Industrial Ethanol Fermentation Research Articles

Attention-based LSTM Block Model Framework based on static and dynamic variables for modeling fuel ethanol fermentation process

In the industrial fuel ethanol process, the initial feed conditions, which are static variables, and each control operation, which is dynamic variable and is changing during the producing process, have an impact on the concentration of ethanol out of the tank. Developing the accurate concentration model of ethanol out of the tank with the static variables and as early as possible dynamic variables is a challenging work and is useful in analyzing and optimizing the production process. Given that the ethanol fermentation process is multiphase and dynamic, a block concentration model of ethanol out of the tank based on multilayer perceptron (MLP) and long short-term memory (LSTM) is proposed to deal with the coexistence state of the static variables and the dynamic variables. A Channel-Squeeze-and-Excitation (CSE) module is constructed to solve the problem of high-dimensional input and low-dimensional output caused by the block model. The proposed model is applied for the industrial ethanol fermentation process. The results show that CSE-MLSTM is significantly different from other prediction models and improves the prediction accuracy. Temperature control experiments and ablation experiments effectively verify the reliability and effectiveness of CSE-MLSTM.

Cloning, expression and properties of trehalase from Pectobacterium cypripedii

Trehalase is widely used in industrial fermentation, food, medicine and other fields. There is a lack of industrial varieties of trehalase with excellent performance in China. Moreover, the applied research on trehalase was not well conducted. In this study, a strain of Pectobacterium cypripedii was screened from nature, and the gene PCTre encoding an acidic trehalase was cloned and expressed in E. coli BL21(DE3). The highest enzyme activity reached 4130 U/mL after fermenting in a 5 L fermenter for 28 h. The enzymatic properties study showed that PCTre hydrolyzed trehalose specifically. The optimum pH and temperature were 5.5 and 35 ℃, respectively. 80% of the enzyme activity was retained after being treated at pH 4.0, 4.5, and 5.0 for 8 h, showing good acid tolerance. Moreover, it has good tolerance to organic solvents, 60% enzyme activity was retained after being treated with 20% (V/V) ethanol solution for 24 h. Furthermore, trehalose could be completely hydrolyzed within 16 h in a simulated fermentation system containing 20% (V/V) ethanol and 7.5% trehalose, with 500 U/L PCTre added. This indicated a good application potential for industrial ethanol fermentation.

Ethanol Production from Colpomenia sinuosa by an Alginate Fermentation Strain Meyerozyma guilliermondii.

With the consumption of energy and the spread of COVID-19, the demand for ethanol production is increasing in the world. The industrial ethanol fermentation microbes cannot metabolize the alginate component of macro algae, which affects the ethanol yield. In this research, the ethanol production process from macro algae by an alginate fermentation yeast Meyerozyma guilliermondii, especially the pretreatment process of Colpomenia sinuosa, was studied. At the same time, the experimental design of Box-Behnken was carried out to achieve the optimum fermentation performance. The concentration of KH2PO4 (A: 2–6 g.L−1), pH (B: 4–7), reaction time (C: 60–120 h) and temperature (D: 24–34 °C) were variable input parameters. During the ethanol production process, the algae powder was firstly mixed with water at 90 °C for 0.5 h. Later the fermentation culture medium was prepared and then it was fermented by the yeast Meyerozyma guilliermondii to produce ethanol. And the optimal fermentation parameters were as follows: fermentation temperature of 28 °C, KH2PO4 dosage of 4.7 g.L−1, initial pH of 6, and fermentation time of 99 h. The ethanol yield reached 0.268 g.g−1 (ethanol to algae), close to the predicted value of model. The generation of alginate lyase during the fermentation of algae was also examined. The highest alginate lyase activity reached 46.42 U.mL−1.

