Hugh G. Lawford

Performance testing of Zymomonas mobilis metabolically engineered for cofermentation of glucose, xylose, and arabinose.

IOGEN Corporation of Ottawa, Canada, has recently built a 40t/d biomass-to-ethanol demonstration plant adjacent to its enzyme production facility. It has partnered with the University of Toronto to test the C6/C5 cofermenta-tion performance characteristics of the National Renewable Energy Labora-torys metabolically engineered Zymomonas mobilis using various biomass hydrolysates. IOGENs feedstocks are primarily agricultural wastes such as corn stover and wheat straw. Integrated recombinant Z. mobilis strain AX101 grows on D-xylose and/or L-arabinose as the sole carbon/energy sources and ferments these pentose sugars to ethanol in high yield. Strain AX101 lacks the tetracycline resistance gene that was a common feature of other recombinant Zm constructs. Genomic integration provides reliable cofermentation performance in the absence of antibiotics, another characteristic making strain AX101 attractive for industrial cellulosic ethanol production. In this work, IOGENs biomass hydrolysate was simulated by a pure sugar medium containing 6% (w/v) glucose, 3% xylose, and 0.35% arabinose. At a level of 3 g/L (dry solids), corn steep liquor with inorganic nitrogen (0.8 g/L of ammonium chloride or 1.2 g/L of diammonium phosphate) was a cost-effective nutritional supplement. In the absence of acetic acid, the maximum volumetric ethanol productivity of a continuous fermentation at pH 5.0 was 3.54 g/L x h. During prolonged continuous fermentation, the efficiency of sugar-to-ethanol conversion (based on total sugar load) was maintained at >85%. At a level of 0.25% (w/v) acetic acid, the productivity decreased to 1.17 g/L x h at pH 5.5. Unlike integrated, xylose-utilizing rec Zm strain C25, strain AX101 produces less lactic acid as byproduct, owing to the fact that the Escherichia coli arabinose genes are inserted into a region of the host chromosome tentatively assigned to the gene for D-lactic acid dehydrogenase. In pH-controlled batch fermentations with sugar mixtures, the order of sugar exhaustion from the medium was glucose followed by xylose and arabinose. Both the total sugar load and the sugar ratio were shown to be important determinants for efficient cofermentation. Ethanol at a level of 3% (w/v) was implicated as both inhibitory to pentose fermentation and as a potentiator of acetic acid inhibition of pentose fermentation at pH 5.5. The effect of ethanol may have been underestimated in other assessments of acetic acid sensitivity. This work underscores the importance of employing similar assay conditions in making comparative assessments of biocatalyst fermentation performance.

Improving Fermentation Performance of Recombinant Zymomonas in Acetic Acid-Containing Media

In the production of ethanol from lignocellulosic biomass, the hydrolysis of the acetylated pentosans in hemicellulose during pretreatment produces acetic acid in the prehydrolysate. The National Renewable Energy Laboratory (NREL) is currently investigating a simultaneous saccharification and cofermentation (SSCF) process that uses a proprietary metabolically engineered strain ofZymomonas mobilis that can coferment glucose and xylose. Acetic acid toxicity represents a major limitation to bioconversion, and cost-effective means of reducing the inhibitory effects of acetic acid represent an opportunity for significant increased productivity and reduced cost of producing fermentation fuel ethanol from biomass. In this study, the fermentation performance of recombinant Z.mobilis 39676:pZB4L, using a synthetic hardwood prehydrolysate containing 1% (w/v) yeast extract, 0.2% KH2PO4, 4% (w/v) xylose, and 0.8% (w/v) glucose, with varying amounts of acetic acid was examine. To minimize the concentration of the inhibitory undissociated form of acetic acid, the pH was controlled at 6.0. The final cell mass concentration decreased linearly with increasing level of acetic acid over the range 0-0.75% (w/v), with a 50% reduction at about 0.5% (w/v) acetic acid. The conversion efficiency was relatively unaffected, decreasing from 98 to 92%. In the absence of acetic acid, batch fermentations were complete at 24 h. In a batch fermentation with 0.75% (w/v) acetic acid, about two-thirds of the xylose was not metabolized after 48 h. In batch fermentations with 0.75% (w/v) acetic acid, increasing the initial glucose concentration did not have an enhancing effect on the rate of xylose fermentation. However, nearly complete xylose fermentation was achieved in 48 h when the bioreactor was fed glucose. In the fed-batch system, the rate of glucose feeding (0.5 g/h) was designed to simulate the rate of cellulolytic digestion that had been observed in a modeled SSCF process with recombinant Zymomonas. In the absence of acetic acid, this rate of glucose feeding did not inhibit xylose utilization. It is concluded that the inhibitory effect of acetic acid on xylose utilization in the SSCF biomass-to-ethanol process will be partially ameliorated because of the simultaneous saccharification of the cellulose.

