Cofermentation of glucose,xylose, and arabinose by mixed cultures of two genetically engineeredZymomonas mobilis strains

Cofermentation of xylose and arabinose, in addition to glucose, is critical for complete bioconversion of lignocellulosic biomass, such as agricultural residues and herbaceous energy crops, to ethanol. A factorial design experiment was used to evaluate the cofermentation of glucose, xylose, and arabinose with mixed cultures of two genetically engineeredZymomonas mobilis strains (one ferments xylose and the other arabinose). The pH range studied was 5.0-6.0, and the temperature range was 30-37°C The individual sugar concentrations used were 30 g/L glucose, 30 g/L xylose, and 20 g/L arabinose. The optimal cofermentation conditions obtained by data analysis, using Design Expert software, were pH 5.85 and temperature 31.5°C. The cofermentation process yield at optimal conditions was 72.5% of theoritical maximum. The results showed that neither the arabinose strain nor arabinose affected the performance of the xylose strain; however, both xylose strain and xylose had a significant effect on the performance of the arabinose strain. Although cofermentation of all three sugars is achieved by the mixed cultures, there is a preferential order of sugar utilization. Glucose is used rapidly, then xylose, followed by arabinose.     

A simultaneous sucrose bioconversion into ethanol and levan byZymomonas mobilis

Two biotechnological systems were developed for sucrose conversion into levan and ethanol withZymomonas mobilis, ensuring a 66.7% transfer of substrate carbon in a batch and 61% carbon transfer in a continuous culture. The effect of glucose, ethanol, and medium pH on sucrose conversion byZ. mobilis was studied. The addition of ethanol to the fermentation medium, in the final conc. of 100 g/L, uncoupled levan synthesis from ethanol fermentation. For a continuous culture, the most efficient conversion of substrate carbon into levan was reached at pH 4.8, giving 64.2 g/L levan, with the levan yield of 0.22 g/g and the productivity of 3.2 g/L/h.     

Operability and feasibility of ethanol production by immobilizedZymomonas mobilis in a fluidized-bed bioreactor

Studies have been carried out using immobilized Z.mobilis in fluidized-bed bioreactors and have emphasized operation during high productivity and conversion. The bacteria are immobilized within small uniform beads (~1 to 1.5-mm diam) of K-carrageenan at cell loadings of 15-50 g (dry wt)/L. Conversion and productivity were measured under a variety of conditions, including feedstocks, flow rates, temperature, pH, and column sizes (up to 2.5 m tall). Volumetric productivities of 50-120 g EtOH/h-L reactor volume have been achieved. Productivities of 60 g/h-L are demonstrated from a 15% feed with residual glucose concentrations of less than 0.1% and 7.4% EtOH in the tallest fermentor. Among feeds of 10, 15, and 20% dextrose, the 15% gave the highest productivity and avoided substrate inhibition. A temperature of 30°C and pH 5 were the optimum conditions. The ethanol yield was shown to be nearly constant at 0.49 g EtOH/g glucose, or 97% of the theoretical under a variety of conditions and transients. The biocatalyst beads have been shown to remain active for two months. Nonsterile feed has been used for weeks without detrimental contamination. The advantages of this advanced bioreactor system over conventional batch technology are discussed.     

Ethanol production usingZymomonas mobilis in a cross-linked immobilized cell reactor

The bacteriumZymomonas mobilis may be utilized to produce ethanol from glucose in a cross-linked immobilized cell reactor. Reactor startup is much more rapid with cross-linkedZymomonas than with the yeastSaccharomyces cerevisiae. Volumetric ethanol productivities (based on liquid holdup) three times those obtained with cross-linked yeast, and comparable to those obtained withZymomonas immobilized by other methods, are possible.     

