Determination of amino acids in cell culture and fermentation broth media using anion-exchange chromatography with integrated pulsed amperometric detection.

Anion-exchange chromatography with integrated pulsed amperometric detection (AE-IPAD) separates and directly detects amino acids, carbohydrates, alditols, and glycols in the same injection without pre- or post-column derivatization. These separations use a combination of NaOH and NaOH/sodium acetate eluents. We previously published the successful use of this technique, also known as AAA-Direct, to determine free amino acids in cell culture and fermentation broth media. We showed that retention of carbohydrates varies with eluent NaOH concentration differently than amino acids, and thus separations can be optimized by varying the initial NaOH concentration and its duration. Unfortunately, some amino acids eluting in the acetate gradient portion of the method were not completely resolved from system-related peaks and from unknown peaks in complex cell culture and fermentation media. In this article, we present changes in method that improve amino acid resolution and system ruggedness. The success of these changes and their compatibility with the separations previously designed for fermentation and cell culture are demonstrated with yeast extract-peptone-dextrose broth, M199, Dulbecco's modified Eagle's (with F-12), L-15 (Leibovitz), and McCoy's 5A cell culture media.     

Determination of amino acids in cell culture and fermentation broth media using anion-exchange chromatography with integrated pulsed amperometric detection

Cell culture and fermentation broth media are used in the manufacture of biotherapeutics and many other biological materials. Characterizing the amino acid composition in cell culture and fermentation broth media is important because deficiencies in these nutrients can reduce desired yields or alter final product quality. Anion-exchange (AE) chromatography using sodium hydroxide (NaOH) and sodium acetate gradients, coupled with integrated pulsed amperometric detection (IPAD), determines amino acids without sample derivatization. AE-IPAD also detects carbohydrates, glycols, and sugar alcohols. The presence of these compounds, often at high concentrations in cell culture and fermentation broth media, can complicate amino acid determinations. To determine whether these samples can be analyzed without sample preparation, we studied the effects of altering and extending the initial NaOH eluent concentration on the retention of 42 different carbohydrates and related compounds, 30 amino acids and related compounds, and 3 additional compounds. We found that carbohydrate retention is impacted in a manner different from that of amino acid retention by a change in [NaOH]. We used this selectivity difference to design amino acid determinations of diluted cell culture and fermentation broth media, including Bacto yeast extract-peptone-dextrose (yeast culture medium) broth, Luria-Bertani (bacterial culture medium) broth, and minimal essential medium and serum-free protein-free hybridoma medium (mammalian cell culture media). These media were selected as representatives for both prokaryotic and eukaryotic culture systems capable of challenging the analytical technique presented in this paper. Glucose up to 10mM (0.2%, w/w) did not interfere with the chromatography, or decrease recovery greater than 20%, for the common amino acids arginine, lysine, alanine, threonine, glycine, valine, serine, proline, isoleucine, leucine, methionine, histidine, phenylalanine, glutamate, aspartate, cystine, and tyrosine.     

A modified Saccharomyces cerevisiae strain that consumes L-Arabinose and produces ethanol

Metabolic engineering is a powerful method to improve, redirect, or generate new metabolic reactions or whole pathways in microorganisms. Here we describe the engineering of a Saccharomyces cerevisiae strain able to utilize the pentose sugar L-arabinose for growth and to ferment it to ethanol. Expanding the substrate fermentation range of S. cerevisiae to include pentoses is important for the utilization of this yeast in economically feasible biomass-to-ethanol fermentation processes. After overexpression of a bacterial L-arabinose utilization pathway consisting of Bacillus subtilis AraA and Escherichia coli AraB and AraD and simultaneous overexpression of the L-arabinose-transporting yeast galactose permease, we were able to select an L-arabinose-utilizing yeast strain by sequential transfer in L-arabinose media. Molecular analysis of this strain, including DNA microarrays, revealed that the crucial prerequisite for efficient utilization of L-arabinose is a lowered activity of L-ribulokinase. Moreover, high L-arabinose uptake rates and enhanced transaldolase activities favor utilization of L-arabinose. With a doubling time of about 7.9 h in a medium with L-arabinose as the sole carbon source, an ethanol production rate of 0.06 to 0.08 g of ethanol per g (dry weight). h(-1) under oxygen-limiting conditions, and high ethanol yields, this yeast strain should be useful for efficient fermentation of hexoses and pentoses in cellulosic biomass hydrolysates.     