Complex yeast\u2013bacteria interactions affect the yield of industrial ethanol fermentation

Sugarcane ethanol fermentation represents a simple microbial community dominated by S. cerevisiae and co-occurring bacteria with a clearly defined functionality. In this study, we dissect the microbial interactions in sugarcane ethanol fermentation by combinatorically reconstituting every possible combination of species, comprising approximately 80% of the biodiversity in terms of relative abundance. Functional landscape analysis shows that higher-order interactions counterbalance the negative effect of pairwise interactions on ethanol yield. In addition, we find that Lactobacillus amylovorus improves the yeast growth rate and ethanol yield by cross-feeding acetaldehyde, as shown by flux balance analysis and laboratory experiments. Our results suggest that Lactobacillus amylovorus could be considered a beneficial bacterium with the potential to improve sugarcane ethanol fermentation yields by almost 3%. These data highlight the biotechnological importance of comprehensively studying microbial communities and could be extended to other microbial systems with relevance to human health and the environment.

Comparative proteomic analyses reveal the metabolic aspects and biotechnological potential of nitrate assimilation in the yeast Dekkera bruxellensis.

The yeast Dekkera bruxellensis is well-known for its adaptation to industrial ethanol fermentation processes, which can be further improved if nitrate is present in the substrate. To date, the assimilation of nitrate has been considered inefficient because of the apparent energy cost imposed on cell metabolism. Recent research, however, has shown that nitrate promotes growth rate and ethanol yield when oxygen is absent from the environment. Given this, the present work aimed to identify the biological mechanisms behind this physiological behaviour. Proteomic analyses comparing four contrasting growth conditions gave some clues on how nitrate could be used as primary nitrogen source by D. bruxellensis GDB 248 (URM 8346) cells in anaerobiosis. The superior anaerobic growth in nitrate seems to be a consequence of increased cell metabolism (glycolytic pathway, production of ATP and NADPH and anaplerotic reactions providing metabolic intermediates) regulated by balanced activation of TORC1 and NCR de-repression mechanisms. On the other hand, the poor growth observed in aerobiosis is likely due to an oxidative stress triggered by nitrate when oxygen is present. These results represent a milestone regarding the knowledge about nitrate metabolism and might be explored for future use of D. bruxellensis as an industrial yeast. KEY POINTS: • Nitrate can be regarded as preferential nitrogen source for D. bruxellensis. • Oxidative stress limits the growth of D. bruxellensis in nitrate in aerobiosis. • Nitrate is a nutrient for novel industrial bioprocesses using D. bruxellensis.

Gene regulation of the Lactobacillus vini in response to industrial stress in the fuel ethanol production

The industrial ethanol fermentation imposes several stresses to microorganisms. However, some bacterial species are well adapted and manage to endure these harmful conditions. Lactobacillus vini is one of the most found bacteria in these environments, indicating the existence of efficient tolerance mechanisms. In view of this premise, the present study aimed to describe the tolerance of L. vini to several stressing agents encounter in industrial environments and the genetic components of the stress response. In general, L. vini showed significant tolerance to stressors commonly found in fuel-ethanol fermentations, and only doses higher than normally reached in processes restrained its growth. The lag phase and the growth rate were the most responsive kinetic parameter affected. Gene expression analysis revealed that uspII gene positively responded to all conditions tested, a typical profile of a general stress response gene. In addition, the results also revealed aspects of regulatory modules of co-expressed genes responding to different stresses, and also the similarities of response mechanism with basis in common cellular damages. Altogether, these data contribute to uncover the factors that could favour L. vini in the industrial fermentation which could be shared with other well adapted species and reports the first stress response genes in this bacterium.

Identification and characterisation of two high-affinity glucose transporters from the spoilage yeast Brettanomyces bruxellensis.

ABSTRACTThe yeast Brettanomyces bruxellensis (syn. Dekkera bruxellensis) is an emerging and undesirable contaminant in industrial low-sugar ethanol fermentations that employ the yeast Saccharomyces cerevisiae. High-affinity glucose import in B. bruxellensis has been proposed to be the mechanism by which this yeast can outcompete S. cerevisiae. The present study describes the characterization of two B. bruxellensis genes (BHT1 and BHT3) believed to encode putative high-affinity glucose transporters. In vitro-generated transcripts of both genes as well as the S. cerevisiae HXT7 high-affinity glucose transporter were injected into Xenopus laevis oocytes and subsequent glucose uptake rates were assayed using 14C-labelled glucose. At 0.1 mM glucose, Bht1p was shown to transport glucose five times faster than Hxt7p. pH affected the rate of glucose transport by Bht1p and Bht3p, indicating an active glucose transport mechanism that involves proton symport. These results suggest a possible role for BHT1 and BHT3 in the competitive ability of B. bruxellensis.

