Simultaneous Saccharification and Ethanol Fermentation of Corn Stover at High Temperature and High Solids Loading by a Thermotolerant Strain Saccharomyces cerevisiae DQ1

The thermotolerant strain Saccharomyces cerevisiae DQ1 was applied to the simultaneous saccharification and fermentation (SSF) at high temperature and high solids loading of the dilute acid-pretreated corn stover in the present study. The SSF using S. cerevisiae DQ1 was operated at 30?% solids loading of the pretreated corn stover with three-step SSF mode and achieved up to ethanol titer of 48?g/L and yield of 65.6?%. S. cerevisiae DQ1 showed strong thermotolerance in both the regular one-step SSF and the three-step SSF with changing temperature in each step. The three-step SSF at 40°C using S. cerevisiae DQ1 tolerated the greater cellulase dosage and solids loading of the pretreated corn stover and resulted in increased ethanol production. The present study provided a practical potential for the future SSF of lignocellulose feedstock at high temperature to reach high ethanol titer.     

Onsite bio-detoxification of steam-exploded corn stover for cellulosic ethanol production

In the process of ethanol production from steam-exploded corn stover (SECS), a cellulose-degradation strain of Aspergillus nidulans (FLZ10) was investigated whether it could remove the inhibitors released from steam exploded pretreatment , and thereby be used for biological detoxification on Saccharomycescerevisiae. The results showed that FLZ10 removed 75.2% formic acid, 53.6% acetic acid, and 100% hydroxymethyl furfural (5-HMF) and furfural from the hydrolysate washed from SECS after 72 h cultivation. A cellulase activity of 0.49 IU/ml was simultaneously produced while the biological detoxification occurred. An ethanol yield of 0.45 g/g on glucose was obtained in the hydrolysate biodetoxified by FLZ10. The glucose consumption rate of FLZ10 was much lower than that of S. cerevisiae, thereby it had little competition with S. cerevisiae on glucose consumption. Based on SECS to ethanol mass balance analysis, with the onsite bio-detoxification, fermentation using S. cerevisiae effectively converted monomeric glucose with 94.4% ethanol yield.     

Biological pretreatment of corn stover with Phlebia brevispora NRRL‐13108 for enhanced enzymatic hydrolysis and efficient ethanol production

Biological pretreatment of lignocellulosic biomass by white‐rot fungus can represent a low‐cost and eco‐friendly alternative to harsh physical, chemical, or physico‐chemical pretreatment methods to facilitate enzymatic hydrolysis. In this work, solid‐state cultivation of corn stover with Phlebia brevispora NRRL‐13018 was optimized with respect to duration, moisture content and inoculum size. Changes in composition of pretreated corn stover and its susceptibility to enzymatic hydrolysis were analyzed. About 84% moisture and 42 days incubation at 28°C were found to be optimal for pretreatment with respect to enzymatic saccharification. Inoculum size had little effect compared to moisture level. Ergosterol data shows continued growth of the fungus studied up to 57 days. No furfural and hydroxymethyl furfural were produced. The total sugar yield was 442 ± 5 mg/g of pretreated corn stover. About 36 ± 0.6 g ethanol was produced from 150 g pretreated stover per L by fed‐batch simultaneous saccharification and fermentation (SSF) using mixed sugar utilizing ethanologenic recombinant Eschericia coli FBR5 strain. The ethanol yields were 32.0 ± 0.2 and 38.0 ± 0.2 g from 200 g pretreated corn stover per L by fed‐batch SSF using Saccharomyces cerevisiae D5A and xylose utilizing recombinant S. cerevisiae YRH400 strain, respectively. This research demonstrates that P. brevispora NRRL‐13018 has potential to be used for biological pretreatment of lignocellulosic biomass. This is the first report on the production of ethanol from P. brevispora pretreated corn stover. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:365–374, 2017     

Simultaneous saccharification and fermentation of steam-exploded corn stover at high glucan loading and high temperature

Background

Simultaneous saccharification and fermentation (SSF) is a promising process for bioconversion of lignocellulosic biomass. High glucan loading for hydrolysis and fermentation is an efficient approach to reduce the capital costs for bio-based products production. The SSF of steam-exploded corn stover (SECS) for ethanol production at high glucan loading and high temperature was investigated in this study.

