Long-term repeated fed-batch ethanol fermentation in aerated condition

In this study, we attempted to assess the process stability of long-term fed-batch ethanol fermentation in the absence and presence of aeration (0.33 vvm). To examine the effect of aeration, a long-term repeated fed-batch operation was conducted for 396 h to mimic a long-term industrial bioethanol production process. In this long-term repeated fed-batch ethanol fermentation experiments, withdrawal-fill operation were conducted every 36 h for 10 repeat cycles. The whole operation was stably sustained in a quasi-steady state. The average maximal cell concentration and the average maximal ethanol production during operation were increased by 81.63 and 12.12%, respectively, when aeration was used. In addition, since aeration was carried out, the average ethanol yield slightly decreased by 4.03% and the average specific ethanol production rate decreased by 46.75% during operation. However, the average ethanol productivity increased by 17.53% when aeration was carried out. After 396 h of long-term repeated fed-batch ethanol fermentation, 1,908.9 g of ethanol was cumulatively produced when aeration was used, which was 12.47%, higher than when aeration was not used (1,697.2 g). Meanwhile, glycerol production was greatly decreased during long-term repeated fed-batch ethanol fermentation, in which the glycerol concentration in the culture broth decreased from about 34∼15 g/L. Thus, we can conclude that cell growth was greatly improved by overcoming ethanol inhibition and glycerol production was remarkably decreased when aeration was carried out, although aeration in ethanol fermentation decreased the specific ethanol production rate and ethanol yield.     

Screening for yeast with antibacterial properties from an ethanol distillery

A general screening for the expression of antibacterial activity and non-flocculating type of yeast strains from must and fermented broth of alcohol distilleries was performed. From 60 strains only Saccharomyces sp. M26 presented a inhibitory halo in Lactobacillus fermentum culture and significant reduction in the culture turbidity (71%) and specific growth rate (56%) when compared to the control. Freezing did not affect the antibacterial activity of the Saccharomyces sp. M26 extract and heating at 90 degrees C for 20 min completely destroyed this activity. It is expected the decrease of lactic acid bacteria growth in the S. cerevisiae alcoholic fermentation should allow for better control of these bacteria in the process.     

Highly efficient bioethanol production by a Saccharomyces cerevisiae strain with multiple stress tolerance to high temperature, acid and ethanol

Use of super strains exhibiting tolerance to high temperature, acidity and ethanol is a promising way to make ethanol production economically feasible. We describe here the breeding and performance of such a multiple-tolerant strain of Saccharomyces cerevisiae generated by a spore-to-cell hybridization technique without recombinant DNA technology. A heterothallic strain showing a high-temperature (41°C) tolerant (Htg(+)) phenotype, a derivative from a strain isolated from nature, was crossed with a homothallic strain displaying high-ethanol productivity (Hep(+)), a stock culture at the Thailand Institute of Scientific and Technological Research. The resultant hybrid TJ14 displayed ability to rapidly utilize glucose, and produced ethanol (46.6g/l) from 10% glucose fermentation medium at high temperature (41°C). Not only ethanol productivity at 41°C but also acid tolerance (Acd(+)) was improved in TJ14 as compared with its parental strains, enabling TJ14 to grow in liquid medium even at pH 3. TJ14 maintained high ethanol productivity (46.0g/l) from 10% glucose when fermentation was done under multiple-stress conditions (41°C and pH 3.5). Furthermore, when TJ14 was subjected to a repeated-batch fermentation scheme, the growth and ethanol production of TJ14 were maintained at excellent levels over ten cycles of fermentation. Thus, the multiple-stress (Htg(+) Hep(+) Acd(+)) resistant strain TJ14 should be useful for cost-effective bioethanol production under high-temperature and acidic conditions.     

