Effect of pH and lactic or acetic acid on ethanol productivity by Saccharomyces cerevisiae in corn mash

The effects of lactic and acetic acids on ethanol production by Saccharomyces cerevisiae in corn mash, as influenced by pH and dissolved solids concentration, were examined. The lactic and acetic acid concentrations utilized were 0, 0.5, 1.0, 2.0, 3.0 and 4.0% w/v, and 0, 0.1, 0.2, 0.4, 0.8 and 1.6% w/v, respectively. Corn mashes (20, 25 and 30% dry solids) were adjusted to the following pH levels after lactic or acetic acid addition: 4.0, 4.5, 5.0 or 5.5 prior to yeast inoculation. Lactic acid did not completely inhibit ethanol production by the yeast. However, lactic acid at 4% w/v decreased (P<0.05) final ethanol concentration in all mashes at all pH levels. In 30% solids mash set at pH ≤5, lactic acid at 3% w/v reduced (P<0.05) ethanol production. In contrast, inhibition by acetic acid increased as the concentration of solids in the mash increased and the pH of the medium declined. Ethanol production was completely inhibited in all mashes set at pH 4 in the presence of acetic acid at concentrations ≥0.8% w/v. In 30% solids mash set at pH 4, final ethanol levels decreased (P<0.01) with only 0.1% w/v acetic acid. These results suggest that the inhibitory effects of lactic acid and acetic acid on ethanol production in corn mash fermentation when set at a pH of 5.0–5.5 are not as great as that reported thus far using laboratory media.     

Effects of acetic acid and lactic acid on the growth of Saccharomyces cerevisiae in a minimal medium

Specific growth rates (μ) of two strains of Saccharomyces cerevisiae decreased exponentially (R ²>0.9) as the concentrations of acetic acid or lactic acid were increased in minimal media at 30°C. Moreover, the length of the lag phase of each growth curve (h) increased exponentially as increasing concentrations of acetic or lactic acid were added to the media. The minimum inhibitory concentration (MIC) of acetic acid for yeast growth was 0.6% w/v (100 mM) and that of lactic acid was 2.5% w/v (278 mM) for both strains of yeast. However, acetic acid at concentrations as low as 0.05–0.1% w/v and lactic acid at concentrations of 0.2–0.8% w/v begin to stress the yeasts as seen by reduced growth rates and decreased rates of glucose consumption and ethanol production as the concentration of acetic or lactic acid in the media was raised. In the presence of increasing acetic acid, all the glucose in the medium was eventually consumed even though the rates of consumption differed. However, this was not observed in the presence of increasing lactic acid where glucose consumption was extremely protracted even at a concentration of 0.6% w/v (66 mM). A response surface central composite design was used to evaluate the interaction between acetic and lactic acids on the specific growth rate of both yeast strains at 30C. The data were analysed using the General Linear Models (GLM) procedure. From the analysis, the interaction between acetic acid and lactic acid was statistically significant (P≤0.001), i.e., the inhibitory effect of the two acids present together in a medium is highly synergistic. Journal of Industrial Microbiology & Biotechnology (2001) 26, 171–177. Received 06 June 2000/ Accepted in revised form 21 September 2000     

Buffering capacity of whole corn mash alters concentrations of organic acids required to inhibit growth of Saccharomyces cerevisiae and ethanol production

Growth of Saccharomyces cerevisiae and fermentative ethanol production in the presence of acetic and lactic acids was measured in whole corn mash. In this industrial medium, as compared to glucose minimal medium, the yeast had increased tolerance to organic acid stress. It was concluded that the increased buffering capacity of whole corn mash, resulting in decreased concentration of undissociated acid, was responsible for this phenomenon.     

Anaerobic fermentative production of lactic acid using cheese whey and corn steep liquor

Cheese whey was the most suitable substrate for production of lactic acid under anaerobic conditions by Entercoccus flavescens which, on supplementating with corn steep liquor (5% v/v) and 10 mM CaCO₃ at pH 5.5, 37°C, yielded 12.6 g lactic acid/l in 36 h. Production was scaled up to a 10 l bioreactor under controlled pH and continuous CO₂ supply and gave 28 g lactic acid/l in 30 h resulting in a net 8.7-fold increase in production as compared to unoptimized conditions.     

