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-tory's metabolically engineered Zymomonas mobilis using various biomass hydrolysates. IOGEN's 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, IOGEN's 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.
Iogen Corporation of Ottawa, Canada, has recently built a 50 t/d biomass-to-ethanol demonstration plant adjacent to its enzyme production facility. Iogen has partnered with the University of Toronto ...to test the C6/C5 cofermentation performance characteristics of National Renewable Energy Laboratory's metabolically engineered Zymomonas mobilis using its biomass hydrolysates. In this study, the biomass feedstock was an agricultural waste, namely oat hulls, which was hydrolyzed in a proprietary two-stage process involving pretreatment with dilute sulfuric acid at 200-250 degrees C, followed by cellulase hydrolysis. The oat hull hydrolysate (OHH) contained glucose, xylose, and arabinose in a mass ratio of about 8:3:0.5. Fermentation media, prepared from diluted hydrolysate, were nutritionally amended with 2.5 mL/L of corn steep liquor (50% solids) and 1.2 g/L of diammonium phosphate. The estimated cost for large-scale ethanol production using this minimal level of nutrient supplementation was 4.4cents/gal of ethanol. This work examined the growth and fermentation performance of xylose-utilizing, tetracycline-resistant, plasmid-bearing, patented, recombinant Z. mobilis cultures: CP4:pZB5, ZM4:pZB5, 39676:pZB4L, and a hardwood prehydrolysate-adapted variant of 39676:pZB4L (designated as the "adapted" strain). In pH-stat batch fermentations with unconditioned OHH containing 6% (w/v) glucose, 3% xylose, and 0.75% acetic acid, rec Zm ZM4:pZB5 gave the best performance with a fermentation time of 30 h, followed by CP4:pZB5 at 48 h, with corresponding volumetric productivities of 1.4 and 0.89 g/ (L x h), respectively. Based on the available glucose and xylose, the process ethanol yield for both strains was 0.47 g/g (92% conversion efficiency). At 48 h, the process yield for rec Zm 39676:pZB4L and the adapted strain was 0.32 and 0.34 g/g, respectively. None of the test strains was able to ferment arabinose. Acetic acid tolerance appeared to be a major determining factor in cofermentation performance.
Lignocellulosic biomass is a potential feedstock for bioethanol production. Biomass hydrolysates, prepared with a procedure including pretreatment and hydrolysis, are considered to be used as ...fermentation media for microorganisms, such as yeast. During the hydrolysate preparation procedure, toxic compounds are released or formed which may inhibit the growth of the microorganism and thus the product formation. To study the effects of these compounds on fermentation performance, the production of various hydrolysates with diverse inhibitory effects is of importance. A platform of methods that generates hydrolysates through four different ways and tests their inhibitory effects using Bioscreen C Analyzer growth tests is described here. The four methods, based on concentrated acid, dilute acid, mild alkaline and alkaline/oxidative conditions, were used to prepare hydrolysates from six different biomass sources. The resulting 24 hydrolysates showed great diversity on growth rate in Bioscreen C Analyzer growth tests. The approach allows the prediction of a specific hydrolysate's performance and helps to select biomass type and hydrolysate preparation method for a specific production strain, or vice versa.
In pH-controlled batch fermentations with pure sugar synthetic hardwood hemicellulose (1% w/v glucose and 4% xylose) and corn stover hydrolysate (8% glucose and 3.5% xylose) lacking acetic acid, the ...xylose-utilizing, tetracycline (Tc)-sensitive, genomically integrated variant of Zymomonas mobilis ATCC 39676 (designated strain C25) exhibited growth and fermentation performance that was inferior to National Renewable Energy Laboratory's first-generation, Tc-resistant, plasmid-bearing Zymomonas recombinants. With C25, xylose fermentation following glucose exhaustion was markedly slower, and the ethanol yield (based on sugars consumed) was lower, owing primarily to an increase in lactic acid formation. There was an apparent increased sensitivity to acetic acid inhibition with C25 compared with recombinants 39676:pZB4L, CP4:pZB5, and ZM4:pZB5. However, strain C25 performed well in continuous fermentation with nutrient-rich synthetic corn stover medium over the dilution range 0.03-0.06/h, with a maximum process ethanol yield at D = 0.03/h of 0.46 g/g and a maximum ethanol productivity of 3 g/(L(.)h). With 0.35% (w/v) acetic acid in the medium, the process yield at D = 0.04/h dropped to 0.32 g/g, and the maximum productivity decreased by 50% to 1.5 g/(L(.)h). Under the same operating conditions, rec Zm ZM4:pZB5 performed better; however, the medium contained 20 mg/L of Tc to constantly maintain selective pressure. The absence of any need for antibiotics and antibiotic resistance genes makes the chromosomal integrant C25 more compatible with current regulatory specifications for biocatalysts in large-scale commercial operations.