Use of mixed cultures for the fermentation of cereal-based substrates with potential probiotic properties

The production of a new cereal-based probiotic foods with suitable aroma, flavor and pH using mixed culture fermentation has been investigated. This required the selection of suitable types of cereal grains and probiotic microorganisms. In a medium of 5% (w/v) malt suspension the effects of yeast presence on the fermentation of a lactic acid bacterium (LAB), Lactobacillus reuteri, was studied. With different inoculum ratios between the yeast and the LAB, the characteristics of the fermentation broth including pH and the contents of free amino nitrogen (FAN), reducing sugar, lactic acid and ethanol were investigated. It was found that LAB growth was enhanced by the introduction of the yeast. In mixed culture broth pH was lowered and the production of lactic acid and ethanol were increased in comparison against pure LAB culture.     

Yeasts in sustainable bioethanol production: A review

Bioethanol has been identified as the mostly used biofuel worldwide since it significantly contributes to the reduction of crude oil consumption and environmental pollution. It can be produced from various types of feedstocks such as sucrose, starch, lignocellulosic and algal biomass through fermentation process by microorganisms. Compared to other types of microoganisms, yeasts especially Saccharomyces cerevisiae is the common microbes employed in ethanol production due to its high ethanol productivity, high ethanol tolerance and ability of fermenting wide range of sugars. However, there are some challenges in yeast fermentation which inhibit ethanol production such as high temperature, high ethanol concentration and the ability to ferment pentose sugars. Various types of yeast strains have been used in fermentation for ethanol production including hybrid, recombinant and wild-type yeasts. Yeasts can directly ferment simple sugars into ethanol while other type of feedstocks must be converted to fermentable sugars before it can be fermented to ethanol. The common processes involves in ethanol production are pretreatment, hydrolysis and fermentation. Production of bioethanol during fermentation depends on several factors such as temperature, sugar concentration, pH, fermentation time, agitation rate, and inoculum size. The efficiency and productivity of ethanol can be enhanced by immobilizing the yeast cells. This review highlights the different types of yeast strains, fermentation process, factors affecting bioethanol production and immobilization of yeasts for better bioethanol production.     

Characterization of the impact of acetate and lactate on ethanolic fermentation by Thermoanaerobacter ethanolicus

Ethanolic fermentation of simple sugars is an important step in the production of bioethanol as a renewable fuel. Significant levels of organic acids, which are generally considered inhibitory to microbial metabolism, could be accumulated during ethanolic fermentation, either as a fermentation product or as a by-product generated from pre-treatment steps. To study the impact of elevated concentrations of organic acids on ethanol production, varying levels of exogenous acetate or lactate were added into cultures of Thermoanaerobacter ethanolicus strain 39E with glucose, xylose or cellobiose as the sole fermentation substrate. Our results found that lactate was in general inhibitory to ethanolic fermentation by strain 39E. However, the addition of acetate showed an unexpected stimulatory effect on ethanolic fermentation of sugars by strain 39E, enhancing ethanol production by up to 394%. Similar stimulatory effects of acetate were also evident in two other ethanologens tested, T. ethanolicus X514, and Clostridium thermocellum ATCC 27405, suggesting the potentially broad occurrence of acetate stimulation of ethanolic fermentation. Analysis of fermentation end product profiles further indicated that the uptake of exogenous acetate as a carbon source might contribute to the improved ethanol yield when 0.1% (w/v) yeast extract was added as a nutrient supplement. In contrast, when yeast extract was omitted, increases in sugar utilization appeared to be the likely cause of higher ethanol yields, suggesting that the characteristics of acetate stimulation were growth condition-dependent. Further understanding of the physiological and metabolic basis of the acetate stimulation effect is warranted for its potential application in improving bioethanol fermentation processes.     

