Recent Developments in the Zymomonas Process for Ethanol Production

Abstract

The bacterium Zymomonas mobilis, which is used in the tropics to make pulque and alcoholic palm wines, appears to have considerable potential for industrial alcohol fermentations. Some of the advantages of the Zymomonas process reported in studies from our laboratory^1-24 are

1. There are significantly higher specific rates of sugar uptake and ethanol production compared to those found for yeasts.

2. Considerably higher volumetric ethanol productivities found in continuous cell recycle systems (up to 120 to 200 g/hr).

3. There are higher ethanol yields and lower biomass production than for yeasts. The lower biomass concentrations would seem to be a consequence of the lower metabolic energy available for growth. Zymomonas metabolize glucose via the Entner-Doudoroff pathway while yeasts convert glucose to ethanol via glycolysis.

4. Zymomonas cultures grow anaerobically and, unlike yeasts, do not require the controlled addition of oxygen to maintain viability at high cell concentrations.

5. The ethanol tolerance of some selected strains of Zymomonas is comparable if not higher than strains of Saccharomyces cerevisiae. Ethanol concentrations of 85 g/(up to 11% v/v) have been achieved in continuous culture and up to 130 g/(16% v/v) in batch culture.     

A holistic high-throughput screening framework for biofuel feedstock assessment that characterises variations in soluble sugars and cell wall composition in <Emphasis Type="Italic">Sorghum bicolor</Emphasis>

Background

A major hindrance to the development of high yielding biofuel feedstocks is the ability to rapidly assess large populations for fermentable sugar yields. Whilst recent advances have outlined methods for the rapid assessment of biomass saccharification efficiency, none take into account the total biomass, or the soluble sugar fraction of the plant. Here we present a holistic high-throughput methodology for assessing sweet Sorghum bicolor feedstocks at 10 days post-anthesis for total fermentable sugar yields including stalk biomass, soluble sugar concentrations, and cell wall saccharification efficiency.

Results

A mathematical method for assessing whole S. bicolor stalks using the fourth internode from the base of the plant proved to be an effective high-throughput strategy for assessing stalk biomass, soluble sugar concentrations, and cell wall composition and allowed calculation of total stalk fermentable sugars. A high-throughput method for measuring soluble sucrose, glucose, and fructose using partial least squares (PLS) modelling of juice Fourier transform infrared (FTIR) spectra was developed. The PLS prediction was shown to be highly accurate with each sugar attaining a coefficient of determination (R ² ) of 0.99 with a root mean squared error of prediction (RMSEP) of 11.93, 5.52, and 3.23 mM for sucrose, glucose, and fructose, respectively, which constitutes an error of <4% in each case. The sugar PLS model correlated well with gas chromatography–mass spectrometry (GC-MS) and brix measures. Similarly, a high-throughput method for predicting enzymatic cell wall digestibility using PLS modelling of FTIR spectra obtained from S. bicolor bagasse was developed. The PLS prediction was shown to be accurate with an R ² of 0.94 and RMSEP of 0.64 μg.mgDW^-1.h^-1.

Conclusions

This methodology has been demonstrated as an efficient and effective way to screen large biofuel feedstock populations for biomass, soluble sugar concentrations, and cell wall digestibility simultaneously allowing a total fermentable yield calculation. It unifies and simplifies previous screening methodologies to produce a holistic assessment of biofuel feedstock potential.

     

Xylitol production from a mutant strain of Candida tropicalis

Aims: To characterize the kinetics of growth, sugar uptake and xylitol production in batch and fed‐batch cultures for a xylitol assimilation‐deficient strain of Candida tropicalis isolated via chemical mutagenesis. Methods and Results: Chemical mutagenesis using nitrosoguanidine led to the isolation of the xylitol‐assimilation deficient strain C. tropicalis SS2. Shake‐flask fermentations with this mutant showed a sixfold higher xylitol yield than the parent strain in medium containing 25 g l^?1 glucose and 25 g l^?1 xylose. With 20 g l^?1 glycerol, replacing glucose for cell growth, and various concentrations of xylose, the studies indicated that the mutant strain resulted in xylitol yields from xylose close to theoretical. Under fully aerobic conditions, fed‐batch fermentation with repeated addition of glycerol and xylose resulted in 3·3 g l^?1 h^?1 xylitol volumetric productivity with the final concentration of 220 g l^?1 and overall yield of 0·93 g g^?1 xylitol. Conclusions: The xylitol assimilation‐deficient mutant isolated in this study showed the potential for high xylitol yield and volumetric productivity under aerobic conditions. In the evaluation of glycerol as an alternative low‐cost nonfermentable carbon source, high biomass and xylitol yields under aerobic conditions were achieved; however, the increase in initial xylose concentrations resulted in a reduction in biomass yield based on glycerol consumption. This may be a consequence of the role of an active transport system in the yeast requiring increasing energy for xylose uptake and possible xylitol secretion, with little or no energy available from xylose metabolism. Significance and Impact of the Study: The study confirms the advantage of using a xylitol assimilation‐deficient yeast under aerobic conditions for xylitol production with glycerol as a primary carbon source. It illustrates the potential of using the xylose stream in a biomass‐based bio‐refinery for the production of xylitol with further cost reductions resulting from using glycerol for yeast growth and energy production.     