Effect of yeast inoculation rate on the metabolism of contaminating lactobacilli during fermentation of corn mash

Two separate 4 (bacterial concentrations)×6 (yeast concentrations) full factorial experiments were conducted in an attempt to identify a novel approach to minimize the effects caused by bacterial contamination during industrial production of ethanol from corn. Lactobacillus plantarum and Lactobacillus paracasei, commonly occurring bacterial contaminants in ethanol plants, were used in separate fermentation experiments conducted in duplicate using an industrial strain of Saccharomyces cerevisiae, Allyeast Superstart. Bacterial concentrations were 0, 1×10⁶, 1×10⁷ and 1×10⁸ cells/ml mash. Yeast concentrations were 0, 1×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, and 4×10⁷ cells/ml mash. An increased yeast inoculation rate of 3×10⁷ cells/ml resulted in a greater than 80% decrease (P<0.001) and a greater than 55% decrease (P<0.001) in lactic acid production by L. plantarum and L. paracasei, respectively, when mash was infected with 1×10⁸ lactobacilli/ml. No differences (P>0.25) were observed in the final ethanol concentration produced by yeast at any of the inoculation rates studied, in the absence of lactobacilli. However, when the mash was infected with 1×10⁷ or 1×10⁸ lactobacilli/ml, a reduction of 0.7–0.9% v/v (P<0.005) and a reduction of 0.4–0.6% v/v (P<0.005) in the final ethanol produced was observed in mashes inoculated with 1×10⁶ and 1×10⁷ yeast cells/ml, respectively. At higher yeast inoculation rates of 3×10⁷ or 4×10⁷ cells/ml, no differences (P>0.35) were observed in the final ethanol produced even when the mash was infected with 1×10⁸ lactobacilli/ml. The increase in ethanol corresponded to the reduction in lactic acid production by lactobacilli. This suggests that using an inoculation rate of 3×10⁷ yeast cells/ml reduces the growth and metabolism of contaminating lactic bacteria significantly, which results in reduced lactic acid production and a concomitant increase in ethanol production by yeast.     

Isolation and Identification of Acetic Acid Bacteria for Submerged Acetic Acid Fermentation at High Temperature

Industrial vinegar production by submerged acetic acid fermentation has been carried out using Acetobacter strains at about 30°C. To obtain strains suitable for acetic acid fermentation at higher temperature, about 1,100 strains of acetic acid bacteria were isolated from vinegar mash, soils in vinegar factories and fruits, and their activities to oxidize ethanol at high temperature were examined. One of these strains, No. 1023, identified as Acetobacter aceti, retained full activity to produce acetic acid in continuous submerged culture at 35°C and produced 45% of activity at 38°C, while the usual strain of A. aceti completely lost its activity at 35°C. Thus the use of this strain may reduce the cooling costs of industrial vinegar production.     

Improvement of robustness and ethanol production of ethanologenic Saccharomyces cerevisiae under co-stress of heat and inhibitors

Bioethanol is an attractive alternative to fossil fuels. Saccharomyces cerevisiae is the most important ethanol producer. However, yeast cells are challenged by various environmental stresses during the industrial process of ethanol production. The robustness under heat, acetic acid, and furfural stresses was improved for ethanologenic S. cerevisiae in this work using genome shuffling. Recombinant yeast strain R32 could grow at 45°C, and resist 0.55% (v/v) acetic acid and 0.3% (v/v) furfural at 40°C. When ethanol fermentation was conducted at temperatures ranging from 30 to 42°C, recombinant strain R32 always gave high ethanol production. After 42 h of fermentation at 42°C, 187.6 ± 1.4 g/l glucose was utilized by recombinant strain R32 to produce 81.4 ± 2.7 g/l ethanol, which were respectively 3.4 and 4.1 times those of CE25. After 36 h of fermentation at 40°C with 0.5% (v/v) acetic acid, 194.4 ± 1.2 g/l glucose in the medium was utilized by recombinant strain R32 to produce 84.2 ± 4.6 g/l of ethanol. The extent of glucose utilization and ethanol concentration of recombinant strain R32 were 6.3 and 7.9 times those of strain CE25. The ethanol concentration produced by recombinant strain R32 was 8.9 times that of strain CE25 after fermentation for 48 h under 0.2% (v/v) furfural stress at 40°C. The strong physiological robustness and fitness of yeast strain R32 support its potential application for industrial production of bioethanol from renewable resources such as lignocelluloses.     

