Date of Award

Summer 2012

Document Type


Degree Name

Doctor of Philosophy (PhD)


Civil, Construction, and Environmental Engineering

First Advisor

Zitomer, Daniel H

Second Advisor

King, Michael

Third Advisor

Maki, James


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.