Conditions that promote production of lactic acid byZymomonas mobilis in batch and continuous culture

This study documents the similar pH-dependent shift in pyruvate metabolism exhibited byZymomonas mobilis ATCC 29191 and ATCC 39676 in response to controlled changes in their steady-state growth environment. The usual high degree of ethanol selectivity associated with glucose fermentation by Z.mobilis is associated with conditions that promote rapid and robust growth, with about 95% of the substrate (5% w/v glucose) being converted to ethanol and CO₂, and the remaining 5% being used for the synthesis of cell mass. Conditions that promote energetic uncoupling cause the conversion efficiency to increase to 98% as a result of the reduction in growth yield (cell mass production). Under conditions of glucose-limited growth in a chemostat, with the pH controlled at 6.0, the conversion efficiency was observed to decrease from 95% at a specific growth rate of 0.2/h to only 80% at 0.042/h. The decrease in ethanol yield was solely attributable to the pH-dependent shift in pyruvate metabolism, resulting in the production of lactic acid as a fermentation byproduct. At a dilution rate (D) of 0.042/h, decreasing from pH 6.0 to 5.5 resulted in a decrease in lactic acid from 10.8 to 7.5 g/L. Lactic acid synthesis depended on the presence of yeast extract (YE) or tryptone in the 5% (w/v) glucose-mineral salts medium. At D = 0.15/h, reduction in the level of YE from 3 to 1 g/L caused a threefold decrease in the steady-state concentration of lactic acid at pH 6. No lactic acid was produced with the same mineral salts medium, with ammonium chloride as the sole source of assimilable nitrogen. With the defined salts medium, the conversion efficiency was 98% of theoretical maximum. When chemostat cultures were used as seed for pH-stat batch fermentations, the amount of lactic acid produced correlated well with the activity of the chemostat culture; however, the mechanism of this prolonged induction     

Continuous fermentation of cellulosic biomass to ethanol

Experimental results are presented for continuous conversion of pretreated hardwood flour to ethanol. A simultaneous saccharification and fermentation (SSF) system comprised ofTrichoderma reesei cellulase supplemented with additional β-glucosidase and fermentation bySaccharomyces cerevisiae was used for most experiments, with data also presented for a direct microbial conversion (DMC) system comprised ofClostridium thermocellum. Using a batch SSF system, dilute acid pretreatment of mixed hardwood at short residence time(10 s, 220°C, 1% H₂SO₄) was compared to poplar wood pretreated at longer residence time (20 min, 160°C, 0.45% H₂SO₄). The short residence time pretreatment resulted in a somewhat (10–20%) more reactive substrate, with the reactivity difference particularly notable at low enzyme loadings and/or low agitation. Based on a preliminary screening, inhibition of SSF by byproducts of short residence time pretreatment was measurable, but minor. Both SSF and DMC were carried out successfully in well-mixed continuous systems, with steady-state data obtained at residence times of 0.58–3 d for SSF as well as 0.5 and 0.75 d for DMC. The SSF system achieved substrate conversions varying from 31% at a 0.58-d residence time to 86% at a 2-d residence time. At comparable substrate concentrations (4–5 g/l) and residence times (0.5–0.58 d), substrate conversion in the DMC system (77%) was significantly higher than that in the SSF system (31%). Our results suggest that the substrate conversion in SSF carried out in CSTR is relatively insensitive to enzyme loading in the range 7–25 U/g cellulose and to substrate concentration in the range of 5–60 g/L cellulose in the feed.     

Continuous fermentation studies with xylose-utilizing recombinant Zymomonas mobilis

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 coferm entation performance. Using declared assumptions, the cost of using CSL and Mg was estimated to be 12.5c/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).     

Continuous potable alcohol production by immobilizedSaccharomyces cerevisiae on mineral kissiris

A biocatalyst prepared by the immobilization of Saccharomyces cerevisiae on the surface of the mineral kissiris was used in the present study for continuous potable-alcohol production. An ethanol productivity (calculated on the basis of liquid volume) of 10.5 g/L/h was obtained at a 0.7/h dilution rate, 121 g/L sucrose content, and 29.6% conversion employing molasse as feed material. Glucose, raisin extracts, and molasse were successively used as feed materials without stopping the operation of the reactor for 6 mo. The ethanol productivity and yield remained constant during the operational-stability study of the reactor, carried out for 44 d. Biomass productivity, yield, and free-cell concentration in glucose, raisin extracts, and molasse were examined. Finally, a system with two continuous reactors joined successively was also studied in the present investigation.     