Results

Glucan conversion of corn stover biomass pretreated by steam explosion was maintained at approximately 71 to 79% at an enzyme loading of 30 filter paper units (FPU)/g glucan, and 74 to 82% at an enzyme loading of 60 FPU/g glucan, with glucan loading varying from 3 to 12%. Glucan conversion decreased obviously with glucan loading beyond 15%. The results indicated that the mixture was most efficient in enzymatic hydrolysis of SECS at 3 to 12% glucan loading. The optimal SSF conditions of SECS using a novel Saccharomyces cerevisiae were inoculation optical density (OD)₆₀₀?=?4.0, initial pH 4.8, 50% nutrients added, 36 hours pre-hydrolysis time, 39°C, and 12% glucan loading (20% solid loading). With the addition of 2% Tween 20, glucan conversion, ethanol yield, final ethanol concentration reached 78.6%, 77.2%, and 59.8 g/L, respectively, under the optimal conditions. The results suggested that the solid and degradation products’ inhibitory effect on the hydrolysis and fermentation of SECS were also not obvious at high glucan loading. Additionally, glucan conversion and final ethanol concentration in SSF of SECS increased by 13.6% and 18.7%, respectively, compared with separate hydrolysis and fermentation (SHF).

Conclusions

Our research suggested that high glucan loading (6 to 12% glucan loading) and high temperature (39°C) significantly improved the SSF performance of SECS using a thermal- and ethanol-tolerant strain of S. cerevisiae due to the removal of degradation products, sugar feedback, and solid’s inhibitory effects. Furthermore, the surfactant addition obviously increased ethanol yield in SSF process of SECS.

     

分别利用产甘油假丝酵母抗逆转录因子CgSTB5和CgSEF1对酿酒酵母菌株糠醛耐受改造

【背景】纤维素是生物转化解决能源问题的主要原料之一,其水解物中存在严重影响抑制菌株生长的糠醛,需脱毒才可应用于发酵,提高菌株耐受性是解决纤维素水解液实际生产应用的关键。【目的】酿酒酵母(Saccharomyces cerevisiae)是主要的纤维素水解液发酵工业菌株,但糠醛耐受性较低,通过分子改造获得具有高糠醛耐受性的菌株。【方法】利用新获得的产甘油假丝酵母(Candidaglycerinogenes)的相关抗逆转录因子CgSTB5、CgSEF1和CgCAS5,通过分子技术进行S.cerevisiae改造,考察其对酿酒酵母糠醛耐受性的影响,并尝试应用于未脱毒纤维素乙醇发酵。【结果】单个表达CgSTB5和CgSEF1的酿酒酵母,通过菌株点板实验表明菌株的糠醛耐受性提高25%以上,并且摇瓶发酵结果显示糠醛降解性能明显提高,生长延滞期明显缩短,S.cerevisiae W303/p414-CgSTB5的未脱毒纤维素乙醇发酵生产效率提高12.5%左右。【结论】转录因子CgSTB5和CgSEF1均能对提高酿酒酵母糠醛耐受性起到重要作用,并且有助于提高酿酒酵母菌株未脱毒纤维素乙醇发酵性能。     

A high-throughput transient gene expression system for switchgrass (Panicum virgatum L.) seedlings

Background

Fermentations using Escherichia coli KO11, Saccharomyces cerevisiae 424A(LNH-ST), and Zymomonas mobilis AX101 are compared side-by-side on corn steep liquor (CSL) media and the water extract and enzymatic hydrolysate from ammonia fiber expansion (AFEX)-pretreated corn stover.

Results

The three ethanologens are able produce ethanol from a CSL-supplemented co-fermentation at a metabolic yield, final concentration and rate greater than 0.42 g/g consumed sugars, 40 g/L and 0.7 g/L/h (0-48 h), respectively. Xylose-only fermentation of the tested ethanologenic bacteria are five to eight times faster than 424A(LNH-ST) in the CSL fermentation. All tested strains grow and co-ferment sugars at 15% w/v solids loading equivalent of ammonia fiber explosion (AFEX)-pretreated corn stover water extract. However, both KO11 and 424A(LNH-ST) exhibit higher growth robustness than AX101. In 18% w/w solids loading lignocellulosic hydrolysate from AFEX pretreatment, complete glucose fermentations can be achieved at a rate greater than 0.77 g/L/h. In contrast to results from fermentation in CSL, S. cerevisiae 424A(LNH-ST) consumed xylose at the greatest extent and rate in the hydrolysate compared to the bacteria tested.