Acetic acid production by Dekkera/Brettanomyces yeasts

Yeast belonging to the genera Brettanomyces and Dekkera are noted for spoiling cellar and bottled wine through the production of haze, turbidity and acetic acid. However, I was unable to find information on the use of these yeasts for the expressed purpose of acetic acid production. Sixty yeast strains belonging to these, and several other genera, from the ARS Culture Collection, Peoria, IL, were screened for their ability to produce both ethanol and/or acetic acid. For ethanol production, the strains were grown anaerobically at 24 and 30 °C in batch culture using glucose (100 g/l) as the carbon/energy source. For acetic acid production, the strains were grown aerobically in batch culture using either glucose (100 g/l) or ethanol (35 g/l) as the carbon/energy source. In the initial ethanol production screen, 19 strains produced at least 45 g ethanol/l. In the initial acetic acid screen, 28 of the yeast strains produced at least 5 g acetic acid/l from 100 g glucose/l, while 23 strains produced at least 5 g acetic acid/l from 35 g ethanol/l.     

Production of acetic acid by Dekkera/Brettanomyces yeasts under conditions of constant pH

Sixty yeast strains were previously screened for their ability to produce acetic acid, in shaken flask batch culture, from either glucose or ethanol. Seven of the strains belonging to the Brettanomyces and Dekkera genera, from the ARS Culture Collection, Peoria, IL, were further evaluated for acetic acid production in bioreactor batch culture at 28 °C, constant aeration (0.75 v/v/m) and pH (6.5). The medium contained either 100 g glucose/l or 35 g ethanol/l as the carbon/energy source. Dekkera intermedia NRRL YB-4553 produced 42.8 and 14.9 g acetic acid/l from the two carbon sources, respectively, after 64.5 h. The optimal pH was determined to be 5.5. When the initial glucose concentration was 150 or 200 g/l, the yeast produced 57.5 and 65.1 g acetic acid/l, respectively.     

Production of propionic acid by mixed cultures ofPropionibacterium shermanii andLactobacillus casei in autoclave-sterilized whey

Summary Pure cultures ofPropionibacterium freudenreichii ss.shermanii did not grow in autoclave-sterilized cheese whey (121°C, 15 psi, 20 min) at whey concentrations greater than 2% (w/v) spray-dried sweet dairy whey. Propionic acid was produced from autoclave-sterilized whey by growingP. shermanii in mixed culture withLactobacillus casei. In medium containing 5–12% autoclaved whey solids and 1% yeast extract, the mixed culture produced 1.3–3.0% propionic acid, 0.5–1.0% acetic acid, and 0.05–0.80% lactic acid. All the lactose was consumed. Using pH-controlled fermentors (pH=7.0), mixed cultures produced at least 30% more propionic acid than cultures in which pH was not controlled.     

Growth rates of Dekkera/Brettanomyces yeasts hinder their ability to compete with Saccharomyces cerevisiae in batch corn mash fermentations

Growth rates determined by linear regression analysis revealed that Saccharomyces cerevisiae consistently grew more rapidly than Brettanomyces yeasts under a wide array of batch fermentative conditions, including acetic acid stress, in normal gravity (ca. 20°Plato) mashes made from ground corn. Brettanomyces yeasts only grew more rapidly than S. cerevisiae when acetic acid concentrations were elevated to industrially irrelevant levels (>0.45%, w/v). Furthermore, the three Brettanomyces isolates used in this study failed to produce significant quantities of acetic acid under pure culture fermentative conditions. In fact, the small amounts of acetic acid which accumulated in pure culture fermentations of whole corn mash were below the concentration required to inhibit the growth and metabolism of S. cerevisiae. Acetic acid concentrations in pure culture Brettanomyces fermentations exceeded 0.05% (w/v) only in media containing low levels of glucose (<4%, w/v) or when aeration rates were elevated to at least 0.03 vol. air vol.^–1 mash min^–1. Consequently, it was concluded that Brettanomyces yeasts would not be capable of competing with S. cerevisiae in industrial batch fermentations of whole corn mash based solely on growth rates, nor would they be capable of producing inhibitory concentrations of acetic acid in such fermentations.     