Growth inhibition in animal cell culture

Eight independent cell lines accumulated ammonia in culture to concentrations between 1.3 and 2.9 mM. The growth inhibition of such concentrations of ammonium chloride when added to culture medium was variable. The cell lines tested could be divided into 3 groups depending on their growth response to 2 mM added NH4Cl. In the first group (293, HDF, Vero, and PQXB1/2) little (less than 14%) or no growth inhibition occurred. In the second group (McCoy and MDCK) a reduction in final cell yield of 50-60% was observed. The third group (HeLa and BHK) was most sensitive to the effects of NH4Cl with growth inhibition (greater than 75%) compared to controls. The growth inhibitory effect of added lactate up to 20 mM was negligible (less than 10%) for 3 cell lines, although one cell line (PQXB1/2) showed greater sensitivity. The interactive effects of ammonia and lactate were determined in a matrix experiment. At lactate (greater than 12 mM) and ammonia (1-4 mM), the growth inhibitory effects of the two components were synergistic. However, at low concentrations of lactate (less than 12 mM) the toxic effect of ammonia was reduced. A proposed mechanism for the sparing effect of lactate on ammonia toxicity is discussed. This may have importance in developing strategies for the optimal growth of ammonia-sensitive cell lines.     

Xylanase recovery by ethanol and Na2SO4 precipitation

Xylans are the major components of the hemicellulosic fraction of lignocellulosic biomass and their hydrolysis can be obtained using xylanases fromPenicillium janthinellum. In this work, sugarcane bagasse hemicellulosic hydrolysate was used as the substrate for producing xylanase. The precipitation of these enzymes was studied using ethanol and Na₂SO₄ as precipitating agents. Ethanol precipitation experiments were performed batchwise in concentrations ranging from 10 to 80%, pH 4.0 to 7.0, at 4áC. The concentrations used in the precipitations with Na₂SO₄ were from 5 to 60% at pH 5.5 and 25áC. Solubility curves as a function of xylanase activity and total protein for both precipitating agents were made. According to the results, Na₂SO₄ is not appropriate for precipitating xylanases in this medium since at salt concentrations higher than 25%, the enzyme was denaturated and at this concentration less than 80% of the enzyme and total protein were precipitated. Because of differences in xylanase and total protein solubility, a fractionated precipitation using ethanol can be performed, since with 40% ethanol, 49% of the total protein was precipitated and more than 95% of the enzyme was kept in solution. On the other hand approx 100% of the xylanases were recovered by precipitation after adding 80% ethanol.     

Comparison of yellow poplar pretreatment between NREL digester and sunds hydrolyzer

Single-stage cocurrent dilute acid pretreatments were carried out on yellow poplar (Liriodendron tulipifera) sawdust using an as-installed and short residence time modified pilot-scale Sunds hydrolyzer and a 4-L bench-scale NREL digester (steam explosion reactor). Pretreatment conditions for the Sunds hydrolyzer, installed in the NREL process development unit (PDU), which operates at 1 t/d (bone-dry t) feed rate, spanned the temperature range of 160 – 210°C, 0.1 – 1.0% (w/w) sulfuric acid, and 4-10-min residence times. The batch pretreatments of yellow poplar sawdust in the bench-scale digester were carried out at 210 and 230°C, 0.26% (w/w) sulfuric acid, and 1-, 3-, and 4-min residence times. The dilute acid prehydrolysis solubilized more than 90% of the hemicellulose, and increased the enzymatic digestibility of the cellulose that remained in the solids. Compositional analysis of the pretreated solids and liquors and mass balance data show that the two pretreatment devices had similar pretreatment performance.     

Fermentation of xylose to glycerol byRhizopus javanicus

Glycerol production from xylose fermentation usingRhizopus javanicus (ATCC 22581) has been investigated in shake flasks. The medium composition (xylose concentration, nitrogen sources), aeration rate, and temperature have been found to affect the accumulation and yield of glycerol. Some of these effects are explained in terms of the critical parameters, osmotic pressure, and dissolved oxygen levels in the medium. Relatively high glycerol yields and concentrations have been obtained at high sugar concentration with high level of aeration at room temperature. The addition of polyethylene glycol or sulfite can improve the yield and accumulation of glycerol.     

Continuous attrition bioreactor with enzyme recycling for the bioconversion of cellulose

A new type of reactor, the attrition bioreactor, has been developed to increase the rate of the enzymatic hydrolysis of cellulose and also to cut pretreatment costs. It was found that the attrition bioreactor could be operated continuously or semicontinuously in conjunction with a membrane filter to produce a high cellulose conversion rate and low enzyme consumption. The membrane filter served to contain the enzyme and cellulose within the reactor while allowing sugar to permeate as a product.     