Cellulosic Fuel Ethanol Alternative Fermentation Process Designs with Wild-Type and Recombinant Zymomonas mobilis

Iogen (Canada) is a major manufacturer of industrial cellulase and hemicellulase enzymes for the textile, pulp and paper, and poultry feed industries. Iogen has recently constructed a 40 t/d biomass-to-ethanol demonstration plant adjacent to its enzyme production facility. The integration of enzyme and ethanol plants results in significant reduction in production costs and offers an alternative use for the sugars generated during biomass conversion. Iogen has partnered with the University of Toronto to test the fermentation performance characteristics of metabolically engineered Zymomonas mobilis created at the National Renewable Energy Laboratory. This study focused on strain AX101, a xylose- and arabinose-fermenting stable genomic integrant that lacks the selection marker gene for antibiotic resistance. The “Iogen Process” for biomass depolymerization consists of a dilute-sulpfuric acid-catalyzed steam explosion, followed by enzymatic hydrolysis. This work examined two process design options for fermentation, first, continuous cofermentation of C5 and C6 sugars by Zm AX101, and second, separate continuous fermentations of prehydrolysate by Zm AX101 and cellulose hydrolysate by either wildtype Z. mobilis ZM4 or an industrial yeast commonly used in the production of fuel ethanol from corn. Iogen uses a proprietary process for conditioning the prehydrolysate to reduce the level of inhibitory acetic acid to at least 2.5 g/L. The pH was controlled at 5.5 and 5.0 for Zymomonas and yeast fermentations, respectively. Neither 2.5 g/L of acetic acid nor the presence of pentose sugars (C6:C5 = 2:1) appreciably affected the high-performance glucose fermentation of wild-type Z. mobilis ZM4. By contrast, 2.5 g/L of acetic acid significantly reduced the rate of pentose fermentation by strain AX101. For single-stage continuous fermentation of pure sugar synthetic cellulose hydrolysate (60 g/L of glucose), wild-type Zymomonas exhibited a four-fold higher volumetric productivity compared with industrial yeast. Low levels of acetic acid stimulated yeast ethanol productivity. The glucose-to-ethanol conversion efficiency for Zm and yeast was 96 and 84%, respectively.

Corn Steep Liquor as a Cost-Effective Nutrition Adjunct in High-Performance Zymomonas Ethanol Fermentations

The ethanologenic bacteriumZymomonas mobilis has been demonstrated to possess several fermentation performance characteristics that are superior to yeast. In a recent survey conducted by the National Renewable Energy Laboratory (NREL),Zymomonas was selected as the most promising host for improvement by genetic engineering directed to pentose metabolism for the production of ethanol from lignocellulosic biomass and wastes. Minimization of costs associated with nutritional supplements and seed production is essential for economic large-scale production of fuel ethanol. Corn steep liquor (CSL) is a byproduct of corn wet-milling and has been used as a fermentation nutrient supplement in several different fermentations. This study employed pH-controlled batch fermenters to compare the growth and fermentation performance ofZ. mobilis in glucose media with whole and clarified corn steep liquor as sole nutrient source, and to determine minimal amounts of CSL required to sustain high-performance fermentation.It was concluded that CSL can be used as a cost-effective single-source nutrition adjunct forZymomonas fermentations. Supplementation with inorganic nitrogen significantly reduced the requirement for CSL. Depending on the type of process and mode of operation, there can be a significant contribution of nutrients from the seed culture, and this would also reduce the requirement for CSL. Removal of the insolubles (40% of the total solids) from CSL did not detract significantly from its nutritional effectiveness. On an equal-volume basis, clarified CSL was 1.33 times more “effective” (in terms of cell mass yield and fermentation time) than whole CSL. For fermentations at sugar loading of >5% (w/v), the recommended level of supplementation with clarified CSL is 1.0% (v/v). Based on CSL at US

