Mark S. Ou

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.

Biochemical and Physical Properties of the Methanococcus jannaschii 20S Proteasome and PAN, a Homolog of the ATPase (Rpt) Subunits of the Eucaryal 26S Proteasome

The 20S proteasome is a self-compartmentalized protease which degrades unfolded polypeptides and has been purified from eucaryotes, gram-positive actinomycetes, and archaea. Energy-dependent complexes, such as the 19S cap of the eucaryal 26S proteasome, are assumed to be responsible for the recognition and/or unfolding of substrate proteins which are then translocated into the central chamber of the 20S proteasome and hydrolyzed to polypeptide products of 3 to 30 residues. All archaeal genomes which have been sequenced are predicted to encode proteins with up to approximately 50% identity to the six ATPase subunits of the 19S cap. In this study, one of these archaeal homologs which has been named PAN for proteasome-activating nucleotidase was characterized from the hyperthermophile Methanococcus jannaschii. In addition, the M. jannaschii 20S proteasome was purified as a 700-kDa complex by in vitro assembly of the alpha and beta subunits and has an unusually high rate of peptide and unfolded-polypeptide hydrolysis at 100 degrees C. The 550-kDa PAN complex was required for CTP- or ATP-dependent degradation of beta-casein by archaeal 20S proteasomes. A 500-kDa complex of PAN(Delta1-73), which has a deletion of residues 1 to 73 of the deduced protein and disrupts the predicted N-terminal coiled-coil, also facilitated this energy-dependent proteolysis. However, this deletion increased the types of nucleotides hydrolyzed to include not only ATP and CTP but also ITP, GTP, TTP, and UTP. The temperature optimum for nucleotide (ATP) hydrolysis was reduced from 80 degrees C for the full-length protein to 65 degrees C for PAN(Delta1-73). Both PAN protein complexes were stable in the absence of ATP and were inhibited by N-ethylmaleimide and p-chloromercuriphenyl-sulfonic acid. Kinetic analysis reveals that the PAN protein has a relatively high V(max) for ATP and CTP hydrolysis of 3.5 and 5.8 micromol of P(i) per min per mg of protein as well as a relatively low affinity for CTP and ATP with K(m) values of 307 and 497 microM compared to other proteins of the AAA family. Based on electron micrographs, PAN and PAN(Delta1-73) apparently associate with the ends of the 20S proteasome cylinder. These results suggest that the M. jannaschii as well as related archaeal 20S proteasomes require a nucleotidase complex such as PAN to mediate the energy-dependent hydrolysis of folded-substrate proteins and that the N-terminal 73 amino acid residues of PAN are not absolutely required for this reaction.

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.

Metabolic Flux Control at the Pyruvate Node in an Anaerobic Escherichia coli Strain with an Active Pyruvate Dehydrogenase

ABSTRACT During anaerobic growth of Escherichia coli, pyruvate formate-lyase (PFL) and lactate dehydrogenase (LDH) channel pyruvate toward a mixture of fermentation products. We have introduced a third branch at the pyruvate node in a mutant of E. coli with a mutation in pyruvate dehydrogenase (PDH*) that renders the enzyme less sensitive to inhibition by NADH. The key starting enzymes of the three branches at the pyruvate node in such a mutant, PDH*, PFL, and LDH, have different metabolic potentials and kinetic properties. In such a mutant (strain QZ2), pyruvate flux through LDH was about 30%, with the remainder of the flux occurring through PFL, indicating that LDH is a preferred route of pyruvate conversion over PDH*. In a pfl mutant (strain YK167) with both PDH* and LDH activities, flux through PDH* was about 33% of the total, confirming the ability of LDH to outcompete the PDH pathway for pyruvate in vivo. Only in the absence of LDH (strain QZ3) was pyruvate carbon equally distributed between the PDH* and PFL pathways. A pfl mutant with LDH and PDH* activities, as well as a pfl ldh double mutant with PDH* activity, had a surprisingly low cell yield per mole of ATP (YATP) (about 7.0 g of cells per mol of ATP) compared to 10.9 g of cells per mol of ATP for the wild type. The lower YATP suggests the operation of a futile energy cycle in the absence of PFL in this strain. An understanding of the controls at the pyruvate node during anaerobic growth is expected to provide unique insights into rational metabolic engineering of E. coli and related bacteria for the production of various biobased products at high rates and yields.

Complete Genome Sequence of a thermotolerant sporogenic lactic acid bacterium, Bacillus coagulans strain 36D1

Bacillus coagulans is a ubiquitous soil bacterium that grows at 50–55 °C and pH 5.0 and ferments various sugars that constitute plant biomass to L (+)-lactic acid. The ability of this sporogenic lactic acid bacterium to grow at 50–55 °C and pH 5.0 makes this organism an attractive microbial biocatalyst for production of optically pure lactic acid at industrial scale not only from glucose derived from cellulose but also from xylose, a major constituent of hemicellulose. This bacterium is also considered as a potential probiotic. Complete genome sequence of a representative strain, B. coagulans strain 36D1, is presented and discussed.

Fermentation of sweet sorghum derived sugars to butyric acid at high titer and productivity by a moderate thermophile Clostridium thermobutyricum at 50°C.

In this study, a moderate thermophile Clostridium thermobutyricum is shown to ferment the sugars in sweet sorghum juice treated with invertase and supplemented with tryptone (10 g L(-1)) and yeast extract (10 g L(-1)) at 50°C to 44 g L(-1) butyrate at a calculated highest volumetric productivity of 1.45 g L(-1)h(-1) (molar butyrate yield of 0.85 based on sugars fermented). This volumetric productivity is among the highest reported for batch fermentations. Sugars from acid and enzyme-treated sweet sorghum bagasse were also fermented to butyrate by this organism with a molar yield of 0.81 (based on the amount of cellulose and hemicellulose). By combining the results from juice and bagasse, the calculated yield of butyric acid is approximately 90 kg per tonne of fresh sweet sorghum stalk. This study demonstrates that C. thermobutyricum can be an effective microbial biocatalyst for production of bio-based butyrate from renewable feedstocks at 50°C.