Study on Technological Parameters of Pilot Production of Ethanol

Cassava and corn are the two main ingredients in the process of producing fuel ethanol. This paper is mainly based on experiments. In this experiment, the traditional ethanol fermentation with cassava and corn as raw materials was compared with the the industrial ethanol fermentation with the same raw material, and the technological parameters of ethanol fermentation in traditional ethanol fermentation and industrial ethanol fermentation were compared and studied. The traditional double enzyme method was used for ethanol fermentation. Liquefaction temperature (70 plus or minus 1)°C, Saccharification temperature (60 plus or minus 1)°C, Fermentation temperature (30 plus or minus 1)°C. Experimental results show that: The average alcoholic production rate of corn was 36.64% and the average alcoholic production rate of cassava was 42.46% in the traditional ethanol fermentation, the average alcoholic production rate of corn was 38.22% and the average alcoholic production rate of cassava was 44.76% in the industrial ethanol fermentation. The industrial ethanol fermentation experiment is better than the traditional ethanol fermentation by comparison, because the former has better sealed anaerobic environment and greater capacity. It is suitable for large-scale production parameter study, in order to obtain higher utilization rate of raw materials, it shows a higher rate of alcohol production.

Application of Computer Monitoring Technology in Industrial Ethanol Production and Fermentation

Factors that influence the industrial ethanol fermentation process are complex. The development of computer monitoring technology can optimize fermentation conditions to obtain the maximum yield. This paper discusses the application of computer control technology in industrial ethanol production and fermentation, and studies the dynamics of ethanol fermentation. It finds that the monitoring system includes hardware and software. Under the computer control, the system can automatically complete the environment control of fermenter without human intervention, acquire and process field data, greatly increasing the yield of ethanol fermentation. The number and activity of yeast cells, the function of substrate and the production of ethanol are very important in the fermentation process.

Removal of Bacterial Contamination from Bioethanol Fermentation System Using Membrane Bioreactor

A major issue hindering efficient industrial ethanol fermentation from sugar-based feedstock is excessive unwanted bacterial contamination. In industrial scale fermentation, reaching complete sterility is costly, laborious, and difficult to sustain in long-term operation. A physical selective separation of a co-culture of Saccharomyces cerevisiae and an Enterobacter cloacae complex from a buffer solution and fermentation media at dilution rates of 0.1–1 1/h were examined using an immersed membrane bioreactor (iMBR). The effect of the presence of yeast, inoculum size, membrane pore size, and surface area, backwashing and dilution rate on bacteria removal were assessed by evaluating changes in the filtration conditions, medium turbidity, and concentration of compounds and cell biomass. The results showed that using the iMBR with dilution rate of 0.5 1/h results in successful removal of 93% of contaminating bacteria in the single culture and nearly complete bacteria decontamination in yeast-bacteria co-culture. During continuous fermentation, application of lower permeate fluxes provided a stable filtration of the mixed culture with enhanced bacteria washout. This physical selective separation of bacteria from yeast can enhance final ethanol quality and yields, process profitability, yeast metabolic activity, and decrease downstream processing costs.

Biofilm formation and antimicrobial sensitivity of lactobacilli contaminants from sugarcane-based fuel ethanol fermentation.

