. Methanogen communities in all digesters before toxicant exposure and bioaugmentation were similar. However, bioaugmented and non-augmented digester communities were significantly different during the recovery phase after toxicant exposure. Sequences similar to Methanospirillum were related to improved performance of the FDMB bioaugmented digesters. FDMB could be developed as a standard inoculum for BMP analyses and to bioaugment anaerobic digesters for improved performance. These results may encourage developing customized, dried methanogenic biomass for specific anaerobic biotechnology applications

Activity of Methanogenic Biomass After Heat and Freeze Drying in Air

It would be beneficial if methanogenic cultures could be preserved for anaerobic digester bioaugmentation or as seed for standard tests such as biochemical methane potential. However, storage of wet culture or drying in anaerobic atmosphere may not be economically feasible. In this study, the effect of heat and freeze drying in ambient air on the methanogenic activity of an anaerobic culture was determined. The anaerobic culture was dried in air at 104 °C for 12 h, and by freezing at −196 °C in air with subsequent drying at subzero temperatures. The rehydrated culture consistently produced CH4 from H2:CO2 and acetate after drying. Drying caused a greater decrease in acetate methanogenic activity compared to H2:CO2 methanogenic activity. Transcript qPCR results for a functional gene in methanogens (mcrA) also revealed significant survivability of rehydrated methanogenic populations. Inactivation due to drying differed among genera, with least to most inactivation in the order Methanospirillum \u3c Methanosaeta \u3c Methanoculleus