Characteristics of some lactic Acid bacteria used as starter cultures in dry sausage production

The fermentation characteristics of two commercial (Duploferment 66 and Saga II) and five Norwegian lactic acid bacteria used in dry sausage production were compared with those of Lactobacillus plantarum ATCC 8014. The Norwegian strains lacked the ability to ferment mannitol, sorbitol, lactose, and d-(+)-raffinose and grew at 8 but not at 42 degrees C, in contrast to the ATCC culture and the two commercial strains. The lactate dehydrogenase activity of the Norwegian strains was not stimulated by pyruvate. All strains were capable of peroxide destruction when grown in the presence of myoglobin.     

Enhanced production of bioethanol from waste of beer fermentation broth at high temperature through consecutive batch strategy by simultaneous saccharification and fermentation

Malt hydrolyzing enzymes and yeast glycolytic and fermentation enzymes in the waste from beer fermentation broth (WBFB) were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A new ‘one-pot consecutive batch strategy’ was developed for efficient bio-ethanol production by simultaneous saccharification and fermentation (SSF) using WBFB without additional enzymes, microbial cells, or carbohydrates. Bio-ethanol production was conducted in batches using WBFB supernatant in the first phase at 25–67 °C and 50 rpm, followed by the addition of 3% WBFB solid residue to the existing culture broth in the second phase at 67 °C. The ethanol production increased from 50 to 102.5 g/L when bare supernatant was used in the first phase, and then to 219 g ethanol/L in the second phase. The amount of ethanol obtained using this strategy was almost equal to that obtained using the original WBFB containing 25% solid residue at 33 °C, and more than double that obtained when bare supernatant was used. Microscopic and gel electrophoresis studies revealed yeast cell wall degradation and secretion of cellular material into the surrounding medium. Scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) supported the existence of enzymes in WBFB involved in bioethanol production at elevated temperatures. The results of this study will provide insight for the development of new strategies for biofuel production.     

Genotypic and phenotypic characterization of industrial autochthonous Saccharomyces cerevisiae for the selection of well-adapted bioethanol-producing strains

In northwestern Argentina, sugarcane-derived industrial fermentation is being extensively used for bioethanol production, where highly adaptive native strains compete with the baker's yeast Saccharomyces cerevisiae traditionally used as starter culture. Yeast populations of 10 distilleries from Tucumán (Argentina) were genotypic and phenotypic characterized to select well-adapted bioethanol-producing autochthonous strains to be used as starter cultures for the industrial production of bioethanol fuel. From the 192 isolates, 69.8% were identified as S. cerevisiae, 25.5% as non-Saccharomyces, and 4.7% as Saccharomyces sp. wild yeasts. The majority of S. cerevisiae isolates (68.5%) were non-flocculating yeasts, while the flocculating strains were all obtained from the only continuous fermentation process included in the study. Simple Sequence Repeat analysis revealed a high genetic diversity among S. cerevisiae genotypes, where all of them were very different from the original baker's strain used as starter. Among these, 38 strains multi-tolerant to stress by ethanol (8%), temperature (42.5 °C) and pH (2.0) were obtained. No major differences were found among these strains in terms of ethanol production and residual sugars in batch fermentation experiments with cell recycling. However, only 10 autochthonous strains maintained their viability (more than 80%) throughout five consecutive cycles of sugarcane-based fermentations. In summary, 10 autochthonous isolates were found to be superior to baker's yeast used as starter culture (S. cerevisiae Calsa) in terms of optimal technological, physiological and ecological properties. The knowledge generated on the indigenous yeast populations in industrial fermentation processes of bioethanol-producing distilleries allowed the selection of well-adapted bioethanol-producing strains.     