Fermentation of xylose into ethanol by a new fungus strain <Emphasis Type="Italic">Pestalotiopsis</Emphasis> sp. XE-1

A new fungus, Pestalotiopsis sp. XE-1, which produced ethanol from xylose with yield of 0.47 g ethanol/g of consumed xylose was isolated. It also produced ethanol from arabinose, glucose, fructose, mannose, galactose, cellobiose, maltose, and sucrose with yields of 0.38, 0.47, 0.45, 0.46, 0.31, 0.25, 0.31, and 0.34 g ethanol/g of sugar consumed, respectively. It produced maximum ethanol from xylose at pH 6.5, 30°C under a semi-aerobic condition. Acetic acid produced in xylose fermenting process inhibited ethanol production of XE-1. The ethanol yield in the pH-uncontrolled batch fermentation was about 27% lower than that in the pH-controlled one. The ethanol tolerance of XE-1 was higher than most xylose-fermenting, ethanol-producing microbes, but lower than Saccharomyces cerevisiae and Hansenula polymorpha. XE-1 showed tolerance to high concentration of xylose, and was able to grow and produce ethanol even when it was cultivated in 97.71 g/l xylose.     

Fed-batch production of a bioflocculant from <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

The constant-rate fed-batch production of the polygalacturonic acid bioflocculant REA-11 was studied. A controlled sucrose-feeding strategy resulted in a slight improvement in biomass and a 7% reduction in flocculating activity compared with the batch process. When fed with a 3 g l⁻¹ urea solution, the flocculating activity was enhanced to 720 U ml⁻¹ in 36 h. High cell density (2.12 g l⁻¹) and flocculating activity (820 U ml⁻¹) were obtained in a 10-l fermentor by feeding with a sucrose-urea solution, with values of nearly two times and 50% higher than those of the batch process, respectively. Moreover, the residual sucrose declined to 2.4 g l⁻¹, and residual urea decreased to 0.03 g l⁻¹. Even higher flocculating activity of 920 U ml⁻¹ and biomass of 3.26 g l⁻¹ were obtained by feeding with a sucrose-urea solution in a pilot scale fermentation process, indicating the potential industrial utility of this constant-rate feeding strategy in bioflocculant production by Corynebacterium glutamicum.     

Quantification of the contribution of surface outgrowth to biocatalysis in sol-gels: oxytetracycline production by <Emphasis Type="Italic">Streptomyces rimosus</Emphasis>

A technique was developed for differentiating the activity of microbes solely within sol gels by using the contribution of biomass outgrowth. Streptomyces rimosus was immobilised in colloidal silica gels and biomass growth, oxytetracycline synthesis, pH and carbohydrate consumption were compared for UV surface-sterilised gels, untreated gels, and liquid cultures. Absolute and biomass specific oxytetracycline yields were higher for non-sterile gels than for liquid culture. Biomass solely within colloidal silica gels (1.7 mg ml^–1), and gels obtained from colloidal silica modified by addition of larger silica particles (1.2 mg ml^–1) yielded 27 and 21 g ml^–1 oxytetracycline compared with 97 and 104 g ml^–1 for unsterilised gels (3.6 and 5.2 mg ml^–1 biomass) displaying outgrowth. It was therefore apparent that biomass and antibiotic production within the gels was limited and that optimisation requires gel modification.     

Production of pediocin SM-1 by Pediococcus pentosaceus Mees 1934 in fed-batch fermentation: Effects of sucrose concentration in a complex medium and process modeling

Production of pediocin SM-1 by Pediococcus pentosaceus Mees 1934 was investigated in semi-aerobic, pH-controlled, batch and fed-batch fermentations using a complex medium containing sucrose as the main source of carbon. The effects of sucrose concentration were studied in fed-batch fermentations in which a sucrose solution was added at stable feeding rates (5, 7, 9 and 10 g/l/h). The results showed that pediocin is produced as a product of the primary metabolism and its titer could be greatly improved by adjusting the sucrose feeding rate in fed-batch fermentation. The maximum titer of pediocin of 145 AU/ml was obtained in the fed-batch culture with 7 g/l/h feeding rate and that was 119% higher compared to the titer obtained in batch culture. Higher feeding rates (9 and 10 g/l/h) resulted in decreased pediocin yields while biomass levels appeared to be rather unaffected. The specific rate of pediocin formation was also sensitive to sucrose concentration levels. A mathematical model developed on the basis of well-known rate equations for batch and fed-batch cultures and growth associated production, described successfully cell growth, sucrose assimilation, lactate production and pediocin production in fed-batch culture.     

