K.T. Shanmugam

Isolation and Characterization of Acid-Tolerant, Thermophilic Bacteria for Effective Fermentation of Biomass-Derived Sugars to Lactic Acid

ABSTRACT Biomass-derived sugars, such as glucose, xylose, and other minor sugars, can be readily fermented to fuel ethanol and commodity chemicals by the appropriate microbes. Due to the differences in the optimum conditions for the activity of the fungal cellulases that are required for depolymerization of cellulose to fermentable sugars and the growth and fermentation characteristics of the current industrial microbes, simultaneous saccharification and fermentation (SSF) of cellulose is envisioned at conditions that are not optimal for the fungal cellulase activity, leading to a higher-than-required cost of cellulase in SSF. We have isolated bacterial strains that grew and fermented both glucose and xylose, major components of cellulose and hemicellulose, respectively, to l(+)-lactic acid at 50°C and pH 5.0, conditions that are also optimal for fungal cellulase activity. Xylose was metabolized by these new isolates through the pentose-phosphate pathway. As expected for the metabolism of xylose by the pentose-phosphate pathway, [13C]lactate accounted for more than 90% of the total 13C-labeled products from [13C]xylose. Based on fatty acid profile and 16S rRNA sequence, these isolates cluster with Bacillus coagulans, although the B. coagulans type strain, ATCC 7050, failed to utilize xylose as a carbon source. These new B. coagulans isolates have the potential to reduce the cost of SSF by minimizing the amount of fungal cellulases, a significant cost component in the use of biomass as a renewable resource, for the production of fuels and chemicals.

Methylglyoxal Bypass Identified as Source of Chiral Contamination in l(+) and d(−)-lactate Fermentations by Recombinant Escherichia coli

Two new strains of Escherichia coli B were engineered for the production of lactate with no detectable chiral impurity. All chiral impurities were eliminated by deleting the synthase gene (msgA) that converts dihydroxyacetone-phosphate to methylglyoxal, a precursor for both l(+)- and d(−)-lactate. Strain TG113 contains only native genes and produced optically pure d(−)-lactate. Strain TG108 contains the ldhL gene from Pediococcus acidilactici and produced only l(+)-lactate. In mineral salts medium containing 1xa0mM betaine, both strains produced over 115xa0g (1.3xa0mol) lactate from 12% (w/v) glucose, >95% theoretical yield.

Production of D(-)-lactate from sucrose and molasses

Escherichia coli W3110 derivatives, strains SZ63 and SZ85, were previously engineered to produce optically pure d(−) and l(+)-lactate from hexose and pentose sugars. To expand the substrate range, a cluster of sucrose genes (cscR′ cscA cscKB) was cloned and characterized from E. coli KO11. The resulting plasmid was functionally expressed in SZ63 but was unstable in SZ85. Over 500xa0mm d(−)-lactate was produced from sucrose and from molasses by SZ63(pLOI3501).

Fermentation of 10% (w/v) sugar to D: (-)-lactate by engineered Escherichia coli B.

Derivatives of ethanologenic Escherichia coli K011 were constructed for d(−)-lactate production by deleting genes encoding competing pathways followed by metabolic evolution, a growth-based selection for mutants with improved performance. Resulting strains, SZ132 and SZ186, contain native genes for sucrose utilization. No foreign genes are present in SZ186. Strain SZ132 also contains a chromosomally integrated endoglucanase gene (Erwinia chrysanthemi celY). Strain SZ132 produced over 1xa0mol lactate per liter of complex medium containing 10% (w/v) sugar (fermentation times of 48xa0h for glucose, 120xa0h for sucrose). Both strains produced 667–700xa0mmol lactate per liter of mineral salts medium. Yields for metabolized sugar ranged from 88% to 95% in both media.

Fermentation of 12% (w/v) Glucose to 1.2 m Lactate by Escherichia coli Strain SZ194 using Mineral Salts Medium

A non-recombinant mutant of Escherichia coli B, strain SZ194, was developed that produces over 1xa0md-lactate from glucose (or sucrose) in 72xa0h using mineral salts medium supplemented with 1xa0mm betaine in simple anaerobic fermentations. Rates and yields were highest at pH 7.5. Yields approached the theoretical maximum with only trace amounts of co-products. Chiral purity of d-lactate was estimated to be 95%. Specific and volumetric productivities for SZ194 in mineral salts medium (pH 7.5) with betaine were equivalent to those in Luria broth.

Simultaneous saccharification and co-fermentation of crystalline cellulose and sugar cane bagasse hemicellulose hydrolysate to lactate by a thermotolerant acidophilic Bacillus sp.

