This Small Business Technology Transfer (STTR) Phase I project will develop a facultative anaerobic bacterium that ferments sugars obtained from cellulosic sources into isoprene, a valuable feedstock used in the production of latex rubber, plastics, and pharmaceuticals. Isoprene also is used for the production of chemicals, or polymerized into replacements for petroleum-derived jet fuel. This work will build on prototype E. coli work by redesigning gene constructs and expressing them in the facultative anaerobic host Bacillus coagulans. Phase I of this research will entail synthesizing and cloning a set of isoprene synthase (IspS) genes into B. coagulans; this work will first evaluate ten separate potential IPS molecules to determine the best one for isoprene production in bacteria. The engineered B. coagulans strains will be evaluated for isoprene production by GC-MS headspace analysis. Achieving anaerobic isoprene production from lignocellulosic sources within at least an order of magnitude of the rates achieved in E. coli at the end of Phase I will demonstrate feasibility. Phase II will then focus on improving the flux of metabolites through the pathway to this enzyme.
The broader impact/commercial potential of this project will be to develop a high efficiency bacterial strain of Bacillus coagulans to ferment sugars derived from cellulosic materials, such as pulp and pulp mill sludge into isoprene. The paper industry produces an estimated 2 million tons of fiber sludge annually. This material is either discarded in a landfill or burned, adding millions of dollars to the cost of paper production. The composition of this sludge is ~50% cellulose and hemicellulose on a dry weight basis, making it an excellent fermentation feedstock when converted into its constituent five and six carbon sugars. Isoprene is a platform chemical that is capable of playing a central role in the future bio-economy. Annual global consumption of isoprene is 1.7 billion pounds per year, of which more than 95% is used in the production of isoprene rubber, styrene-isoprene-styrene block copolymer (SIS), and butyl rubber, with an estimated price of $1.30 per pound. The balance is used in the production of high-value isoprenoid derivatives such as vitamins, nutraceuticals, pesticides, fragrances, flavors and pharmaceuticals. The project also will provide an improved understanding of the biosynthetic pathway of isoprene and other high-value isoprenoid compounds.
The Phase I project aimed to create a genetically engineered anaerobic bacterium of the species Bacillus coagulans to produce isoprene for the fine chemicals market from sugars derived from pulp and paper mill residuals. This work followed on from successful development of isoprene producing Escherichia coli strains as prototypes. The PIs had developed a promising new transformation technology for B. coagulans, which promised an opportunity to translate the prototype work into a more commercially viable fermentation system. Unfortunately we were unable to produce a stable genetically engineered B coagulans for unknown reasons. We shifted the focus towards engineering isoprene production of Lactobacillus rhamnosus ATCC 10863, which also failed to yield stable transformants. In anticipation of obtaining an isoprene producing Bacillus or Lactobacillus, we began fermentation trials with wild type strains of both species to assess their utility in bio-based product production from pulp and paper residuals. We also tested isoprene production from engineered E. coli using paper mill residuals as a carbon source. We grew microbes on pulp mill sludge hydrolysate and diluted red liquor substrates containing 5-20% mixed C5 and C6 sugars. We monitored culture turbidity, sugar consumption and lactic acid production. We assayed for isoprene production by non-genetically engineered Bacillus coagulans in headspace vial experiments. Cells were grown in 10mL of LB with addition of 2% glucose, 2% xylose or no additional. Isoprene was not detected in any of the headspace vials. Bacillus coagulans grew best when using glucose as the carbon source. We also investigated lactic acid production by simultaneous saccharification and fermentation (SSF) using B. coagulans. Fermentations were done in 1L fermenter vessels using mineral salts and pulp mill sludge as a carbon source with Novozymes CTEC2 cellulase at 40°C. Lactic acid levels increased during the fermentation reaching a peak of 4g /L. After an increase in glucose content in the media during the first hour the glucose levels were low or zero from 6 hours until the end of the experiment. As a proof of concept for isoprene production from pulp mill residuals we used a prototype E. coli strain transformed for isoprene production by SSF. Isoprene levels were detected in the headspace gas after 3hrs incubation and rose quickly until 6.5 hours were the level was 44.4ppm, leveled out and the highest level detected was 50.7 at 23 hrs. After 23hrs the levels dropped. After an increase in glucose content in the media over the first 2 hours the glucose levels dropped and were low or zero from 4.5 hours onward till the end of the experiment. Dissolved oxygen dropped from 15.9% at time 0 to below 1% by 4.5hrs and dropped to 0.4% by the end of the fermentation. Lactobacillus rhamnosus grew and produced lactic acid in media containing any of the residuals stream sugar syrups. The greatest lactic acid production was from combined rejects (17.4 g L-1) while the least lactic acid production was from heavy red liquor (10.4 g L-1). Lactobacillus did not grow in media containing either xylose or arabinose as the main sugar source but grew well in media containing glucose, mannose or galactose. Lactic acid was produced from media containing glucose, mannose or galactose and very little lactic acid was produced when Lactobacillus was grown in media containing xylose or arabinose. Ultimately, we failed to produce a bacterial strain of either B. coagulans or L. rhamnosus as a platform for commercial isoprene production from pulp and paper mill residuals. We did, however, demonstrate the feasibility of using these residuals as a low-cost substrate for bio-based chemical production. We demonstrated conversion of residuals to lactic acid with wild-type strains and to isoprene with engineered E. coli. These fermentations can be economically done using SSF. While commercial production of isoprene from residuals awaits a commercially viable strain with isoprene yields at least 10x those of our prototype E. coli strain these results suggest that there is an opportunity to use pulp and paper residuals as a low-cost biorefinery feedstock.