Simultaneous saccharification and fermentation of lignocellulose

Simultaneous saccharification and fermentation (SSF) processes for producing ethanol from lignocellulose are capable of improved hydrolysis rates, yields, and product concentrations compared to separate hydrolysis and fermentation (SHF) systems, because the continuous removal of the sugars by the yeasts reduces the end-product inhibition of the enzyme complex. Recent experiments using Genencor 150L cellulase and mixed yeast cultures have produced yields and concentrations of ethanol from cellulose of 80% and 4.5%, respectively. The mixed culture was employed because B.clausenii has the ability to ferment cellobiose (further reducing end-product inhibition), while the brewing yeastS. cerevisiae provides a robust ability to ferment the monomeric sugars. These experimental results are combined with a process model to evaluate the economics of the process and to investigate the effect of alternative processes, conditions, and organisms.     

Dilute acid pretreatment of softwoods

Selective thinning of forests in the western United States will generate a large, sustainable quantity of softwood residues that can be an attractive feedstock for fuel ethanol production. The major species available from thinning of forests in northern California and the eastern Rocky Mountains include white fir (Abies concolor), Douglas fir (Pseudotsuga menziesii), and Ponderosa pine (Pinus ponderosa). Douglas fir chips were soaked in 0.4% sulfuric acid solution, then pretreated with steam at 200 – 230°C for 1 – 5 min. After pretreatment, 90 – 95% of the hemicellulose and as much as 20% of the cellulose was solubilized in water, and 90% of the remaining cellulose can be hydrolyzed to glucose by cellulase enzyme. The prehydrolysates, at as high as 10% total solid concentration, can be readily fermented by the unadapted yeastSaccharomyces cerevisiae D₅A.     

Xylose fermentation

The economic impact of conversion of xylose to ethanol for a wood-to-ethanol plant was examined, and the maximum potential reduction in the price of ethanol from utilization of xylose is estimated to be 0.42 per gallon from a base case price of0.42 per gallon from a base case price of 1.65. The sensitivity of the price of ethanol to the yield, ethanol concentration and rate of the xylose fermentation was also examined, and the price of ethanol is most affected by changes in yield and ethanol concentration, with rate of lesser importance. Current performances of various xylose conversion biocatalysts were analyzed, andC. shehatae andP. stipitis appear to be the best yeasts.     

Continuous production of mixed alcohols and acids from carbon monoxide

Continuous, steady-state fermentations using carbon monoxide gas as the sole carbon and energy source have been achieved with the CO strain ofButyribacterium methylotrophicum. Fermentation pH was found to regulate carbon monoxide metabolism over the pH range of 6.8 to 5.0. Cell growth diminished at low pH, with washout occurring at pH 5.0. As observed previously in batch culture, lower pH values favored production of butyrate over acetate. The mechanism responsible for this trend is currently being investigated by quantification of key intracellular enzyme activities. At low pH values, direct, steady-state fermentation of carbon monoxide to alcohols has been verified. Of major significance is the production of butanol from carbon monoxide in pure culture. This newly identified pathway provides a potential mechanism for direct bioconversion of synthesis gas to butanol.     

Yeast adaptation on softwood prehydrolysate

Several strains and genera of yeast, includingSaccharomyces cerevisiae D₅A,Pachysolen tannophilus, S. cerevisiae K-l,Brettanomyces custersii, Candida shehatae, andCandida acidothermophilum, are screened for growth on dilute acid-pretreated softwood prehydrolysate. Selected softwood species found in forest underbrush of the western United States, which contain predominantly hexosan hemicellulose, were studied. This phase of the work emphasized debarked Douglas fir. The two best initial isolates were gradually selected for improved growth by adaptation to increasing prehydrolysate concentrations in batch culture, with due consideration of nutrient requirements. Microaerophilic conditions were evaluated to encourage tolerance of pretreatment hydrolysate, as well as ethanol product. Adaptation and simultaneous saccharification and fermentation (SSF) results are used to illustrate improved performance with an adapted strain, compared to the wild type.