Polylactides produced from renewable feedstocks, such as corn starch, are being developed as alternatives to plastics derived from petroleum. In addition to corn, other less expensive biomass resources can be readily converted to component sugars (glucose, xylose, etc.) by enzyme and/or chemical treatment for fermentation to optically pure lactic acid to reduce the cost of lactic acid. Lactic acid bacteria used by the industry lack the ability to ferment pentoses (hemicellulose‐derived xylose and arabinose), and their growth and fermentation optima also differ from the optimal conditions for the activity of fungal cellulases required for depolymerization of cellulose. To reduce the overall cost of simultaneous saccharification and fermentation (SSF) of cellulose, we have isolated bacterial biocatalysts that can grow and ferment all sugars in the biomass at conditions that are also optimal for fungal cellulases. SSF of Solka Floc cellulose by one such isolate, Bacillus sp. strain 36D1, yielded l(+)‐lactic acid at an optical purity higher than 95% with cellulase (Spezyme CE; Genencor International) added at about 10 FPU/g cellulose, with a product yield of about 90% of the expected maximum. Volumetric productivity of SSF to lactic acid was optimal between culture pH values of 4.5 and 5.5 at 50 °C. At a constant pH of 5.0, volumetric productivity of lactic acid was maximal at 55 °C. Strain 36D1 also co‐fermented cellulose‐derived glucose and sugar cane bagasse hemicellulose‐derived xylose simultaneously (SSCF). In a batch SSCF of 40% acid‐treated hemicellulose hydrolysate (over‐limed) and 20 g/L Solka Floc cellulose, strain 36D1 produced about 35 g/L lactic acid in about 144 h with 15 FPU of Spezyme CE/g cellulose. The maximum volumetric productivity of lactic acid in this SSCF was 6.7 mmol/L (h). Cellulose‐derived lactic acid contributed to about 30% of this total lactic acid. These results show that Bacillus sp. strain 36D1 is well‐suited for simultaneous saccharification and co‐fermentation of all of the biomass‐derived sugars to lactic acid.

Betaine tripled the volumetric productivity of D(-)-lactate by Escherichia coli strain SZ132 in mineral salts medium

Osmotic stress restricts glycolytic flux, growth (rate and yield), d-lactate productivity, and d-lactate tolerance in Escherichia coli B strain SZ132 during batch fermentation in mineral salts medium with 10% (w/v) sugar. Addition of 1xa0mm betaine, a non-metabolized protective osmolyte, doubled cell yield, increased specific productivity of d-lactate and glycolytic flux by 50%, and tripled volumetric productivity (from 8.6 to 25.7xa0mmolxa0l−1xa0h−1; 0.8 to 2.3xa0gxa0l−1xa0h−1). Glycolytic flux and specific productivity in mineral salts medium with betaine exceeded that in Luria broth, substantially eliminating the need for complex nutrients during d-lactate production. In mineral salts medium supplemented with betaine, SZ132 produced approximately 1xa0mol d-lactate (90xa0g) per 100xa0g sugar (glucose or sucrose).

Fermentation of sugar cane bagasse hemicellulose hydrolysate to l(+)-lactic acid by a thermotolerant acidophilic Bacillus sp.**

Sugar cane bagasse hemicellulose, hydrolyzed by dilute H2SO4, supplemented with mineral salts and 0.5% corn steep liquor, was fermented to l(+)-lactic acid using a newly isolated strain of Bacillusxa0sp. In batch fermentations at 50xa0°C and pHxa05, over 5.5% (w/v) l(+)-lactic acid was produced (89% theoretical yield; 0.9xa0g lactate perxa0g sugar) with an optical purity of 99.5%.

Techno-economic analysis of ethanol production from sugarcane bagasse using a Liquefaction plus Simultaneous Saccharification and co-Fermentation process.

A techno-economic analysis was conducted for a simplified lignocellulosic ethanol production process developed and proven by the University of Florida at laboratory, pilot, and demonstration scales. Data obtained from all three scales of development were used with Aspen Plus to create models for an experimentally-proven base-case and 5 hypothetical scenarios. The model input parameters that differed among the hypothetical scenarios were fermentation time, enzyme loading, enzymatic conversion, solids loading, and overall process yield. The minimum ethanol selling price (MESP) varied between 50.38 and 62.72 US cents/L. The feedstock and the capital cost were the main contributors to the production cost, comprising between 23-28% and 40-49% of the MESP, respectively. A sensitivity analysis showed that overall ethanol yield had the greatest effect on the MESP. These findings suggest that future efforts to increase the economic feasibility of a cellulosic ethanol process should focus on optimization for highest ethanol yield.

Complete genome sequence of Paenibacillus sp. strain JDR-2

Paenibacillus sp. strain JDR-2, an aggressively xylanolytic bacterium isolated from sweetgum (Liquidambar styraciflua) wood, is able to efficiently depolymerize, assimilate and metabolize 4-O-methylglucuronoxylan, the predominant structural component of hardwood hemicelluloses. A basis for this capability was first supported by the identification of genes and characterization of encoded enzymes and has been further defined by the sequencing and annotation of the complete genome, which we describe. In addition to genes implicated in the utilization of β-1,4-xylan, genes have also been identified for the utilization of other hemicellulosic polysaccharides. The genome of Paenibacillus sp. JDR-2 contains 7,184,930 bp in a single replicon with 6,288 protein-coding and 122 RNA genes. Uniquely prominent are 874 genes encoding proteins involved in carbohydrate transport and metabolism. The prevalence and organization of these genes support a metabolic potential for bioprocessing of hemicellulose fractions derived from lignocellulosic resources.