Fermentative production of<Emphasis Type="SmallCaps"> l</Emphasis>-(+)-lactic acid by an alkaliphilic marine microorganism

Of six strains of lactic acid-producing alkaliphilic microorganisms, Halolactibacillus halophilus was most efficient. It produced the highest concentration and yield of lactic acid, with minimal amounts of acetic and formic acid when sucrose and glucose were used as substrate. Mannose and xylose were poorly utilized. In batch fermentation at 30°C, pH 9 with 4 and 8% (w/v) sucrose, lactic acid was produced at 37.7 and 65.8 g l⁻¹, with yields of 95 and 83%, respectively. Likewise, when 4 and 8% (w/v) glucose were used, 33.4 and 59.6 g lactic acid l⁻¹ were produced with 85 and 76% yields, respectively. l-(+)-lactic acid had an optical purity of 98.8% (from sucrose) and 98.3% (from glucose).     

一株耐受低pH、高浓度乳酸及琥珀酸菌株的筛选与鉴定

【目的】通过构建的人工耐酸系统,筛选耐受低pH值、乳酸及琥珀酸的菌株。【方法】构建人工耐酸系统长期驯化菌株,利用不同p H的酸性平板进行筛选,从环境中筛选出一株对低p H值、高浓度乳酸以及琥珀酸有很好耐受性的菌株。通过形态学特征、生理生化特征研究,并结合18S rDNA基因序列分析及分子系统发育树的构建结果,确定菌株的种类。【结果】经过酸性人工系统的长期驯化,筛选分离出一株耐受低pH值、高浓度乳酸以及琥珀酸的菌株WJ-2,经鉴定该菌株为酿酒酵母(Saccharomyces cerevisiae),其最适生长温度为30°C。酸性平板实验显示该菌株能够耐受pH2.5的酸性环境,同时对9%的乳酸及8%的琥珀酸也有很好的耐受性。另外,耐酸菌株WJ-2在pH 2.5、9%乳酸和8%琥珀酸的培养环境中仍能保持相对中性的细胞内pH值。【结论】通过构建人工酸性系统,成功筛选出一株对低pH值、高浓度乳酸以及琥珀酸具有耐受性的菌株——酿酒酵母菌WJ-2,该方法可为筛选具有特定耐受能力菌株提供一个新思路。     

Acid hydrolysis of sugarcane bagasse for lactic acid production

In order to use sugarcane bagasse as a substrate for lactic acid production, optimum conditions for acid hydrolysis of the bagasse were investigated. After lignin extraction, the conditions were varied in terms of hydrochloric (HCl) or sulfuric (H₂SO₄) concentration (0.5–5%, v/v), reaction time (1–5 h) and incubation temperature (90–120 °C). The maximum catalytic efficiency (E) was 10.85 under the conditions of 0.5% of HCl at 100 °C for 5 h, which the main components (in g l⁻¹) in the hydrolysate were glucose, 1.50; xylose, 22.59; arabinose, 1.29; acetic acid, 0.15 and furfural, 1.19. To increase yield of lactic acid production from the hydrolysate by Lactococcus lactis IO-1, the hydrolysate was detoxified through amberlite and supplemented with 7 g l⁻¹ of xylose and 7 g l⁻¹ of yeast extract. The main products (in g l⁻¹) of the fermentation were lactic acid, 10.85; acetic acid, 7.87; formic acid, 6.04 and ethanol, 5.24.     