Industrial ethanol fermentation is subject to bacterial contamination that causes significant economic losses in ethanol fuel plants. Chronic contamination has been associated with biofilms that are normally more resistant to antimicrobials and cleaning efforts than planktonic cells. In this study, contaminant species of Lactobacillus isolated from biofilms (source of sessile cells) and wine (source of planktonic cells) from industrial and pilot-scale fermentations were compared regarding their ability to form biofilms and their sensitivity to different antimicrobials. Fifty lactobacilli were isolated and the most abundant species were Lactobacillus casei, Lactobacillus fermentum and Lactobacillus plantarum. The majority of the isolates (87.8%) were able to produce biofilms in pure culture. The capability to form biofilms and sensitivity to virginiamycin, monensin and beta-acids from hops, showed inter- and intra-specific variability. In the pilot-scale fermentation, Lactobacillus brevis, L. casei and the majority of L. plantarum isolates were less sensitive to beta-acids than their counterparts from wine; L. brevis isolates from biofilms were also less sensitive to monensin when compared to the wine isolates. Biofilm formation and sensitivity to beta-acids showed a positive and negative correlation for L. casei and L. plantarum, respectively.

Improvement of oxidative stress tolerance in Saccharomyces cerevisiae through global transcription machinery engineering

Excessive oxidative stress poses significant damage to yeast cells during fermentation process, and finally affects fermentation efficiency and the quality of products. In this paper, global transcription machinery engineering was employed to elicit Saccharomyces cerevisiae phenotypes of higher tolerance against oxidative stress caused by H2O2. Two strains from two plasmid-based mutagenesis libraries (Spt15 and Taf25), which exhibited significant increases in oxidative stress tolerance, were successfully isolated. At moderate H2O2 shock (≤3.5 mM), a positive correlation was found between the outperformance in cell growth of the oxidation-tolerate strains and H2O2 concentration. Several mutations were observed in the native transcription factors, which resulted in a different transcriptional profile compared with the control. Catalase and superoxide dismutase activities of the two mutants increased under H2O2 stress conditions. Fermentation experiments revealed that the mutant strain taf25-3 has a shorter lag phase compared to the control one, indicating that taf25-3 had improved adaptation ability to H2O2-induced oxidative stress and higher fermentation efficiency. Our study demonstrated that several amino acid substitutions in general transcription factors (Spt15 and Taf25) could modify the cellular oxidation defense systems and improve the anti-oxidation ability of S. cerevisiae. It could make the industrial ethanol fermentation more efficient and cost-effective by using the strain of higher stress tolerance.

Interaction of Lactobacillus vini with the ethanol‐producing yeasts Dekkera bruxellensis and Saccharomyces cerevisiae

Lactobacillus vini was recently described as a contaminant in industrial ethanol fermentations and its co-occurrence with Dekkera bruxellensis was noted. We investigated the growth characteristics of L. vini in cocultivation together with either Saccharomyces cerevisiae or D. bruxellensis. Lower cell numbers of both the yeasts and L. vini as well as a decrease in ethanol and lactate formation in mixed batch cultures compared with pure cultures were noted. L. vini formed cell aggregates (flocs) in all cultivation media with different shapes in Man–Rogosa–Sharpe and yeast extract–peptone–dextrose media. Flocs’ size and proportion of cells bound to flocs increased with increasing ethanol concentration. In coculture, formation of lactic acid bacteria–yeast cell aggregates consisting of a bacterial core with an outer layer of yeast cells was observed. L. vini–D. bruxellensis flocs had a bigger surface, due to cells protruding from the pseudomycelium. The involvement of mannose residues in the flocculation between L. vini and yeasts was tested. The presence of mannose induced deflocculation in a concentration-dependent manner. Less mannose was required for the deflocculation of D. bruxellensis as compared with S. cerevisiae.

Homo- and heterofermentative lactobacilli differently affect sugarcane-based fuel ethanol fermentation

Bacterial contamination during industrial yeast fermentation has serious economic consequences for fuel ethanol producers. In addition to deviating carbon away from ethanol formation, bacterial cells and their metabolites often have a detrimental effect on yeast fermentative performance. The bacterial contaminants are commonly lactic acid bacteria (LAB), comprising both homo- and heterofermentative strains. We have studied the effects of these two different types of bacteria upon yeast fermentative performance, particularly in connection with sugarcane-based fuel ethanol fermentation process. Homofermentative Lactobacillus plantarum was found to be more detrimental to an industrial yeast strain (Saccharomyces cerevisiae CAT-1), when compared with heterofermentative Lactobacillus fermentum, in terms of reduced yeast viability and ethanol formation, presumably due to the higher titres of lactic acid in the growth medium. These effects were only noticed when bacteria and yeast were inoculated in equal cell numbers. However, when simulating industrial fuel ethanol conditions, as conducted in Brazil where high yeast cell densities and short fermentation time prevail, the heterofermentative strain was more deleterious than the homofermentative type, causing lower ethanol yield and out competing yeast cells during cell recycle. Yeast overproduction of glycerol was noticed only in the presence of the heterofermentative bacterium. Since the heterofermentative bacterium was shown to be more deleterious to yeast cells than the homofermentative strain, we believe our findings could stimulate the search for more strain-specific antimicrobial agents to treat bacterial contaminations during industrial ethanol fermentation.