This study examined the continuous cofermentation performance characteristics of a dilute-acid "prehydrolysate-adapted" recombinant Zymomonas 39676:pZB4L and builds on the pH-stat batch fermentations ...with this recombinant that we reported on last year. Substitution of yeast extract by 1% (w/v) corn steep liquor (CSL) (50% solids) and Mg (2 mM) did not alter the cofermentation performance. Using declared assumptions, the cost of using CSL and Mg was estimated to be 12.5 cents/gal of ethanol with a possibility of 50% cost reduction using fourfold less CSL with 0.1% diammonium phosphate. Because of competition for a common sugar transporter that exhibits a higher affinity for glucose, utilization of glucose was complete whereas xylose was always present in the chemostat effluent. The ethanol yield, based on sugar used, was 94% of theoretical maximum. Altering the sugar ratio of the synthetic dilute acid hardwood prehydrolysate did not appear to significantly change the pattern of xylose utilization. Using a criterion of 80% sugar utilization for determining the maximum dilution rate (D(max)), changing the composition of the feed from 4% xylose to 3%, and simultaneously increasing the glucose from 0.8 to 1.8% shifted D(max) from 0.07 to 0.08/h. With equal amounts of both sugars (2.5%), D(max) was 0.07/h. By comparison to a similar investigation with rec Zm CP4:pZB5 with a 4% equal mixture of xylose and glucose, we observed that at pH 5.0, the D(max) was 0.064/h and shifted to 0.084/h at pH 5.75. At a level of 0.4% (w/v) acetic acid in the CSL-based medium with 3% xylose and 1.8% glucose at pH 5.75, the D(max) for the adapted recombinant shifted from 0.08 to 0.048/h, and the corresponding maximum volumetric ethanol productivity decreased 45%, from 1.52 to 0.84 g/(L.h). Under these conditions of continuous culture, linear regression of a Pirt plot of the specific rate of sugar utilization vs D showed that 4 g/L of acetic acid did not affect the maximum growth yield (0.030 g dry cell mass/g sugar), but did increase the maintenance coefficient twofold, from 0.46 to 1.0 g of sugar/(g of cell(.)h).
The effect of oxygenation on ethanol production from xylose or glucose by
Pichia stipitis in batch culture was investigated. The fermentation parameters with respect to ethanol production by
P. ...stipitis were strongly dependent on the oxygen transfer rate (OTR). On the basis of OTR values, the relationship between the optimum specific oxygen uptake rate (
qO
2) and maximum specific ethanol production rate ((
q
p)
max) was evaluated. The optimum
qO
2 for ethanol production was 14.3 or 66.7 mg·(g cell)
−1·
−1 when xylose or glucose was used as a carbon source, respectively. When a mixture of glucose and xylose was used as the carbon source in a culture of
P. stipitis alone, the yield and productivity of ethanol were significantly enhanced when
qO
2 was adjusted to an optimum level dependent upon the type of sugar consumed. In a co-culture of
Saccharomyces cerevisiae and
P. stipitis, it was impossible to control
qO
2 at an optimum value for xylose fermentation by
P. stipitis because oxygen was consumed by the
S. cerevisiae. In a co-culture of
P. stipitis and a respiratory-deficient mutant of
S. cerevisiae, the optimum
qO
2 for xylose fermentation by
P. stipitis was successfully maintained due to the low oxygen consumption of the mutant yeast strain. Co-culture of these two strains resulted in the maximum yield and the highest productivity of ethanol from a mixture of glucose and xylose.
•Negative retention occurs due to intermolecular interactions in the feed solutions.•Presence of gluconic acid induces negative retention of acetic acid.•Formation of salt hydration shells lead to ...negative HMF retention.•Results obtained for binary mixtures can be applied to complex mixtures.
Separation of sugars and aldonic acids from aromatic compounds and short-chain organic acids is a crucial issue in various biorefinery concepts based on wood or agricultural byproducts, which can be resolved by nanofiltration. Model solutions containing up to six compounds, representing major components of biomass hydrolysates, were used to investigate interactions between the individual chemical species, with particular emphasis on the negative retention of 5-hydroxymethylfurfural (HMF) and acetic acid (AcOH).
Various factors were found to explain the negative retention of these substances in binary and multicomponent mixtures. The presence of gluconic acid led to the negative retention of AcOH due to an additional electric potential. For HMF retention, the hydration shell formed during the solvation of magnesium sulfate was determined as the main influencing factor. For the first time, the obtained results provide a mechanistic explanation for the negative retention of biomass hydrolysates. In addition, a novel methodology based on a revised concept of retention to quantitatively describe such effects in binary mixtures is provided, which can be qualitatively applied to any biomass hydrolysate. Our results are expected to make nanofiltration for the separation of biomass hydrolysates more predictable, thereby facilitating process development.
Abstract
The recent start-up of several full-scale ‘second generation’ ethanol plants marks a major milestone in the development of Saccharomyces cerevisiae strains for fermentation of ...lignocellulosic hydrolysates of agricultural residues and energy crops. After a discussion of the challenges that these novel industrial contexts impose on yeast strains, this minireview describes key metabolic engineering strategies that have been developed to address these challenges. Additionally, it outlines how proof-of-concept studies, often developed in academic settings, can be used for the development of robust strain platforms that meet the requirements for industrial application. Fermentation performance of current engineered industrial S. cerevisiae strains is no longer a bottleneck in efforts to achieve the projected outputs of the first large-scale second-generation ethanol plants. Academic and industrial yeast research will continue to strengthen the economic value position of second-generation ethanol production by further improving fermentation kinetics, product yield and cellular robustness under process conditions.
This minireview discusses how academic and industrial research yielded the robust, engineered yeast strains that are now used in the first large-scale factories for fuel-ethanol production from non-food agricultural residues.
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