Characterization of Extremophilic Bacteria for Potential in the Biofuel and Bioprocess Industries

Industrial bioprocesses are constrained by the availability of microbes that are optimized for harsh bioprocess conditions. Over 500 soil and sediment samples collected from 77 saline and/or thermal sites in the continental United States, Hawaii, and Puerto Rico were used to inoculate fermentations in an effort to optimize the microbial communities for a biofuel process developed at Texas A&M (MixAlco?).

A diverse bacterial isolate library (N= 1,866 isolates) was constructed by employing a variety of culturing techniques across thirty-four of the fermentation communities selected based on superior performance in the biofuel platform. Phylogenetic analysis of partial 16S rDNA sequences placed 1,200 of these isolates in the genus Bacillus. Other genera include, but are not limited to: Ureibacillus, Bacillus, Geobacillus, and Caldalkalibacillus.

The central hypothesis of this dissertation was that selection due to the original site conditions of the natural inocula, and also, survival during fermentation likely favored isolates in the library that possess biofuel/bioenergy relevant traits (e.g. hydrolysis of lignocellulosic biomass, utilization of hydrolysis products, and tolerance to inhibitory compounds released during hydrolysis).

The phylogeny for this library was used to identify and prioritize a diverse subsample of the library (n=207) for high-throughput screens of extracellular cellulase activity, n-butanol tolerance, vanillin utilization, and lignin degradation, as indicated by decolorization of the surrogate Congo red. Many isolates exhibited the capacities screened, including several isolates that were positive for more than one of these traits. Subsequently, a subset of the 207 screened isolates were studied further for tolerance and/or utilization of bioprocess byproducts (e.g. lignocellulosic hydrolysate, bio-oil) that are known to harbor both compounds with inhibitory effects on growth and pentose sugars released after hydrolysis. Polyhydroxyalkanoates (PHA) are bio-plastics produced by some bacteria. There is interest in industry in the identification of bacteria that can utilize wastes from some bioprocesses while creating bio-plastics, thus, a subset of the library (n=43 isolates) was screened for PHA accumulation associated with growth with the bio-diesel byproduct glycerol as the carbon source. Several isolates from the library are PHA producers with glycerol based on a fluorescence screen conducted. Microfluidic microbial fuel cells (MFCs) take advantage of microbial metabolism to convert organic matter to electricity. Microbial communities collected directly from soils were screened in a MFC array developed at Texas A&M. The screening of natural microbial communities identified electricigens with enhanced power generation abilities.

The variation identified in these industrially relevant traits across isolates provides a proof of concept for both the existence of this variation in nature and the efficacy of employing fermentation and culturing approaches to enrich for these phenotypes. Also, it became clear this library could serve as a resource for bioprocess isolates for industry, either directly or as a starting point to advance the bioprocess optimization prior to some form of genetic engineering targeting a particular function.