Modeling and dynamic optimization of fuel-grade ethanol fermentation using fed-batch process

This paper investigates optimization of operational strategies of an industrial ethanol fermentation process. One of the challenges associated with this type or process is that most of the measurements are only taken sporadically, thereby complicating process monitoring and optimization. The one exception to this rule involves temperature measurements, which are readily available. However, an existing model of the plant investigated in this paper does not include an energy balance and, accordingly, the temperature measurements cannot be used to estimate model parameters. This paper addresses these deficiencies and proposes modifications to an existing ethanol fermentation model. The proposed changes include the derivation of an energy balance, modification of the reaction kinetics to include additional inhibition terms, and also estimation of model parameters from industrial data. The new model is validated against plant data and then used for optimization of the process operations. It is shown that modifications of the input profiles for the cooling rate and the glucoamylase addition can lead to an approximately 10% increase in ethanol yield. These are promising results, even though these findings will ultimately need to be validated during real plant operations.

Comparative lipidomic analysis of S. cerevisiae cells during industrial bioethanol fermentation

Variations in the composition and level of phospholipids (PLs) in yeast cells during industrial ethanol fermentation processes were analyzed. A comparative lipidomic method was used to investigate the changes in total cellular PLs during continuous and fed-batch/batch processes. The phospholipid metabolism in yeast changed during both processes, mainly due to the presence of longchain poly unsaturated fatty acids (PUFA) that contained phosphatidyglycerol (PG), phosphatidylethanolamine (PE) and phosphatidylserine (PS). The complexity of the media affected the growth of the yeast and the membrane composition. Yeast incorporated lots of exogenous saturated and PUFAs from the feedstock during the fermentations. During the continuous fermentation, there was an increase in PLs with shorter chains as the fermentation progressed and early in process there were more longchains. During the fed-batch/batch process, the PG species increased as the fermentation progressed. This is probably due to an inositol deficiency in the earlier part of the fermentation.

Transcriptome profiling of Zymomonas mobilis under ethanol stress

BackgroundHigh tolerance to ethanol is a desirable characteristics for ethanologenic strains used in industrial ethanol fermentation. A deeper understanding of the molecular mechanisms underlying ethanologenic strains tolerance of ethanol stress may guide the design of rational strategies to increase process performance in industrial alcoholic production. Many extensive studies have been performed in Saccharomyces cerevisiae and Escherichia coli. However, the physiological basis and genetic mechanisms involved in ethanol tolerance for Zymomonas mobilis are poorly understood on genomic level. To identify the genes required for tolerance to ethanol, microarray technology was used to investigate the transcriptome profiling of the ethanologenic Z. mobilis in response to ethanol stress.ResultsWe successfully identified 127 genes which were differentially expressed in response to ethanol. Ethanol up- or down-regulated genes related to cell wall/membrane biogenesis, metabolism, and transcription. These genes were classified as being involved in a wide range of cellular processes including carbohydrate metabolism, cell wall/membrane biogenesis, respiratory chain, terpenoid biosynthesis, DNA replication, DNA recombination, DNA repair, transport, transcriptional regulation, some universal stress response, etc.ConclusionIn this study, genome-wide transcriptional responses to ethanol were investigated for the first time in Z. mobilis using microarray analysis.Our results revealed that ethanol had effects on multiple aspects of cellular metabolism at the transcriptional level and that membrane might play important roles in response to ethanol. Although the molecular mechanism involved in tolerance and adaptation of ethanologenic strains to ethanol is still unclear, this research has provided insights into molecular response to ethanol in Z. mobilis. These data will also be helpful to construct more ethanol resistant strains for cellulosic ethanol production in the future.

