Date of Award

Spring 2018

Document Type


Degree Name

Master of Science (MS)


Civil Engineering

First Advisor

Zitomer, Daniel

Second Advisor

Venkiteshwaran, Kaushik

Third Advisor

McNamara, Patrick


This thesis describes three research projects on anaerobic digestion (AD) that investigated functional differences and relationships between microbial composition and digester function. Both archaeal and bacterial communities were characterized using high throughput (Illumina) sequencing technology with universal 16S rRNA gene primers. In the first project, limited aeration, and limited aeration with an aerotolerant enrichment culture were investigated as possible methods to increase digester functional performance in comparison to a strictly anaerobic control digester. No functional differences were observed at quasi-steady state between the limited aerated digester (LAD), limited aerated anaerobic digester augmented with an aerotolerant enrichment culture (LADE), and strictly anaerobic control digester (ACD). After limited aeration, Total Kjeldahl Nitrogen (TKN) concentration was less for the LAD than the ACD, and percent difference between the LAD and ACD total TKN concentrations was 6.5%, (p=0.044, n=5). Specific methanogenic activity (SMA) tests for acetate, propionate, non-fat dried milk, and non-fat dried milk and air determined that the LAD did not show an increased methanogenic rate in comparison to the ACD. However, the LADE had a greater SMA with propionate than the ACD. At quasi-steady state operation, differently operated digesters were shown to contain similar archaeal communities during steady state operation. The LAD, LADE, and ACD had a high relative abundance of Methanosaeta, an acetoclastic methanogen, which ranged from 58 to 77% in each digester. In the second project, a novel enrichment technique, that involved exposing methanogenic sludge to >100°C for 24 hours, was used to develop a culture dominated by hydrogenotrophic rather than acetoclastic methanogens and potentially establish the presence of syntrophic acetate oxidizing bacteria (SAOB). The enrichment technique was used on sludge samples obtained from the LAD, LADE, and ACD. LAD, LADE and ACD cultures exposed to high temperature drying were referred to LAD-D, LADE-D and ACD-D, respectively. These dried cultures were then rehydrated and used to seed new enrichment cultures. In SMA tests with non-fat dried milk and acetate, the LAD, LADE, and ACD (wet cultures) had statistically greater SMA than the LAD-D, LADE-D, and ACD-D (dried cultures) enrichments. In contrast, the dry culture enrichments had significantly greater SMA than wet cultures in the H2/CO2 SMA tests. The SMA decrease with non-fat dried milk was correlated to an increased relative abundance of Rikenella. Increased SMA with H2/CO2 was correlated to an increase in relative abundance of hydrogenotrophic methanogens such as Methanobacterium and Methanothermobacter, and a decreased Methanosaeta relative abundance. The third project investigated the effect of ammonia-nitrogen (NH3-N) concentration on the methanogenic rate of wet and dried cultures. Wet cultures had a higher SMA than dried cultures at 0, 3, 6, and 9 g/L NH3-N. However, heat drying increased the subsequent SMA value with H2/CO2. Dried cultures also had a higher relative abundance of Methanosarcina siciliae. Further research is needed to investigate enrichment and survivability of Methanosarcina siciliae at higher temperatures. Methanosarcina siciliae has potential use as an augment to reduce and extract energy from methanol, trimethylamine, and dimethyl sulfide (DMS) present in AD systems.