Derepression of galactose metabolism in melibiase producing bakers' and distillers' yeast

Beet molasses is widely used as a growth substrate for bakers' and distillers' yeast in the production of biomass and ethanol. Most commercial yeasts do not fully utilise the carbohydrates in molasses since they are incapable of hydrolysing the disaccharide melibiose to glucose and galactose. Also, expression of genes encoding enzymes for the utilisation of carbon sources that are alternatives to glucose is tightly regulated, sometimes rates of yeast growth and/or ethanol production. The GAL genes are regulated by specific induction by galactose and repression during growth on glucose. In an industrial distillers' yeast, two genes interacting synergistically in glucose repression of galactose utilization, MIG1 and GAL80, have been disrupted with MEL1, encoding melibiase. The physiology of the wild-type strain and the recombinant strains was investigated on mixtures of glucose and galactose and on molasses. The recombinant strain started to ferment galactose when 9.7 g 1(-1) glucose was still present during a batch fermentation, whereas the wild-type strain did not consume any galactose in the presence of glucose. The ethanol yield in the recombinant strain was 0.50 g ethanol g sugar (-1) in an ethanol fermentation on molasses, compared with 0.48 g ethanol g sugar (-1) for the wild-type strain. The increased ethanol yield was due to utilization of melibiose in the molasses.     

Promotive action of ceramics on yeast ethanol production,and its relationship to pH,glycerol and alcohol dehydrogenase activity

Summary A yeast strain, Saccharomyces cerevisiae KPY32 isolated from pito, a traditional West-African alcoholic beverage, was immobilized in porous ceramic beads as a means of improving its ethanol production. Stationary fermentation cultures at different temperatures were made using semi-synthetic medium and fermentation parameters including ethanol production, sugar consumption, cell growth and pH were monitored. Glycerol production, and the activity of alcohol dehydrogenase (ADH) of the various systems were monitored. It was found that immobilization of the yeast resulted in improved ethanol production, at conversion rates above 93% of the theoretical value. The pH of the immobilized systems was also stabilized at around 4.0, glycerol production was higher, and the ADH activities were higher than those of free-cell systems. Ethanol production at the high temperature of 37° C was also improved by immobilization. The promotive action was found to be related to the pH, presence of glycerol and the enhancement of ADH activity.Offprint requests to: B. Demuyakor     

Bacteria engineered for fuel ethanol production: current status

The lack of industrially suitable microorganisms for converting biomass into fuel ethanol has traditionally been cited as a major technical roadblock to developing a bioethanol industry. In the last two decades, numerous microorganisms have been engineered to selectively produce ethanol. Lignocellulosic biomass contains complex carbohydrates that necessitate utilizing microorganisms capable of fermenting sugars not fermentable by brewers' yeast. The most significant of these is xylose. The greatest successes have been in the engineering of Gram-negative bacteria: Escherichia coli, Klebsiella oxytoca, and Zymomonas mobilis. E. coli and K. oxytoca are naturally able to use a wide spectrum of sugars, and work has concentrated on engineering these strains to selectively produce ethanol. Z. mobilis produces ethanol at high yields, but ferments only glucose and fructose. Work on this organism has concentrated on introducing pathways for the fermentation of arabinose and xylose. The history of constructing these strains and current progress in refining them are detailed in this review.     

Contaminant occurrence, identification and control in a pilot-scale corn fiber to ethanol conversion process

While interest in bioethanol production from lignocellulosic feedstocks is increasing, there is still relatively little pilot-plant data and operating experience available for this emerging industry. A series of batch and continuous fermentation runs were performed in a pilot-plant, some lasting up to six weeks, in which corn fiber-derived sugars were fermented to ethanol using glucose-fermenting and recombinant glucose/xylose-fermenting yeasts. However, contamination by Lactobacillus bacteria was a common occurrence during these runs. These contaminating microorganisms were found to readily consume arabinose, a sugar not utilized by the yeast, producing acetic and lactic acids that had a detrimental effect on fermentation performance. The infections were ultimately controlled with the antibiotic virginiamycin, but routine use of antibiotics is cost prohibitive. The severity of the problem encountered during this work is probably due to use of a highly contaminated feedstock. Lignocellulosic conversion facilities will not employ aseptic designs. Instead, techniques similar to those employed in the corn-based fuel ethanol industry to control infections will be used. Effective control may also be possible by using fermentative microorganisms that consume all biomass-derived sugars.     