Effects of pH and acetic acid on glucose and xylose metabolism by a genetically engineered ethanologenic Escherichia coli

50/t, the cost associated with using clarified CSL at 1.0% (v/v) is 88¢/1000 L of medium and 5.3¢/gal of undenatured ethanol for fermentation of 10% (w/v) glucose. This cost compares favorably to estimates for using inorganic nutrients. The cost impact is reduced to 3.1¢/gal if there is a byproduct credit for selling the insolubles as animal feed at a price of about US

Production of ethanol from pulp mill hardwood and softwood spent sulfite liquors by genetically engineered E. coli

100/t. Therefore, the disposition of the CSL insolubles can significantly impact the calculations of cost associated with the use of CSL as a nutritional adjunct in large-scale fermentations.

Ethanol production from xylose by Thermoanaerobacter ethanolicus in batch and continuous culture

Efficient utilization of the pentosan fraction of hemicellulose from lignocellulosic feedstocks offers an opportunity to increase the yield and to reduce the cost of producing fuel ethanol. The patented, genetically engineered, ethanologenEscherichia coli B (pLOI297) exhibits high-performance characteristics with respect to both yield and productivity in xylose-rich lab media. In addition to producing monomer sugar residues, thermochemical processing of biomass is known to produce substances that are inhibitory to both yeast and bacteria. During prehydrolysis, acetic acid is formed as a consequence of the deacetylation of the acetylated pentosan. Our investigations have shown that the acetic acid content of hemicellulose hydrolysates from a variety of biomass/waste materials was in the range 2–10 g/L (33–166 mM). Increasing the reducing sugar concentration by evaporation did not alter the acetic acid concentration. Acetic acid toxicity is pH dependent. By virtue of its ability to traverse the cell membrane freely, the undissociated (protonated) form of acetic acid (HAc) acts as a membrane protonophore and causes its inhibitory effect by bringing about the acidification of the cytoplasm. With recombinantE. coli B, the pH range for optimal growth with glucose and xylose was 6.4–6.8. With glucose, the pH optimum for ethanol yield and volumetric productivity was 6.5, and for xylose it was 6.0 and 6.5, respectively. However, the decrease in growth and fermentation efficiency at pH 7 is not significant. At pH 7, only 0.56% of acetic acid is undissociated, and at 10 g/L, neither the ethanol yield nor the maximum volumetric productivity, with glucose or xylose, is significantly decreased. The “uncoupling” effect of HAc is more pronounced with xylose and the potency of HAc is potentiated in a minimal salts medium. Controlling the pH at 7 provided an effective means of circumventing acetic acid toxicity without significant loss in fermentation performance of the recombinant biocatalyst.

A new approach to improving the performance ofZymomonas in continuous ethanol fermentations

Although lignocellulosic biomass and wastes are targeted as an attractive alternative fermentation feedstock for the production of fuel ethanol, cellulosic ethanol is not yet an industrial reality because of problems in bioconversion technologies relating both to depolymerization and fermentation. In the production of wood pulp by the sulfite process, about 50% of the wood (hemicellulose and lignin) is dissolved to produce cellulose pulp, and the pulp mill effluent (“spent sulfite liquor” SSL) represents the only lignocellulosic hydrolysate available today in large quantities (about 90 billion liters annually worldwide). Although softwoods have been the traditional feedstock for pulping operations, hardwood pulping is becoming more popular, and the pentose sugars in hardwood SSL (principally xylose) are not fermented by the yeasts currently being used in the production of ethanol from softwood SSL.This study assessed the fermentation performance characteristics of a patented (US Pat. 5,000,000), recombinantEscherichia coli B (ATCC 11303 pLOI297) in anaerobic batch fermentations of both nutrient-supplemented soft and hardwood SSL (30–35 g/L total reducing sugars). The pH was controlled at 7.0 to maximize tolerance to acetic acid. In contrast to the high-performance characteristics exhibited in synthetic media, formulated to mimic the composition of softwood and hardwood SSL (yield approaching theoretical maximum), performance in SSL media was variable with conversion efficiencies in the range of 67–84% for hardwood SSL and 53–76% for softwood SSL. Overlimiting treatment of HSSL, using Ca(OH)2, improved overall volumetric productivity two- to sevenfold to a max of 0.42 g/L/h at an initial cell loading of 0.5 g dry wt/L. A conversion efficiency of 92% (6.1 g/L ethanol) was achieved using diluted Ca(OH)2-treated hardwood SSL. The variable behavior of this particular genetic construct is viewed as a major detractant regarding its candidacy as a biocatalyst for SSL fermentations.