Extracellular peroxidase production by Coprinus species from urea-treated soil

Thirteen strains of inky-cap mushroom Coprinus species were evaluated for the production of extracellular peroxidase. The liquid fermentation was carried out in shake flasks containing 1% glucose, 0.5% peptone, 0.3% yeast extract, and 0.3% malt extract broth at 25 degrees C. Peroxidase activity was detected in the liquid culture of several Coprinus species, including C. echinosporus NBRC 30630; C. macrocephalus NBRC 30117; Coprinus spp. UAMH 10065, UAMH 10066, UAMH 10067, and 074, after 10 days of growth. Peroxidase production by Coprinus sp. UAMH 10067, a Coprinus species isolated from urea-treated soil, was comparable to that of C. cinereus and reached 15 U.mL(-1) after 10 days. In addition, the peroxidase from Coprinus sp. UAMH 10067 was apparently more thermally stable than the enzyme produced by C. cinereus.     

Identification and characterization of Lactobacillus species isolated from fillets of vacuum-packed smoked and salted herring (Clupea harengus)

Seventy-eight strains were isolated from fillets of vacuum-packed smoked and salted herring ( Clupea harengus ) on APT glucose medium at 5°C or 20°C . All the isolates belonged to the genus Lactobacillus . CO₂ production and arginine degradation were characteristic of the isolates. A dichotomous key is proposed, using only four characters (growth on Rogosa broth, fermentation of ribose, presence of meso -diaminopimelic acid, isomer of lactate produced) for presumptive identification of lactobacilli from fish. The isolates could be allocated to three groups related to Lactobacillus groups II or III of Kandler and Weiss. Most of the strains possess important technological capacities : tolerance of high levels of NaCl, liquid smoke and bile salts. No lipolytic activity was found but some proteolytic activity was found on casein. Inhibitory activity against pathogenic or spoilage organisms was frequent and probably due to the production of hydrogen peroxide, or organic acids or both. No Carnobacterium strains were isolated despite the use of suitable temperature and isolation medium, probably because of the smoking of the fish.     

有抑菌能力乳杆菌的筛选及其抑菌物质的初步探究

目的 筛选具有抑菌能力的乳杆菌菌株并对其抑菌物质进行初步探究。方法 采用琼脂扩散法进行抑菌试验,筛选出具有抑菌能力的乳杆菌菌株;通过热稳定性试验检测抑菌物质耐高温的能力;通过有机酸排除与过氧化氢排除试验检测这两种物质对抑菌作用是否有影响;用胃蛋白酶、胰蛋白酶和蛋白酶K消化处理各株乳杆菌无细胞发酵液后进行抑菌试验,判断抑菌物质是否为蛋白多肽类物质。结果 2株副干酪乳杆菌与1株保加利亚乳杆菌对大肠埃希菌、铜绿假单胞菌、伤寒沙门菌和痢疾志贺菌有抑菌效果。这3株乳杆菌无细胞发酵液中的抑菌物质经过高温处理后仍具有抑菌能力,但抑菌能力与处理前相比显著降低（P＜0.05）;有机酸对照组未产生明显的抑菌圈;过氧化氢排除后的无细胞发酵液的抑菌能力未受影响;经过蛋白酶作用3株乳杆菌无细胞发酵液的抑菌能力显著降低（P＜0.05）或消失。生物被膜态副干酪乳杆菌2的抑菌能力与浮游态相近,其无细胞发酵液中的抑菌物质可以完全耐受100℃处理与胃蛋白酶的消化作用,可部分耐受胰蛋白酶的消化作用。结论 副干酪乳杆菌1、副干酪乳杆菌2和保加利亚乳杆菌具有良好的抑菌能力,它们产生的主要抑菌物质为蛋白多肽类,此物质具有较好的耐高温能力与耐蛋白酶能力;被膜态副干酪乳杆菌产生的抑菌物质表现出了更强的抗胁迫能力与稳定性。     

液泡蛋白酶B对酿酒酵母高温乙醇发酵效率的影响

【目的】提高酿酒酵母的高耐温性,从而提高菌株在高温下的乙醇发酵性能。【方法】利用染色体整合过表达酿酒酵母液泡蛋白酶B编码基因PRB1。【结果】在41 °C高温条件下进行乙醇发酵,过表达PRB1基因的重组酿酒酵母菌株可在31 h内消耗全部的葡萄糖,而对照菌株在相同时间内仅消耗不到一半的葡萄糖。【结论】利用蛋白酶B基因过表达可构建耐高温酿酒酵母菌株,提高在高温条件下乙醇的发酵效率。     