Conclusions

Our results confirm that glucose fermentations among the tested strains are effective even at high solids loading (18% by weight). However, xylose consumption in the lignocellulosic hydrolysate is the major bottleneck affecting overall yield, titer or rate of the process. In comparison, Saccharomyces cerevisiae 424A(LNH-ST) is the most relevant strains for industrial production for its ability to ferment both glucose and xylose from undetoxified and unsupplemented hydrolysate from AFEX-pretreated corn stover at high yield.     

Improvement of corn stover bioconversion efficiency by using plant glycoside hydrolase

Plant cell wall is the most abundant substrate for bioethanol production, and plants also represent a key resource for glycoside hydrolase (GH). To exploit efficient way for bioethanol production with lower cellulase loading, the potential of plant GH for lignocellulose bioconversion was evaluated. The GH activity for cell wall proteins (CWPs) was detected from fresh corn stover (FCS), and the synergism of which with Trichoderma reesei cellulase was also observed. The properties for the GH of FCS make it a promising enzyme additive for lignocellulose biodegradation. To make use of the plant GH, novel technology for hydrolysis and ethanol fermentation was developed with corn stover as substrate. Taking steam-exploded corn stover as substrate for hydrolysis and ethanol fermentation, compared with T. reesei cellulase loaded alone, the final glucose and ethanol accumulation increased by 60% and 63% respectively with GH of FCS as an addition.     

A Rapid Simultaneous Saccharification and Fermentation (SSF) Technique to Determine Ethanol Yields

We have developed a relatively simple simultaneous saccharification and fermentation (SSF) technique to determine the ethanol production potential for large sets of biomass samples. The technique is based on soaking approximately 0.5 grams of a biomass sample in aqueous ammonia at room temperature and at atmospheric pressure for 24 h, then fermenting with Saccharomyces cerevisiae D₅A for 24 h using Spezyme CP, for enzymatic hydrolysis of structural polysaccharides. We have tested the technique on a set of corn stover samples representing much of the genetic variability in the commercial corn hybrid population. The samples were weighed into modified Ankom filter bags (F57) before soaking to avoid biomass loss during the process. Fermentation samples were analyzed for ethanol after 24 h by HPLC. Percentages of theoretical maximum ethanol yields of the samples ranged between 44.9 and 73%. We observed that percentages of theoretical maximum ethanol yields were highly correlated (r ²?=?0.90) with acid detergent lignin concentration while a low correlation was observed between cellulose concentration and ethanol yield. Near infrared spectra of corn stover samples were also examined. The coefficient of determination (r ²) from regression of predicted versus measured percent theoretical maximum ethanol yield was 0.96. This result suggests that using NIRS is a promising method for predicting ethanol yield, but larger calibration sets are necessary for obtaining improved accuracy for larger sample populations. We conclude that the developed SSF technique could be applied to large numbers of biomass samples to rapidly estimate ethanol yields and to compare different biomass samples in terms of ethanol yields.     

Engineering and Two-Stage Evolution of a Lignocellulosic Hydrolysate-Tolerant Saccharomyces cerevisiae Strain for Anaerobic Fermentation of Xylose from AFEX Pretreated Corn Stover

The inability of the yeast Saccharomyces cerevisiae to ferment xylose effectively under anaerobic conditions is a major barrier to economical production of lignocellulosic biofuels. Although genetic approaches have enabled engineering of S. cerevisiae to convert xylose efficiently into ethanol in defined lab medium, few strains are able to ferment xylose from lignocellulosic hydrolysates in the absence of oxygen. This limited xylose conversion is believed to result from small molecules generated during biomass pretreatment and hydrolysis, which induce cellular stress and impair metabolism. Here, we describe the development of a xylose-fermenting S. cerevisiae strain with tolerance to a range of pretreated and hydrolyzed lignocellulose, including Ammonia Fiber Expansion (AFEX)-pretreated corn stover hydrolysate (ACSH). We genetically engineered a hydrolysate-resistant yeast strain with bacterial xylose isomerase and then applied two separate stages of aerobic and anaerobic directed evolution. The emergent S. cerevisiae strain rapidly converted xylose from lab medium and ACSH to ethanol under strict anaerobic conditions. Metabolomic, genetic and biochemical analyses suggested that a missense mutation in GRE3, which was acquired during the anaerobic evolution, contributed toward improved xylose conversion by reducing intracellular production of xylitol, an inhibitor of xylose isomerase. These results validate our combinatorial approach, which utilized phenotypic strain selection, rational engineering and directed evolution for the generation of a robust S. cerevisiae strain with the ability to ferment xylose anaerobically from ACSH.     

