Multidisciplinary Graduate Education in Bioprocess Engineering

This report describes the accomplishments of the University of Georgia in establishing an academic program geared toward the emerging biobased products industry. By virtue of its strengths and structure, the University of Georgia is particularly well-suited for developing a program focused on plant- and microbial-based bioproducts, and it was in this general area that this program was developed. The program had several unique characteristics. First, we implemented a distinguished lecture series that brought outstanding scientists and engineers to our University to interact with students and share their vision of the biobased economy. Second, we offered industrially-oriented and multidisciplinary courses that provided students with a broad background on various facets of biobased business and technology. Third, we provided the students with opportunities to expand beyond the classroom by engaging in research lab rotations and industrial internships. Fourth, each student was engaged in a creative research project as led by a multidisciplinary faculty team. Throughout the implementation of these activities, we maintained a student-centered, mentoring approach to education. The most tangible outcome of this project was the graduation of two students who participated in a variety of scholarly activities, culminating in research toward the completion of a thesis and dissertation. Both research projects involved the use of microorganisms to produce industrial products from agricultural substrates via fermentation processes. The research advanced our understanding of microorganisms as used for industrial processes and products, as described in several articles published in scholarly journals and presentations made at scientific conferences (see information on pp. 14-15). Another outcome is one graduate course, Fermentation Engineering Laboratory, which is a unique experiential and multidisciplinary course. This course will be offered in the future as an elective to graduate students in several engineering and science degree programs. Other significant developments have arisen as direct or indirect consequences of this project. The University of Georgia has established a B.S. Biochemical Engineering degree and an M.S. Biochemical Engineering degree. A strong component of these degree programs is education toward a biobased economy. We will integrate particularly positive components of this project (such as the distinguished lecture series) into these degree programs. The University of Georgia is establishing a Center for Biorefining and Carbon Cycling. This multidisciplinary Center houses a pilot scale biorefinery, comprising a pyrolysis unit and an ethanol plant. Together with new faculty positions that are currently being advertised, this project has encouraged the University of Georgia to assume a leadership role in the preparation of students in the biobased industries of the future

Process Design for the Biocatalysis of Value-Added Chemicals from Carbon Dioxide

This report describes results toward developing a process to sequester CO{sub 2} centered on the enzyme pyruvate carboxylase. The process involves the use of bacteria to convert CO{sub 2} and glucose as a co-substrate and generates succinic acid as a commodity chemical product. The first phase of this research has focused on strain development and on process development. Progress in strain development has been made in three areas. The gene encoding for alcohol dehydrogenase has been ''knocked out'' of the bacteria, and thereby eliminating the synthesis of the by-product ethanol. The gene for glucokinase has been overexpressed in the production strain with the goal of faster utilization of glucose (and hence CO{sub 2}). Efforts have continued toward integrating pyruvate carboxylase gene (pyc) onto the E. coli chromosome. Progress in process development has come in conducting several dozen fermentation experiments to find a defined medium that would be successful for the growth of the bacteria, while permitting a high rate of CO{sub 2} utilization in a subsequent prolonged production phase. Using this defined medium, the strains that continue to be constructed are being compared for CO{sub 2} utilization, so that we may understand the factors that govern the biological sequestration process

A co-fermentation strategy to consume sugar mixtures effectively

We report a new approach for the simultaneous conversion of xylose and glucose sugar mixtures into products by fermentation. The process simultaneously uses two substrate-selective strains of Escherichia coli, one which is unable to consume glucose and one which is unable to consume xylose. The xylose-selective (glucose deficient) strain E. coli ZSC113 has mutations in the glk, ptsG and manZ genes while the glucose-selective (xylose deficient) strain E. coli ALS1008 has a mutation in the xylA gene. By combining these two strains in a single process, xylose and glucose are consumed more quickly than by a single-organism approach. Moreover, we demonstrate that the process is able to adapt to changing concentrations of these two sugars, and therefore holds promise for the conversion of variable sugar feed streams, such as lignocellulosic hydrolysates

Biorefinery and Hydrogen Fuel Cell Research

In this project we focused on several aspects of technology development that advances the formation of an integrated biorefinery. These focus areas include: [1] establishment of pyrolysis processing systems and characterization of the product oils for fuel applications, including engine testing of a preferred product and its pro forma economic analysis; [2] extraction of sugars through a novel hotwater extaction process, and the development of levoglucosan (a pyrolysis BioOil intermediate); [3] identification and testing of the use of biochar, the coproduct from pyrolysis, for soil applications; [4] developments in methods of atomic layer epitaxy (for efficient development of coatings as in fuel cells); [5] advancement in fermentation of lignocellulosics, [6] development of algal biomass as a potential substrate for the biorefinery, and [7] development of catalysts from coproducts. These advancements are intended to provide a diverse set of product choices within the biorefinery, thus improving the cost effectiveness of the system. Technical effectiveness was demonstrated in the pyrolysis biooil based diesel fuel supplement, sugar extraction from lignocelluose, use of biochar, production of algal biomass in wastewaters, and the development of catalysts. Economic feasibility of algal biomass production systems seems attractive, relative to the other options. However, further optimization in all paths, and testing/demonstration at larger scales are required to fully understand the economic viabilities. The various coproducts provide a clear picture that multiple streams of value can be generated within an integrated biorefinery, and these include fuels and products