Kinetics of enzymatic lysis and disruption of yeast cells: I. Evaluation of two lytic systems with different properties

Many microorganisms produce enzymes which lyse the walls of yeasts, fungi, and bacteria. The proportions of different enzyme activities present in the lytic system, their action patterns, synergism, and dependence on inhibitors, constitute the activity profile of the lytic system. Taken together, the activity profile and process conditions for lysis determine the reaction rate and the distribution of products from lysis of any given type of cells. Kinetics of glucan hydrolysis, proteolysis, and lysis of brewer's yeast were compared for two extracellular yeast-lytic enzyme systems with different properties. The enzyme sources used were filtered culture broths from Cytophaga sp. NCIB 9497 grown in batch culture and from Oerskovia xanthineolytica LL-G109, grown under carbon limitation in continuous culture. Rate and extent of cell hydrolysis, and the accumulation of soluble proteins, peptides, and carbohydrates from the lysed yeast cells, are discussed in terms of the activity profiles and potential applications of the two enzyme systems.     

Cassava as a Cheap Source of Carbon for Rhizobial Inoculant Production using an Amylase-producing Fungus and a Glycerol-producing Yeast

Summary The aim of this research was to develop methods to use low-cost carbon compounds for rhizobial inoculant production. Five raw starch materials; steamed cassava, sticky rice, fresh corn, dry corn and sorghum were tested for sugar production by an amylase-producing fungus. Streamed cassava produced the highest amount of reducing sugar after fermentation. Bradyrhizobium japonicum USDA110, Azorhizobium caulinodans IRBG23, Rhizobium phaseoli TAL1383, Sinorhizobium fredii HH103, and Mesorhizobium ciceri USDA2429 were tested on minimal medium supplemented with reducing sugar obtained from cassava fermentation. All strains, except B. japonicum USDA110, could grow in medium containing cassava sugar derived from 100 g steamed cassava per litre, and the growth rates for these strains were similar to those in medium containing 0.5 (w/v) mannitol. The sugar derived from steamed cassava was further used for production of glycerol using yeast. After 1 day of yeast fermentation, the culture containing glycerol and heat-killed yeast cells, was used to formulate media for culturing bradyrhizobia. A formulation medium, FM4, with a glycerol concentration of 0.6 g/l and yeast cells (OD₆₀₀ = 0.1) supported growth of B. japonicum USDA110 up to 3.61 × 10⁹ c.f.u./ml in 7 days. These results demonstrate that steamed cassava could be used to provide cheap and effective carbon sources for rhizobial inoculant production.     

Yeasts utilizing methyl alcohol]

The ability to utilize methanol was found in the yeast strains isolated from forest substrates (Zygowillia pini IBFM y-655, Torulopsis molicshiana IBFM y-657) and in the collection yeast cultures (Candida boidinii IBFM y-588 and IBFM )-587). These yeasts assimilated not only methanol but other alcohols (ethanol, glycerol, erythrol, mannitol, and sorbitol), nitrates or nitrites. Their fermentation ability was low.     

Microvinification--how small can we go?

High-throughput methodologies to screen large numbers of microorganisms necessitate the use of small-scale culture vessels. In this context, an increasing number of researchers are turning to microtiter plate (MTP) formats to conduct experiments. MTPs are now widely used as a culturing vessel for phenotypic screening of aerobic laboratory cultures, and their suitability has been assessed for a range of applications. The work presented here extends these previous studies by assessing the metabolic footprint of MTP fermentation. A comparison of Chardonnay grape juice fermentation in MTPs with fermentations performed in air-locked (self-induced anaerobic) and cotton-plugged (aerobic) flasks was made. Maximum growth rates and biomass accumulation of yeast cultures grown in MTPs were indistinguishable from self-induced anaerobic flask cultures. Metabolic profiles measured differed depending on the metabolite. While glycerol and acetate accumulation mirrored that of self-induced anaerobic cultures, ethanol accumulation in MTP ferments was limited by the increased propensity of this volatile metabolite for evaporation in microlitre-scale culture format. The data illustrates that microplate cultures can be used as a replacement for self-induced anaerobic flasks in some instances and provide a useful and economical platform for the screening of industrial strains and culture media.     

