The effect of simple and complex on the composition and diversity of microbial communities and on end-product (biogas and VFAs) synthesis was investigated using an anaerobic batch respirometer at 37 °C and pH 7.2. These experiments, simple substrates were chemically pure and contain a single carbon source (glucose or α-cellulose), while complex substrates were chemically “impure” substrates containing a mixture of two or three carbon sources (biodiesel-derived glycerol or wheat straw) with a substrate/inoculum ratio 6g chemical oxygen demand (COD)/ g volatile solids (VS) seed and 100g of pre-treated dairy manure digestate (DMD), respectively. Concentrations of hydrogen, carbon dioxide, acetate, butyrate, propionate, and ethanol synthesized by different communities selected by growth on the different substrates were measured and confirmed the growth of the microbial communities. 16S rDNA illumina sequencing revealed that DMD without substrates was more diverse than the microbiota cultured by fermentation reactions containing D-glucose, glycerol α-cellulose or wheat straw. The data confirmed that substrates play a crucial role in determining the diversity of species in microbial communities. Dominant operational taxonomic units (OTUs) belonging to families Clostridiaceae, Ruminococcaceae, and Enterobacteriaceae, and the genera Clostridium, Ruminococcus, Sporolactobacillus, and Syntrophomonas were potentially responsible for changes in end-product synthesis patterns in communities cultured with simple and complex substrates. / October 2015

Via mixed-culture fermentation, the MixAlcoTM produces carboxylic acids, which are chemically converted into industrial chemicals and hydrocarbon fuels. Using pilot fermentation data, The Continuum Particle Distribution Model (CPDM) overestimated acid concentration (30–90% error) but more closely estimated conversion (<15% error). Incorporating the effect of air into the model reduced the absolute error of all predictions by >50%. To analyze fermentation data with semi-continuous streams, the Slope method calculates the average flowrate of material from the slope of the moving cumulative sum with respect to time. Although the Slope method does not significantly improve accuracy, it dramatically reduces error compared to traditional techniques (>40% vs. <2%). Nutrients are essential for microbial growth and metabolism. For a four-bottle fermentation train, five nutrient contacting patterns (single-point nutrient addition to Fermentors F1, F2, F3, F4, and multi-point parallel addition) were investigated. Compared to the traditional nutrient contacting method (all nutrients fed to F1), the near-optimal feeding strategies improved exit yield, culture yield, process yield, exit acetate-equivalent yield, conversion, and total acid productivity by approximately 31%, 39%, 46%, 31%, 100%, and 19%, respectively. To estimate nitrogen concentration profiles, a segregated-nitrogen model uses separate mass balances for solid- and liquid-phase nitrogen; the nitrogen reaction flux between phases is assumed to be zero. Using five fermentation trains, each with a different nutrient contacting pattern, the model predictions capture basic behavior; therefore, it is a reasonable tool for estimating and controlling nitrogen profiles. To determine the optimal scenario for mixed-acid fermentations, an array of batch fermentations was performed that independently varied the C/N ratio and the blend of carbohydrate (office paper) and nutrient (wet chicken manure (CM)). Reactant was defined as non-acid volatile solids (NAVS). C/N ratios were based on non-acid carbon (CNA). A blend of 93% paper and 7% wet CM (dry basis) with a C/N ratio of 37 g CNA/g N had the highest culture yield (0.21 g acidproduced/g NAVSinitial), total acid productivity (0.84 g acidproduced/(Lliq·d)), and conversion (0.43 g NAVSconsumed/g NAVSinitial).

Soft sensors have been suggested as potent tools for on-line estimations of critical bioprocess variables to be able to control the biological process in an as high extent as possible. The formation of inhibitory by-products in the form of organic acids, caused by an overflow of glucose, is a problem in most bioprocesses expressing recombinant proteins.    In this project a new method of controlling the glucose feeding in an Escherichia coli fed-batch cultivation expressing the green fluorescent protein (GFP) was investigated. The new controller system implemented in the software controlled the feed rate based on on-line HPLC measurements of the concentration of organic acids.      The results showed that the controller managed to down-regulate the inhibitory organic acids to a low level as it tried to keep the glucose uptake rate at an optimum for maximum cell growth. The results suggested that the controller could be a powerful tool to create a more secure reproducibility and to generate high product yields in recombinant protein productions.

Using mixed-culture fermentation, the carboxylate platform produces carboxylic acids, which are chemically converted into chemicals and fuels. To optimize the mixed-acid fermentation, different bioreactor configurations and operating modes were investigated. Intermittent air exposure did not affect fermentation performance and bacterial profiles, but reduced the high-molecular-weight carboxylic acids. The microbial flora contained strict and facultative microbes, suggesting the presence of a facultative anaerobic community existing in a biofilm. Compared to countercurrent trains, propagated fixed-bed fermentations have similar selectivity and acid distribution, but lower yield, conversion, productivity, and acid concentration. One- to six-stage countercurrent fermentations were operated with similar conditions. Fewer stages increased conversion, whereas more stages increased acid concentration and selectivity. One to four stages achieved similar yield, and four to six stages achieved similar maximum acid concentration. Maximum conversion was achieved with a single stage. Recycling residual biomass retained microorganisms and nutrients and increased yield and productivity. Relative to lower biomass reflux, higher reflux increased conversion, decreased selectivity, and did not affect yield. The recommended carbon-nitrogen ratio is ~24 g carbon/g nitrogen. In four-stage fermentations, recycle to the second fermentor and in parallel to the first three fermentors was optimal. Fermentations with excess or insufficient nitrogen had higher selectivity, but decreased yield and conversion. The glucose-utilization assay is a rapid and repeatable method for determining the amount of microbial activity in a sample. This method determined ~25% efficiency of a new cell separation method. In continuous fermentation, compared to no cell recycle, recycling cellular biomass increased selectivity and yield, but decreased conversion. Compared to lower cell reflux, higher reflux increased productivity, yield, and conversion, but decreased selectivity. Compared to residual biomass recycle, cell recycle had increased selectivity and yield, but decreased conversion. A new method to screen and rank inoculum sources from natural environments was successfully developed and tested.