An improved procedure for extraction and analysis of cellular nucleotides

The fermentation of xylose by Thermoanaerobacter ethanolicus ATCC 31938 was studied in pH-controlled batch and continuous cultures. In batch culture, a dependency of growth rate, product yield, and product distribution upon xylose concentration was observed. With 27 mM xylose media, an ethanol yield of 1.3 mol ethanol/mol xylose (78% of maximum theoretical yield) was typically obtained. With the same media, xylose-limited growth in continuous culture could be achieved with a volumetric productivity of 0.50 g ethanol/liter h and a yield of 0.42 g ethanol/g xylose (1.37 mol ethanol/mol xylose). With extended operation of the chemostat, variation in xylose uptake and a decline in ethanol yield was seen. Instability with respect to fermentation performance was attributed to a selection for mutant populations with different metabolic characteristics. Ethanol production in these T. ethanolicus systems was compared with xylose-to-ethanol conversions of other organisms. Relative to the other systems, T. ethanolicus offers the advantages of a high ethanol yield at low xylose concentrations in batch culture and of a rapid growth rate. Its disadvantages include a lower ethanol yield at higher xylose concentrations in batch culture and an instability of fermentation characteristics in continuous culture.

Fuel ethanol from hardwood hemicellulose hydrolysate by genetically engineered Escherichia coli B carrying genes from Zymomonas mobilis.

Although most fermentation ethanol is currently produced in traditional batch processes with yeast, the ethanologenic bacteriumZymomonas mobilis is recognized as an alternative process organism for fuel alcohol production. Different strategies for improving the productivity of ethanol fermentations are reviewed. In batch and open-type continuous fermentations the advantage of replacing yeast byZymomonas relates principally to the 10% higher fermentation efficiency (product yield), whereas in high cell density, closed-type continuous systems (operating with cell recycle or retention) the superior kinetic properties ofZymomonas can be exploited to affect about a five-fold improvement in volumetric productivity. Unlike yeast, the rate of energy supply (conversion of glucose to ethanol) inZymomonas is not strictly regulated by the energy demand and a nongrowing culture exhibits a maintenance energy coefficient that is at least 25 times higher than yeast. As an alternative to process improvement through genetic engineering of the process organism this investigation has taken a biochemical and physiological approach to increasing the kinetic performance ofZ. mobilis through manipulation and control of the chemical environment. Energetically “uncoupled” phenotypes with markedly increased specific rates of ethanol production were generated under conditions of nutritional limitation (nitrogen, phosphate, or potassium) in steady-state continuous culture. The pH was shown to influence energy coupling inZymomonas affecting the maintenance coefficient (me) rather than the max growth yield coefficient (Yxsάx). Whereas the pH for optimal growth ofZ. mobilis (ATCC 29191) in a complex medium was 6.0–6.5, the specific rate of ethanol production in continuous fermentations was maximal in the range 4.0–4.5. Fermentation conditions are specified for maximizing the specific productivity of aZymomonas-based continuous ethanol fermentation where the potential exists for improving the volumetric productivity in dense culture fermentations with an associated 35–40% reduction in capital costs of fermentation equipment and an estimated savings of 10–15% on cost of product recovery (distillation), and 3–7% on overall production costs based on the projected use of inexpensive feedstocks.