Biorefinery and Carbon Cycling Research Project

In this project we focused on several aspects of technology development that advances the formation of an integrated biorefinery. These focus areas include: [ 1] pretreatment of biomass to enhance quality of products from thermochemical conversion; [2] characterization of and development of coproduct uses; [3] advancement in fermentation of lignocellulosics and particularly C5 and C6 sugars simultaneously, and [ 4] development of algal biomass as a potential substrate for the biorefinery. These advancements are intended to provide a diverse set of product choices within the biorefinery, thus improving the cost effectiveness of the system. Technical effectiveness was demonstrated in the thermochemical product quality in the form of lower tar production, simultaneous of use of multiple sugars in fermentation, use ofbiochar in environmental (ammonia adsorption) and agricultural applications, and production of algal biomass in wastewaters. Economic feasibility of algal biomass production systems seems attractive, relative to the other options. However, further optimization in all paths, and testing/demonstration at larger scales are required to fully understand the economic viabilities. The coproducts provide a clear picture that multiple streams of value can be generated within an integrated biorefinery, and these include fuels and products

Process Design for the Biocatalysis of Value-Added Chemicals from Carbon Dioxide

This report describes results toward developing a process to sequester CO{sub 2} centered on the enzyme pyruvate carboxylase. The process involves the use of bacteria to convert CO{sub 2} and glucose as a co-substrate and generates succinic acid as a commodity chemical product. The first phase of this research has focused on strain development and on process development. Progress in strain development has been made in three areas. The gene encoding for alcohol dehydrogenase has been ''knocked out'' of the bacteria, and thereby eliminating the synthesis of the by-product ethanol. The gene for glucokinase has been overexpressed in the production strain with the goal of faster utilization of glucose (and hence CO{sub 2}). Efforts have continued toward integrating pyruvate carboxylase gene (pyc) onto the E. coli chromosome. Progress in process development has come in conducting several dozen fermentation experiments to find a defined medium that would be successful for the growth of the bacteria, while permitting a high rate of CO{sub 2} utilization in a subsequent prolonged production phase. Using this defined medium, the strains that continue to be constructed are being compared for CO{sub 2} utilization, so that we may understand the factors that govern the biological sequestration process

Nutrient-Limited Operational Strategies for the Microbial Production of Biochemicals

Limiting an essential nutrient has a profound impact on microbial growth. The notion of growth under limited conditions was first described using simple Monod kinetics proposed in the 1940s. Different operational modes (chemostat, fed-batch processes) were soon developed to address questions related to microbial physiology and cell maintenance and to enhance product formation. With more recent developments of metabolic engineering and systems biology, as well as high-throughput approaches, the focus of current engineers and applied microbiologists has shifted from these fundamental biochemical processes. This review draws attention again to nutrient-limited processes. Indeed, the sophisticated gene editing tools not available to pioneers offer the prospect of metabolic engineering strategies which leverage nutrient limited processes. Thus, nutrient- limited processes continue to be very relevant to generate microbially derived biochemicals

Simultaneous utilization of glucose, xylose and arabinose in the presence of acetate by a consortium of <it>Escherichia coli</it> strains

<p>Abstract</p> <p>Background</p> <p>The efficient microbial utilization of lignocellulosic hydrolysates has remained challenging because this material is composed of multiple sugars and also contains growth inhibitors such as acetic acid (acetate). Using an engineered consortium of strains derived from <it>Escherichia coli</it> C and a synthetic medium containing acetate, glucose, xylose and arabinose, we report on both the microbial removal of acetate and the subsequent <it>simultaneous</it> utilization of the sugars.</p> <p>Results</p> <p>In a first stage, a strain unable to utilize glucose, xylose and arabinose (ALS1392, strain <it>E. coli</it> C <it>ptsG manZ glk crr xylA araA</it>) removed 3 g/L acetate within 30 hours. In a subsequent second stage, three <it>E. coli</it> strains (ALS1370, ALS1371, ALS1391), which are each engineered to utilize only one sugar, together simultaneously utilized glucose, xylose and arabinose. The effect of non-metabolizable sugars on the metabolism of the target sugar was minimal. Additionally the deletions necessary to prevent the consumption of one sugar only minimally affected the consumption of a desired sugar. For example, the <it>crr</it> deletion necessary to prevent glucose consumption reduced xylose and arabinose utilization by less than 15% compared to the wild-type. Similarly, the <it>araA</it> deletion used to exclude arabinose consumption did not affect xylose- and glucose-consumption.</p> <p>Conclusions</p> <p>Despite the modest reduction in the overall rate of sugar consumption due to the various deletions that were required to generate the consortium of strains, the approach constitutes a significant improvement in any single-organism approach to utilize sugars found in lignocellulosic hydrolysate in the presence of acetate.</p

High Glycolytic Flux Improves Pyruvate Production by a Metabolically Engineered Escherichia coli Strain▿

We report pyruvate formation in Escherichia coli strain ALS929 containing mutations in the aceEF, pfl, poxB, pps, and ldhA genes which encode, respectively, the pyruvate dehydrogenase complex, pyruvate formate lyase, pyruvate oxidase, phosphoenolpyruvate synthase, and lactate dehydrogenase. The glycolytic rate and pyruvate productivity were compared using glucose-, acetate-, nitrogen-, or phosphorus-limited chemostats at a growth rate of 0.15 h−1. Of these four nutrient limitation conditions, growth under acetate limitation resulted in the highest glycolytic flux (1.60 g/g · h), pyruvate formation rate (1.11 g/g · h), and pyruvate yield (0.70 g/g). Additional mutations in atpFH and arcA (strain ALS1059) further elevated the steady-state glycolytic flux to 2.38 g/g · h in an acetate-limited chemostat, with heterologous NADH oxidase expression causing only modest additional improvement. A fed-batch process with strain ALS1059 using defined medium with 5 mM betaine as osmoprotectant and an exponential feeding rate of 0.15 h−1 achieved 90 g/liter pyruvate, with an overall productivity of 2.1 g/liter · h and yield of 0.68 g/g