一株高产黑色素香灰菌菌株的鉴定、筛选及培养条件的优化

为筛选一株产黑色素能力强的菌株并优化其培养条件。通过ITS测序鉴定11株供试菌株,以菌丝生长速度、平板L值等指标筛选出一株产黑能力强的香灰菌,并对其生长所需碳源、氮源、pH等培养条件进行优化。研究表明,11株供试菌株均为香灰菌（Hypoxylon sp.）,其中Hp.sp0006菌丝生长速度较快、菌球大且均匀、L值最低,并且发酵液黑色素含量最高。该菌株最优培养条件是,以葡萄糖为碳源、牛肉浸膏为氮源、碳氮比20∶1并添加10 mg维生素B₁,黑色素含量可达（1.21±0.17）g/L。香灰菌Hp.sp0006是一株产黑色素较高的菌株,优化后的培养基更有利于黑色素的合成,为香灰菌黑色素的开发利用奠定基础。     

红茶菌群中菌株相互作用影响菌体生长和代谢

[背景]红茶菌是一种由细菌和酵母菌共生发酵而成的传统茶饮料.该饮料中含有多种有益人体健康的营养物质,具有促进消化、消炎、抗菌、抗糖尿病等生理作用.这些有益的代谢物是以醋酸菌和酵母菌为主的微生物相互作用而产生的.因此,红茶菌是一个优良的研究微生物相互作用的体系.[目的]分析不同菌株单独培养和混合培养对菌体生长和代谢产物的...     

Zymomonas mobilis Mutants with an Increased Rate of Alcohol Production

Two new derivatives of Zymomonas mobilis CP4 were isolated from enrichment cultures after 18 months of serial transfers. These new strains were selected for the ability to grow and produce ethanol rapidly on transfer into fresh broth containing ethanol and allyl alcohol. Ethanol production by these strains was examined in batch fermentations under three sets of conditions. Both new derivatives were found to be superior to the parent strain CP4 with respect to the speed and completeness of glucose conversion to ethanol. The best of these, strain YO2, produced 9.5% ethanol (by weight; 11.9% by volume) after 17.4 h compared with 31.8 h for the parent strain CP4. The addition of 1 mM magnesium sulfate improved ethanol production in all three strains. Two factors contributed to the decrease in fermentation time required by the mutants: more rapid growth with minimal lag on subculturing and the retention of higher rates of ethanol production as fermentation proceeded. Alcohol dehydrogenase isozymes were altered in both new strains and no longer catalyzed the oxidation of allyl alcohol into the toxic product acrolein. This loss of allyl alcohol-oxidizing capacity is proposed as a primary factor contributing to increased allyl alcohol resistance, although it is likely that other mutations affecting glycolysis also contribute to the improvement in ethanol production.     

Exploring grape marc as trove for new thermotolerant and inhibitor-tolerant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains for second-generation bioethanol production

Background

Robust yeasts with high inhibitor, temperature, and osmotic tolerance remain a crucial requirement for the sustainable production of lignocellulosic bioethanol. These stress factors are known to severely hinder culture growth and fermentation performance.