A Novel Wild-Type Saccharomyces cerevisiae Strain TSH1 in Scaling-Up of Solid-State Fermentation of Ethanol from Sweet Sorghum Stalks

The rising demand for bioethanol, the most common alternative to petroleum-derived fuel used worldwide, has encouraged a feedstock shift to non-food crops to reduce the competition for resources between food and energy production. Sweet sorghum has become one of the most promising non-food energy crops because of its high output and strong adaptive ability. However, the means by which sweet sorghum stalks can be cost-effectively utilized for ethanol fermentation in large-scale industrial production and commercialization remains unclear. In this study, we identified a novel Saccharomyces cerevisiae strain, TSH1, from the soil in which sweet sorghum stalks were stored. This strain exhibited excellent ethanol fermentative capacity and ability to withstand stressful solid-state fermentation conditions. Furthermore, we gradually scaled up from a 500-mL flask to a 127-m³ rotary-drum fermenter and eventually constructed a 550-m³ rotary-drum fermentation system to establish an efficient industrial fermentation platform based on TSH1. The batch fermentations were completed in less than 20 hours, with up to 96 tons of crushed sweet sorghum stalks in the 550-m³ fermenter reaching 88% of relative theoretical ethanol yield (RTEY). These results collectively demonstrate that ethanol solid-state fermentation technology can be a highly efficient and low-cost solution for utilizing sweet sorghum, providing a feasible and economical means of developing non-food bioethanol.     

EFFICIENT PRODUCTION OF BIOETHANOL FROM CORN STOVER BY PRETREATMENT WITH A COMBINATION OF SULFURIC ACID AND SODIUM HYDROXIDE

Corn stover is the most abundant agricultural residue in China and a valuable reservoir for bioethanol production. In this study, we proposed a process for producing bioethanol from corn stover; the pretreatment prior to presaccharification, followed by simultaneous saccharification and fermentation (SSF) by using a flocculating Saccharomyces cerevisiae strain, was optimized. Pretreatment with acid–alkali combination (1% H₂SO₄, 150°C, 10 min, followed by 1% NaOH, 80°C, 60 min) resulted in efficient lignin removal and excellent recovery of xylose and glucose. A glucose recovery efficiency of 92.3% was obtained by enzymatic saccharification, when the pretreated solid load was 15%. SSF was carried out at 35°C for 36 hr after presaccharification at 50°C for 24 hr, and an ethanol yield of 88.2% was achieved at a solid load of 15% and an enzyme dosage of 15 FPU/g pretreated corn stover.     

Co-generation of ethanol and <Emphasis Type="SmallCaps">l</Emphasis>-lactic acid from corn stalk under a hybrid process

Background

Corn stover, as one important lignocellulosic material, has characteristics of low price, abundant output and easy availability. Using corn stover as carbon source in the fermentation of valuable organic chemicals contributes to reducing the negative environmental problems and the cost of production. In ethanol fermentation based on the hydrolysate of corn stover, the conversion rate of fermentable sugars is at a low level because the native S. cerevisiae does not utilize xylose. In order to increase the conversion rate of fermentable sugars deriving from corn stover, an effective and energy saving biochemical process was developed in this study and the residual xylose after ethanol fermentation was further converted to l-lactic acid.

Results

In the hybrid process based on the hydrolysate of corn stover, the ethanol concentration and productivity reached 50.50 g L^?1 and 1.84 g L^?1 h^?1, respectively, and the yield of ethanol was 0.46 g g^?1. The following fermentation of l-lactic acid provided a product titer of 21.50 g L^?1 with a productivity of 2.08 g L^?1 h^?1, and the yield of l-lactic acid was 0.76 g g^?1. By adopting a blank aeration before the inoculation of B. coagulans LA1507 and reducing the final cell density, the l-lactic acid titer and yield reached 24.25 g L^?1 and 0.86 g g^?1, respectively, with a productivity of 1.96 g L^?1 h^?1.