Fructose production by Zymomonas mobilis in fed-batch culture with minimal sorbitol formation

Summary Fed-batch cultures of Zymomonas mobilis (UQM 2864), a mutant unable to metabolise fructose, grown on diluted sugar cane syrup (200 g/l sucrose) achieved yields of 90.5 g/l fructose and 48.3 g/l ethanol with minimal sorbitol formation and complete utilization of the substrate. The effect of inoculum size on sorbitol formation in the batch stage of fed-batch fermentation are reported. Fermentation of sucrose (350 g/l) supplemented with nutrients yielded 142 g/l fructose and 76.5 g/l ethanol. Some fructose product loss at high fructose concentrations was observed. The fed-batch fermentation process offers a method for obtaining high concentrations of fructose and ethanol from sucrose materials.     

Anomalies in the growth kinetics of Saccharomyces cerevisiae strains in aerobic chemostat cultures

Aerobic glucose-limited chemostat cultivations were conducted with Saccharomyces cerevisiae strains NRRL Y132, ATCC 4126 and CBS 8066, using a complex medium. At low dilution rates all three strains utilised glucose oxidatively with high biomass yield coefficients, no ethanol production and very low steady-state residual glucose concentrations in the culture. Above a threshold dilution rate, respiro-fermentative (oxido-reductive) metabolism commenced, with simultaneous respiration and fermentation occurring, which is typical of Crabtree-positive yeasts. However, at high dilution rates the three strains responded differently. At high dilution rates S. cerevisiae CBS 8066 produced 7–8 g ethanol L⁻¹ from 20 g glucose L⁻¹ with concomitant low levels of residual glucose, which increased markedly only close to the wash-out dilution rate. By contrast, in the respiro-fermentative region both S. cerevisiae ATCC 4126 and NRRL Y132 produced much lower levels of ethanol (3–4 g L⁻¹) than S. cerevisiae CBS 8066, concomitant with very high residual sugar concentrations, which was a significant deviation from Monod kinetics and appeared to be associated either with high growth rates or with a fermentative (or respiro-fermentative) metabolism. Supplementation of the cultures with inorganic or organic nutrients failed to improve ethanol production or glucose assimilation. Journal of Industrial Microbiology & Biotechnology (2000) 24, 231–236. Received 09 August 1999/ Accepted in revised form 18 December 1999     

Engineering of carbon catabolite repression in recombinant xylose fermenting<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

Two xylose-fermenting glucose-derepressed Saccharomyces cerevisiae strains were constructed in order to investigate the influence of carbon catabolite repression on xylose metabolism. S. cerevisiae CPB.CR2 (mig1, XYL1, XYL2, XKS1) and CPB.MBH2 (mig1, mig2, XYL1, XYL2, XKS1) were analysed for changes in xylose consumption rate and ethanol production rate during anaerobic batch and chemostat cultivations on a mixture of 20 g l^–1 glucose and 50 g l^–1 xylose, and their characteristics were compared to the parental strain S. cerevisiae TMB3001 (XYL1, XYL2, XKS1). Improvement of xylose utilisation was limited during batch cultivations for the constructed strains compared to the parental strain. However, a 25% and 12% increased xylose consumption rate during chemostat cultivation was achieved for CPB.CR2 and CPB.MBH2, respectively. Furthermore, during chemostat cultivations of CPB.CR2, where the cells are assumed to grow under non-repressive conditions as they sense almost no glucose, invertase activity was lower during growth on xylose and glucose than on glucose only. The 3-fold reduction in invertase activity could only be attributed to the presence of xylose, suggesting that xylose is a repressive sugar for S. cerevisiae.     