Production of Extracellular Acid Proteases by Aspergillus Clavatus

The production of extracellular acid proteases from Aspergillus clavatus was evaluated in a culture filtrate medium, with different carbon and nitrogen sources. The fungus was cultivated at three different temperatures during 10 days. The proteolytic activity was determined on haemoglobin pH 5.0 at 37 °C. The highest acid proteolytic activity (80 U/ml) was observed in culture medium containing glucose and gelatin at 1%(w/v) at 30 °C at the third day of incubation. Cultures developed in Vogel medium with glucose at 2%(w/v) showed at about 45% of proteolytic activity when compared to the cultures with 1% of the same sugar. The optimum pH of enzymatic activity was 2.0 and the enzyme was stable at pH values ranging from 2.0 to 4.0. The optimum temperature was 40 °C and the half-lives at 40, 45 and 50 °C were 30, 10 and 5 min, respectively. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genome shuffling to improve thermotolerance,ethanol tolerance and ethanol productivity of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Genome shuffling is a powerful strategy for rapid engineering of microbial strains for desirable industrial phenotypes. Here we improved the thermotolerance and ethanol tolerance of an industrial yeast strain SM-3 by genome shuffling while simultaneously enhancing the ethanol productivity. The starting population was generated by protoplast ultraviolet irradiation and then subjected for the recursive protoplast fusion. The positive colonies from the library, created by fusing the inactivated protoplasts were screened for growth at 35, 40, 45, 50 and 55°C on YPD-agar plates containing different concentrations of ethanol. Characterization of all mutants and wild-type strain in the shake-flask indicated the compatibility of three phenotypes of thermotolerance, ethanol tolerance and ethanol yields enhancement. After three rounds of genome shuffling, the best performing strain, F34, which could grow on plate cultures up to 55°C, was obtained. It was found capable of completely utilizing 20% (w/v) glucose at 45–48°C, producing 9.95% (w/v) ethanol, and tolerating 25% (v/v) ethanol stress.     

耐乙酸乳杆菌的筛选及其产乳酸特性

【背景】耐受乙酸的乳酸菌是传统谷物醋醋酸发酵过程中产生乳酸及其风味衍生物的重要功能微生物。【目的】从镇江香醋醋醅中分离鉴定具有耐乙酸特性的乳酸菌,并评价不同条件下该菌株的产乳酸能力。【方法】利用4%(体积比)乙酸含量的MRS培养基分离耐乙酸乳酸菌;对其进行16S rRNA基因鉴定、基因组测序、形态观察以及生理生化特性研究;考察不同乙酸浓度、葡萄糖浓度、发酵温度和时间对菌株产乳酸能力的影响。【结果】分离得到一株可耐受6%乙酸的乳杆菌Lactobacillus sp. JN500903;在厌氧静置、接种量5%、乙酸浓度5%、葡萄糖浓度40 g/L、发酵温度37°C、发酵时间10 d条件下,该菌株乳酸产量为16.1 g/L。【结论】乳杆菌JN500903能够耐受6%乙酸浓度,具有在酸性环境下合成乳酸的能力,有一定的应用潜力。     

Production of lactic acid and byproducts from waste potato starch by <Emphasis Type="Italic">Rhizopus arrhizus</Emphasis>: role of nitrogen sources

Summary Lactic acid was produced by Rhizopus arrhizus using waste potato starch as the substrate. The aim of this study was to identify the role of nitrogen sources and their impact on the formation of lactic acid and associated byproducts. Ammonium sulphate, ammonium nitrate, urea, yeast extract and peptone were assessed in conjunction with various ratios of carbon to nitrogen (C:N). Fermentation media with a low C:N ratio enhanced the production of lactic acid, biomass and ethanol, while a high C:N ratio favoured the production of fumaric acid. Ammonium nitrate appeared to be the most suitable nitrogen source for achieving a high and stable lactic acid yield, and minimizing the production of byproducts such as biomass and ethanol, while urea proved to be the least favourable nitrogen source. Yeast extract and peptone appeared to improve fungal cell growth. The kinetics data revealed that a high concentration of ammonium nitrate enhanced the lactic acid productivity. The maximum lactic acid concentration of 36.4 g/l, representing a yield of 91%, was obtained with addition of 0.909 g/l ammonium nitrate in 32 h.     

Effect of salt nutrients on mannitol production by Lactobacillus intermedius NRRL B-3693

The effects of four salt nutrients (ammonium citrate, sodium phosphate, magnesium sulfate, and manganese sulfate) on the production of mannitol by Lactobacillus intermedius NRRL B-3693 in a simplified medium containing 300 g fructose, 5 g soy peptone, and 50 g corn steep liquor per liter in pH-controlled fermentation at 5.0 at 37°C were evaluated using a fractional factorial design. Only manganese sulfate was found to be essential for mannitol production. Added manganese sulfate concentration of 0.033 g/l was found to support maximum production. The bacterium produced 200.6±0.2 g mannitol, 61.9±0.1 g lactic acid, and 40.4±0.3 g acetic acid from 300 g fructose per liter in 67 h.Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.