Conclusions

In this work, the air pumped into the fermentor was used as both the carrier gas for single-pass gas stripping of ethanol and the oxygen provider for the aerobic growth of B. coagulans LA1507. Ethanol was effectively separated from the fermentation broth, while the residual medium containing xylose was reused for l-lactic acid production. As an energy-saving and environmental-friendly process, it introduced a potential way to produce bioproducts under the concept of biorefinery, while making full use of the hydrolysate of corn stover.

     

Industrial yeast strain engineered to ferment ethanol from lignocellulosic biomass

In this study an industrial Saccharomyces cerevisiae yeast strain capable of fermenting ethanol from pretreated lignocellulosic material was engineered. Genes encoding cellulases (endoglucanase, exoglucanase and β-glucosidase) were integrated into the chromosomal ribosomal DNA and delta regions of a derivative of the K1-V1116 wine yeast strain. The engineered cellulolytic yeast produces ethanol in one step through simultaneous saccharification and fermentation of pretreated biomass without the addition of exogenously produced enzymes. When ethanol fermentation was performed with 10% dry weight of pretreated corn stover, the recombinant strain fermented 63% of the cellulose in 96 h and the ethanol titer reached 2.6% v/v. These results demonstrate that cellulolytic S. cerevisiae strains can be used as a platform for developing an economical advanced biofuel process.     

Ethanol production from galactose by a newly isolated Saccharomyces cerevisiae KL17

A wild-type yeast strain with a good galactose-utilization efficiency was newly isolated from the soil, and identified and named Saccharomyces cerevisiae KL17 by 18s RNA sequencing. Its performance of producing ethanol from galactose was investigated in flask cultures with media containing various combination and concentrations of galactose and glucose. When the initial galactose concentration was 20 g/L, it showed 2.2 g/L/h of substrate consumption rate and 0.63 g/L/h of ethanol productivity. Although they were about 70 % of those with glucose, such performance of S. cerevisiae KL17 with galactose was considered to be quite high compared with other strains reported to date. Its additional merit was that its galactose metabolism was not repressed by the existence of glucose. Its capability of ethanol production under a high ethanol concentration was demonstrated by fed-batch fermentation in a bioreactor. A high ethanol productivity of 3.03 g/L/h was obtained with an ethanol concentration and yield of 95 and 0.39 g/L, respectively, when the cells were pre-cultured on glucose. When the cells were pre-cultured on galactose instead of glucose, fermentation time could be reduced significantly, resulting in an improved ethanol productivity of 3.46 g/L/h. The inhibitory effects of two major impurities in a crude galactose solution obtained from acid hydrolysis of galactan were assessed. Only 5-Hydroxymethylfurfural (5-HMF) significantly inhibited ethanol fermentation, while levulinic acid (LA) was benign in the range up to 10 g/L.     

Evaluation of continuous ethanol fermentation of dilute-acid corn stover hydrolysate using thermophilic anaerobic bacterium <Emphasis Type="Italic">Thermoanaerobacter</Emphasis> BG1L1

Dilute sulfuric acid pretreated corn stover is potential feedstock of industrial interest for second generation fuel ethanol production. However, the toxicity of corn stover hydrolysate (PCS) has been a challenge for fermentation by recombinant xylose fermenting organisms. In this work, the thermophilic anaerobic bacterial strain Thermoanaerobacter BG1L1 was assessed for its ability to ferment undetoxified PCS hydrolysate in a continuous immobilized reactor system at 70°C. The tested strain showed significant resistance to PCS, and substrate concentrations up to 15% total solids (TS) were fermented yielding ethanol of 0.39–0.42 g/g-sugars consumed. Xylose was nearly completely utilized (89–98%) for PCS up to 10% TS, whereas at 15% TS, xylose conversion was lowered to 67%. The reactor was operated continuously for 135 days, and no contamination was seen without the use of any agent for preventing bacterial infections. This study demonstrated that the use of immobilized thermophilic anaerobic bacteria for continuous ethanol fermentation could be promising in a commercial ethanol process in terms of system stability to process hardiness and reactor contamination. The tested microorganism has considerable potential to be a novel candidate for lignocellulose bioconversion into ethanol.     