Metabolic Flux Analysis during the Exponential Growth Phase of Saccharomyces cerevisiae in Wine Fermentations

As a consequence of the increase in global average temperature, grapes with the adequate phenolic and aromatic maturity tend to be overripe by the time of harvest, resulting in increased sugar concentrations and imbalanced C/N ratios in fermenting musts. This fact sets obvious additional hurdles in the challenge of obtaining wines with reduced alcohols levels, a new trend in consumer demands. It would therefore be interesting to understand Saccharomyces cerevisiae physiology during the fermentation of must with these altered characteristics. The present study aims to determine the distribution of metabolic fluxes during the yeast exponential growth phase, when both carbon and nitrogen sources are in excess, using continuous cultures. Two different sugar concentrations were studied under two different winemaking temperature conditions. Although consumption and production rates for key metabolites were severely affected by the different experimental conditions studied, the general distribution of fluxes in central carbon metabolism was basically conserved in all cases. It was also observed that temperature and sugar concentration exerted a higher effect on the pentose phosphate pathway and glycerol formation than on glycolysis and ethanol production. Additionally, nitrogen uptake, both quantitatively and qualitatively, was strongly influenced by environmental conditions. This work provides the most complete stoichiometric model used for Metabolic Flux Analysis of S. cerevisiae in wine fermentations employed so far, including the synthesis and release of relevant aroma compounds and could be used in the design of optimal nitrogen supplementation of wine fermentations.     

Production of Fructose and Ethanol from Cane Molasses Using Saccharomyces cerevisiae ATCC 36858

The purpose of this research was to study the possibility of the production of ethanol and enriched fructose syrups from sugar cane molasses using the yeast Saccharomyces cerevisiae ATCC 36858. In batch experiments with a total sugar concentration of between 96.7 g/l and 323.5 g/l, the fructose yield was above 90% of the theoretical value. The ethanol yield and volumetric productivity were in the range of 66% and 77% of the theoretical value, and between 0.53 g ethanol/l × h and 3.15 g ethanol/l × h, respectively. The fructose fraction in the carbohydrates content of the produced syrups was more than 95% when the total initial sugar concentration in the medium was below 273.8 g/l. Some oligosaccharides and glycerol were also produced in all tested media. The maximum amount of produced oligosaccharides including raffinose accounted for 13.4 g/l in the cane molasses medium with 323.5 g/l sugars in the initial phase of the fermentation process. The oligosaccharides produced and raffinose were completely consumed by the end of the fermentation process when the total initial sugar concentration was less than 191.3 g/l. The glycerol concentration was below 9.9 g/l. These findings are useful in the production of ethanol and high fructose syrups using sugar cane molasses.     

Galacturonic acid inhibits the growth of Saccharomyces cerevisiae on galactose, xylose, and arabinose

The efficient fermentation of mixed substrates is essential for the microbial conversion of second-generation feedstocks, including pectin-rich waste streams such as citrus peel and sugar beet pulp. Galacturonic acid is a major constituent of hydrolysates of these pectin-rich materials. The yeast Saccharomyces cerevisiae, the main producer of bioethanol, cannot use this sugar acid. The impact of galacturonic acid on alcoholic fermentation by S. cerevisiae was investigated with anaerobic batch cultures grown on mixtures of glucose and galactose at various galacturonic acid concentrations and on a mixture of glucose, xylose, and arabinose. In cultures grown at pH 5.0, which is well above the pK(a) value of galacturonic acid (3.51), the addition of 10 g · liter(-1) galacturonic acid did not affect galactose fermentation kinetics and growth. In cultures grown at pH 3.5, the addition of 10 g · liter(-1) galacturonic acid did not significantly affect glucose consumption. However, at this lower pH, galacturonic acid completely inhibited growth on galactose and reduced galactose consumption rates by 87%. Additionally, it was shown that galacturonic acid strongly inhibits the fermentation of xylose and arabinose by the engineered pentose-fermenting S. cerevisiae strain IMS0010. The data indicate that inhibition occurs when nondissociated galacturonic acid is present extracellularly and corroborate the hypothesis that a combination of a decreased substrate uptake rate due to competitive inhibition on Gal2p, an increased energy requirement to maintain cellular homeostasis, and/or an accumulation of galacturonic acid 1-phosphate contributes to the inhibition. The role of galacturonic acid as an inhibitor of sugar fermentation should be considered in the design of yeast fermentation processes based on pectin-rich feedstocks.     