Obtaining and selection of hexokinases-less strains of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> for production of ethanol and fructose from sucrose

Saccharomyces cerevisiae hexokinase-less strains were produced to study the production of ethanol and fructose from sucrose. These strains do not have the hexokinases A and B. Twenty-three double-mutant strains were produced, and then, three were selected for presenting a smaller growth in yeast extract–peptone–fructose. In fermentations with a medium containing sucrose (180.3 g L⁻¹) and with cell recycles, simulating industrial conditions, the capacity of these mutant yeasts in inverting sucrose and fermenting only glucose was well characterized. Besides that, we could also see their great tolerance to the stresses of fermentative recycles, where fructose production (until 90 g L⁻¹) and ethanol production (until 42.3 g L⁻¹) occurred in cycles of 12 h, in which hexokinase-less yeasts performed high growth (51.2% of wet biomass) and viability rates (77% of viable cells) after nine consecutive cycles.     

Quasi steady state growth of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> in glucose-limited acceleration stat (A-stat) cultures

Quasi steady state growth of Lactococcus lactis IL 1403 was studied in glucose-limited A-stat cultivation experiments with acceleration rates (a) from 0.003 to 0.06 h⁻² after initial stabilization of the cultures in chemostat at D = 0.2–0.3 h⁻¹. It was shown that the high limit of quasi steady state growth rate depended on the acceleration rate used—at an acceleration rate 0.003 h⁻² the quasi steady state growth was observed until μ _crit = 0.59 h⁻¹, which is also the μ _max value for the culture. Lower values of μ _crit were observed at higher acceleration rates. The steady state growth of bacteria stabilized at dilution rate 0.2 h⁻¹ was immediately disrupted after initiating acceleration at the highest acceleration rate studied—0.06 h⁻². Observation was made that differences [Δ(μ − D)] of the specific growth rates from pre-programmed dilution rates were the lowest using an acceleration rate of 0.003 h⁻² (< 4% of preset changing growth rate). The adaptability of cells to follow preprogrammed growth rate was found to decrease with increasing dilution rate—it was shown that lower acceleration rates should be applied at higher growth rates to maintain the culture in the quasi steady state. The critical specific growth rate and the biomass yields based on glucose consumption were higher if the medium contained S ₀ = 5 g L⁻¹ glucose instead of S ₀ = 10 g L⁻¹. It was assumed that this was due to the inhibitory effect of lactate accumulating at higher concentrations in the latter cultures. Parallel A-stat experiments at the same acceleration and dilution rates showed good reproducibility—Δ(μ − D) was less than 5%, standard deviations of biomass yields per ATP produced (Y _ATP), and biomass yields per glucose consumed (Y _XS) were less than 15%.     

Repeated-batch production of glucoamylase using recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> immobilized in a fibrous bed bioreactor

The recombinant Saccharomyces cerevisiae strain C468/pGAC9 has an unstable hybrid plasmid pGAC9, which directs production of glucoamylase. A fibrous cotton material with a good adsorption capability for recombinant S. cerevisiae cells was used as the immobilization matrix in an internal loop airlift-driven fibrous bed bioreactor (ILALFBB) system. With batch cultures in the ILALFBB, the fraction of plasmid-carrying cells was 72% after more than 2 days cultivation, which was two times higher than that in the conventional free-cell culture. Correspondingly, a high activity of glucoamylase (GA; 113 U/l) was achieved with a high productivity of 43 U/l/h. The ILALFBB system also maintained a high fraction of viable plasmid-carrying of 74% for glucoamylase production during repeated-batch cultures, achieving a high glucoamylase activity of 140 U/l with a productivity of 19–130 U/l/h in all 14 batches studied during 19.8 days. The stable and long-term glucoamylase production from the ILALFBB was attributed to the effect of cell immobilization on plasmid stability. Plasmid-carrying cells were preferentially retained in the fibrous matrix because of their ability to adhere to the fiber surface and to form cell aggregates higher than those of plasmid-free cells. The repeated batch using immobilized cell of recombinant S. cerevisiae in the ALALFBB system thus provides a feasible method for stable, long-term and high-level production of glucoamylase.     

Effect of <Emphasis Type="Italic">HXT</Emphasis> 1 and <Emphasis Type="Italic">HXT</Emphasis> 7 hexose transporter overexpression on wild-type and lactic acid producing <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> cells

Background  

Since about three decades, Saccharomyces cerevisiae can be engineered to efficiently produce proteins and metabolites. Even recognizing that in baker's yeast one determining step for the glucose consumption rate is the sugar uptake, this fact has never been conceived to improve the metabolite(s) productivity.     