Viscosity Reduction During Fuel Ethanol Production by Fresh Sweet PotatoFermentation

Fresh sweet potato is one of the major feedstocks for fuel ethanol production in China.However,high-viscosity syrup is the key factor affecting high gravity fermentation of fresh sweet potato.It easily leads to pipe block,which seriously reduces the ethanol fermentation efficiency,influences the industrial production of ethanol with fresh sweet potato and increases the energy consumption.In this paper,the experiments on the viscosity-reducing enzyme and its optimized conditions were carried out,and the results showed that:(1) The cellulase from Sichuan Habio Bioengineering Co.,Ltd was the best enzyme,reducing the viscosity from 1.7×104 mPa.s to 8.8×102 mPa.s,and greatly reduced the cost of ethanol production.(2) The optimal pretreated condition was 110 ℃ for 20 min.(3) The viscosity-reducing enzyme could be applied to different varieties of fresh sweet potatoes,and the viscosity all reduced to less than 1.0×103mPa.s,with the lowest value of 2.7×102mPa.s.The viscosity reduction rates were all more than 95%.(4) Under optimal conditions,the viscosity of fermented sweet potatoes decreased from 1.9×105mPa.s to 2.7×103mPa.s after treated for 2 h by enzyme,and reduced to 790 mPa.s after fermentation of 23 h.The ethanol concentration reached 10.56%(V/V).The results were validated further that the viscosity-reducing enzyme had potential in industrial scale ethanol fermentation with fresh sweet potato.Tab 8,Ref 19

Microbial contamination of fuel ethanol fermentations

Microbial contamination is a pervasive problem in any ethanol fermentation system. These infections can at minimum affect the efficiency of the fermentation and at their worse lead to stuck fermentations causing plants to shut down for cleaning before beginning anew. These delays can result in costly loss of time as well as lead to an increased cost of the final product. Lactic acid bacteria (LAB) are the most common bacterial contaminants found in ethanol production facilities and have been linked to decreased ethanol production during fermentation. Lactobacillus sp. generally predominant as these bacteria are well adapted for survival under high ethanol, low pH and low oxygen conditions found during fermentation. It has been generally accepted that lactobacilli cause inhibition of Saccharomyces sp. and limit ethanol production through two basic methods; either production of lactic and acetic acids or through competition for nutrients. However, a number of researchers have demonstrated that these mechanisms may not completely account for the amount of loss observed and have suggested other means by which bacteria can inhibit yeast growth and ethanol production. While LAB are the primary contaminates of concern in industrial ethanol fermentations, wild yeast may also affect the productivity of these fermentations. Though many yeast species have the ability to thrive in a fermentation environment, Dekkera bruxellensis has been repeatedly targeted and cited as one of the main contaminant yeasts in ethanol production. Though widely studied for its detrimental effects on wine, the specific species-species interactions between D. bruxellensis and S. cerevisiae are still poorly understood.

English

  The flocculation mechanism of a stable mutant flocculent yeast strainSaccharomyces cerevisiae KRM-1 was quantitatively investigated for potential industrial interest. It was found that the mutant flocculent strain was NewFlo phenotype by means of sugar inhibition test. The flocculation was completely inhibited by treatment with proteinase K, protein-denaturants and carbohydrate modifier. The absence of calcium ions significantly inhibited the flocculation, indicating that Ca2+ was specifically required for flocculation. The flocculation was stable when temperature below 70°C and pH was in the range of 3.0 - 6.0. The flocculation onset of the mutant flocculent strain was in the early stationary growth phase, which coincided with glucose depletion in the batch fermentation for the production of ethanol from kitchen refuse medium. The results are expected to help develop better strategies for the control of mutant flocculent yeast for future large-scale industrial ethanol fermentation.   Key words: Kitchen refuse, Saccharomyces cerevisiae, flocculation, lectin-like, newflo phenotype.