Results

Grape marc was selected as an extreme environment to search for innately robust yeasts because of its limited nutrients, exposure to solar radiation, temperature fluctuations, weak acid and ethanol content. Forty newly isolated Saccharomyces cerevisiae strains gave high ethanol yields at 40°C when inoculated in minimal media at high sugar concentrations of up to 200 g/l glucose. In addition, the isolates displayed distinct inhibitor tolerance in defined broth supplemented with increasing levels of single inhibitors or with a cocktail containing several inhibitory compounds. Both the fermentation ability and inhibitor resistance of these strains were greater than those of established industrial and commercial S. cerevisiae yeasts used as control strains in this study. Liquor from steam-pretreated sugarcane bagasse was used as a key selective condition during the isolation of robust yeasts for industrial ethanol production, thus simulating the industrial environment. The isolate Fm17 produced the highest ethanol concentration (43.4 g/l) from the hydrolysate, despite relatively high concentrations of weak acids, furans, and phenolics. This strain also exhibited a significantly greater conversion rate of inhibitory furaldehydes compared with the reference strain S. cerevisiae 27P. To our knowledge, this is the first report describing a strain of S. cerevisiae able to produce an ethanol yield equal to 89% of theoretical maximum yield in the presence of high concentrations of inhibitors from sugarcane bagasse.

Conclusions

This study showed that yeasts with high tolerance to multiple stress factors can be obtained from unconventional ecological niches. Grape marc appeared to be an unexplored and promising substrate for the isolation of S. cerevisiae strains showing enhanced inhibitor, temperature, and osmotic tolerance compared with established industrial strains. This integrated approach of selecting multiple resistant yeasts from a single source demonstrates the potential of obtaining yeasts that are able to withstand a number of fermentation-related stresses. The yeast strains isolated and selected in this study represent strong candidates for bioethanol production from lignocellulosic hydrolysates.

     

Mechanisms of enhanced cellulosic bioethanol fermentation by co-cultivation of Clostridium and Thermoanaerobacter spp

Engineering microbial consortia capable of efficient ethanolic fermentation of cellulose is a strategy for the development of consolidated bioprocessing for bioethanol production. Co-cultures of cellulolytic Clostridium thermocellum with non-cellulolytic Thermoanaerobacter strains (X514 and 39E) significantly improved ethanol production by 194-440%. Strain X514 enhanced ethanolic fermentation much more effectively than strain 39E in co-cultivation, with ethanol production in X514 co-cultures at least 62% higher than that of 39E co-cultures. Comparative genome sequence analysis revealed that the higher ethanolic fermentation efficiency in strain X514 was associated with the presence of a complete vitamin B(12) biosynthesis pathway, which is incomplete in strain 39E. The significance of the vitamin B(12)de novo biosynthesis capacity was further supported by the observation of improved ethanol production in strain 39E by 203% following the addition of exogenous vitamin B(12). The vitamin B(12) biosynthesis pathway provides a valuable biomarker for selecting metabolically robust strains for bioethanol production.     

New strains of basidiomycetes that produce bioethanol from lignocellulose biomass

Sixty six isolates were screened for ability of bioethanol production; dynamics of product accumulation and substrate utilization were investigated for two selected strains Trametes hirsuta MT-24.24 and Trametes versicolor IT-1. The strains’ efficiency was evaluated as bioethanol production by 1 g biomass. Strain T. versicolor IT-1 producing over 33 g/L of the ethanol for 9 d was selected. Direct conversion of Na-carboxymethyl cellulose, microcrystalline cellulose and straw was shown with ethanol yields of 2.1, 1.6 and 1.7 g/L, respectively, for 9 d fermentation time.     

Mathematical modeling of ethanol production in solid-state fermentation based on solid medium’ dry weight variation

In this work, mathematical modeling of ethanol production in solid-state fermentation (SSF) has been done based on the variation in the dry weight of solid medium. This method was previously used for mathematical modeling of enzyme production; however, the model should be modified to predict the production of a volatile compound like ethanol. The experimental results of bioethanol production from the mixture of carob pods and wheat bran by Zymomonas mobilis in SSF were used for the model validation. Exponential and logistic kinetic models were used for modeling the growth of microorganism. In both cases, the model predictions matched well with the experimental results during the exponential growth phase, indicating the good ability of solid medium weight variation method for modeling a volatile product formation in solid-state fermentation. In addition, using logistic model, better predictions were obtained.