Fermentation characteristics of Dekkera bruxellensis strains

The influence of pH, temperature and carbon source (glucose and maltose) on growth rate and ethanol yield of Dekkera bruxellensis was investigated using a full-factorial design. Growth rate and ethanol yield were lower on maltose than on glucose. In controlled oxygen-limited batch cultivations, the ethanol yield of the different combinations varied from 0.42 to 0.45 g (g glucose)⁻¹ and growth rates varied from 0.037 to 0.050 h⁻¹. The effect of temperature on growth rate and ethanol yield was negligible. It was not possible to model neither growth rate nor ethanol yield from the full-factorial design, as only marginal differences were observed in the conditions tested. When comparing three D. bruxellensis strains and two industrial isolates of Saccharomyces cerevisiae, S. cerevisiae grew five times faster, but the ethanol yields were 0–13% lower. The glycerol yields of S. cerevisiae strains were up to six-fold higher compared to D. bruxellensis, and the biomass yields reached only 72–84% of D. bruxellensis. Our results demonstrate that D. bruxellensis is robust to large changes in pH and temperature and may have a more energy-efficient metabolism under oxygen limitation than S. cerevisiae.     

Influence of Carbon Source on Nitrate Removal by Nitrate-Tolerant Klebsiella oxytoca CECT 4460 in Batch and Chemostat Cultures

The nitrate-tolerant organism Klebsiella oxytoca CECT 4460 tolerates nitrate at concentrations up to 1 M and is used to treat wastewater with high nitrate loads in industrial wastewater treatment plants. We studied the influence of the C source (glycerol or sucrose or both) on the growth rate and the efficiency of nitrate removal under laboratory conditions. With sucrose as the sole C source the maximum specific growth rate was 0.3 h⁻¹, whereas with glycerol it was 0.45 h⁻¹. In batch cultures K. oxytoca cells grown on sucrose or glycerol were able to immediately use sucrose as a sole C source, suggesting that sucrose uptake and metabolism were constitutive. In contrast, glycerol uptake occurred preferentially in glycerol-grown cells. Independent of the preculture conditions, when sucrose and glycerol were added simultaneously to batch cultures, the sucrose was used first, and once the supply of sucrose was exhausted, the glycerol was consumed. Utilization of nitrate as an N source occurred without nitrite or ammonium accumulation when glycerol was used, but nitrite accumulated when sucrose was used. In chemostat cultures K. oxytoca CECT 4460 efficiently removed nitrate without accumulation of nitrate or ammonium when sucrose, glycerol, or mixtures of these two C sources were used. The growth yields and the efficiencies of C and N utilization were determined at different growth rates in chemostat cultures. Regardless of the C source, yield carbon (Y_C) ranged between 1.3 and 1.0 g (dry weight) per g of sucrose C or glycerol C consumed. Regardless of the specific growth rate and the C source, yield nitrogen (Y_N) ranged from 17.2 to 12.5 g (dry weight) per g of nitrate N consumed. In contrast to batch cultures, in continuous cultures glycerol and sucrose were utilized simultaneously, although the specific rate of sucrose consumption was higher than the specific rate of glycerol consumption. In continuous cultures double-nutrient-limited growth appeared with respect to the C/N ratio of the feed medium and the dilution rate, so that for a C/N ratio between 10 and 30 and a growth rate of 0.1 h⁻¹ the process led to simultaneous and efficient removal of the C and N sources used. At a growth rate of 0.2 h⁻¹ the zone of double limitation was between 8 and 11. This suggests that the regimen of double limitation is influenced by the C/N ratio and the growth rate. The results of these experiments were validated by pulse assays.     

High efficiency degradation of tetrahydrofuran (THF) using a membrane bioreactor: identification of THF-degrading cultures of<Emphasis Type="Italic"> Pseudonocardia</Emphasis> sp. strain M1 and<Emphasis Type="Italic"> Rhodococcus ruber</Emphasis> isolate M2

A mixed microbial culture capable of growing aerobically on tetrahydrofuran (THF) as a sole carbon and energy source was used as the inoculum in a 10 l working volume membrane bioreactor. Following start-up, the reactor was operated in batch mode for 24 h and then switched to continuous feed with 100% biomass recycle. On average, greater than 96% of THF fed to the reactor was removed during the 8-month study. THF loading rates ranged from 0.62 to 9.07 g l^–1 day^–1 with a hydraulic retention time of 24 h. THF concentrations as high as 800 mg/l were tolerated by the culture. Biomass production averaged 0.28 kg total suspended solids/kg chemical oxygen demand removed, i.e., comparable to a conventional wastewater treatment process. Periodic batch wasting resulted in a solids retention time of 7–14 days. Reactor biomass typically ranged from 4 to 10 g/l volatile suspended solids and the effluent contained no solids. Pure THF-degrading cultures were isolated from the mixed culture based on morphological characteristics, Gram-staining and THF degradation. Based on 16S rDNA analysis the isolates were identified as Pseudonocardia sp. M1 and Rhodococcus ruber M2.