Lactic Acid Production from Pretreated Hydrolysates of Corn Stover by a Newly Developed Bacillus coagulans Strain

An inhibitor-tolerance strain, Bacillus coagulans GKN316, was developed through atmospheric and room temperature plasma (ARTP) mutation and evolution experiment in condensed dilute-acid hydrolysate (CDH) of corn stover. The fermentabilities of other hydrolysates with B. coagulans GKN316 and the parental strain B. coagulans NL01 were assessed. When using condensed acid-catalyzed steam-exploded hydrolysate (CASEH), condensed acid-catalyzed liquid hot water hydrolysate (CALH) and condensed acid-catalyzed sulfite hydrolysate (CASH) as substrates, the concentration of lactic acid reached 45.39, 16.83, and 18.71 g/L by B. coagulans GKN316, respectively. But for B. coagulans NL01, only CASEH could be directly fermented to produce 15.47 g/L lactic acid. The individual inhibitory effect of furfural, 5-hydroxymethylfurfural (HMF), vanillin, syringaldehyde and p-hydroxybenzaldehyde (pHBal) on xylose utilization by B. coagulans GKN316 was also studied. The strain B. coagulans GKN316 could effectively convert these toxic inhibitors to the less toxic corresponding alcohols in situ. These results suggested that B. coagulans GKN316 was well suited to production of lactic acid from undetoxified lignocellulosic hydrolysates.     

Cause analysis of the effects of acid-catalyzed steam-exploded corn stover prehydrolyzate on ethanol fermentation by Pichia stipitis CBS 5776

The prehydrolyzate obtained from acid-catalyzed steam-exploded corn stover (ASC) mainly contains xylose and a number of inhibitory compounds that inhibit ethanol fermentation by Pichia stipitis. In this study, the effects of the ASC prehydrolyzate, specifically those of the carbohydrate-degradation products, lignin-degradation products (which were extracted from ASC prehydrolyzate using ethyl acetate), and six major phenolic compounds (added to pure-sugar media individually or in combination), on ethanol fermentation were investigated. Results indicate that the effects of the carbohydrate-degradation products were negligible (10 h delayed) compared with those of pure-sugar fermentation, whereas the effects of the lignin-degradation products were significant (52 h delayed). Meanwhile, the inhibitory effects of the major phenolic compounds were not caused by certain types of inhibitors, but were due to the synergistic effects of various inhibitors.     

Evaluation of Saccharomyces cerevisiae Y5 for ethanol production from enzymatic hydrolysate of non-detoxified steam-exploded corn stover

Saccharomyces cerevisiae Y5 was used to produce ethanol from enzymatic hydrolysate of non-detoxified steam-exploded corn stover, with and without a nitrogen source, and decreasing inoculum size. The results indicated that the ethanol concentration of 44.55 g/L, corresponding to 94.5% of the theoretical yield was obtained after 24 h, with an inoculum size of 10% (v/v) and nitrogen source (corn steep liquor, CSL) of 40 mL/L. With the same inoculum size, and without CSL, the ethanol concentration was 43.21 g/L, corresponding to 91.7% of the theoretical value after 60 h. With a decreased inoculum size of 5% (v/v), and without CSL, the ethanol concentration was 40.00 g/L, corresponding to 85.8% of the theoretical value after 72 h. The strain offers the potential to improve the economy of cellulosic ethanol production by simplifying the production process and reducing the costs associated with the process such as water, capital equipment and nutrient supplementation.     

He-Ne laser irradiation of folate-producing Candida utilis and optimization of culture conditions under submerged fermentation

In an attempt to obtain a microbial strain with higher yield of folate for industrial applications, we mutated the wild strain Candida utilis Y1.0 using a novel mutagenic process, i.e., irradiation by a helium–neon (He-Ne) laser with an output power of 20 mW and an exposure time of 20 min. The yield of folate in the mutated cells reached 1,102 ng/mL, which was 20.4-fold that of the wild strain. The mutant strain Y3.636 was relatively stable in terms of folate production through eight successive transfers of cultures and batch fermentation in a 3.7-L stirred-tank fermenter. Optimization further increased the yield of the mutant by 110 %, i.e., to 2,314?±?13 ng/mL. The optimal culture conditions for folate production were: cultivation in fermentation culture medium composed of 62.5 g/L glucose, 15 g/L corn liquor, 3 g/L (NH₄)₂SO₄, 3 g/L MgSO₄, and 1 g/L glutamic acid; inoculum size of 9 %; incubation at 28 °C and 196 rpm for 36 h. A time-course study of cell growth and folate production by mutant strain Y3.636 strongly suggested that folate production in C. utilis is growth-associated.