Evaluation of thermotolerant yeasts in controlled simultaneous saccharifications and fermentations of cellulose to ethanol

Simultaneous saccharification and fermentation (SSF) experiments were performed at selected temperatures (37, 41, and 43 degrees C) to obtain comprehensive material balance and performance data for several promising strains of thermotolerant yeast. Parameters measured were ethanol concentration, yeast cell density, and residual sugar and cellulose concentrations. The three yeasts Saccharomyces uvarum, Candida brassicae, and C. lusitaniae and two mixed cultures of Brettanomyces clausenii with S. cerevisiae (mixed culture I) and C. Iusitaniae with S. uvarum (mixed culture II) exhibited rapid rates of fermentation, high ethanol yields, strong viability, or high cellobiase activity. Overall, mixed culture II at 41 degrees C performed better than either component yeast by themselves because it combined a cellobiose fermenting capability with the high ethanol tolerance and rapid glucose fermentation of conventional industrial yeasts. Thus, the mixed cultures provide good initial rates by preventing buildup of cellobiose (a strong inhibitor of enzyme activity) while attaining high ultimate yields of ethanol for high cellulase concentrations. However, C. brassicae and S. uvarum gave similar results to mixed culture II at 37 degrees C.     

The Composition of Jerusalem Artichoke (Helianthus tuberosus L.) Spirits Obtained from Fermentation with Bacteria and Yeasts

The composition of spirits distilled from fermentation of Jerusalem artichoke (Helianthus tuberosus L.) tubers was compared by means of gas chromatography. The microorganisms used in the fermentation processes were the bacterium Zymomonas mobilis, strains 3881 and 3883, the distillery yeast Saccharomyces cerevisiae, strains Bc16a and D₂ and the Kluyveromyces fragilis yeast with an active inulinase. The fermentation of mashed tubers was conducted using a single culture of the distillery yeast Saccharomyces cerevisiae and the bacterium Zymomonas mobilis (after acid or enzymatic hydrolysis) as well as Kluyveromyces fragilis (sterilized mashed tubers). The tubers were simultaneously fermented by mixed cultures of the bacterium or the distillery yeast with K. fragilis. The highest ethanol yield was achieved when Z. mobilis 3881 with a yeast demonstrating inulinase activity was applied. The yield reached 94 % of the theoretical value. It was found that the distillates resulting from the fermentation of mixed cultures were characterized by a relatively lower amount of by‐products compared to the distillates resulting from the single species process. Ester production of 0.30–2.93 g/L, responsible for the aromatic quality of the spirits, was noticed when K. fragilis was applied for ethanol fermentation both in a single culture process and also in the mixed fermentation with the bacterium. Yeast applied in this study caused the formation of higher alcohols to concentrations of 7.04 g/L much greater than those obtained with the bacterium. The concentrations of compounds other than ethanol obtained from Jerusalem artichoke mashed tubers, which were fermented by Z. mobilis, were lower than those achieved for yeasts.     

Thermophilic,lignocellulolytic bacteria for ethanol production: current state and perspectives

Lignocellulosic biomass contains a variety of carbohydrates, and their conversion into ethanol by fermentation requires an efficient microbial platform to achieve high yield, productivity, and final titer of ethanol. In recent years, growing attention has been devoted to the development of cellulolytic and saccharolytic thermophilic bacteria for lignocellulosic ethanol production because of their unique properties. First of all, thermophilic bacteria possess unique cellulolytic and hemicellulolytic systems and are considered as potential sources of highly active and thermostable enzymes for efficient biomass hydrolysis. Secondly, thermophilic bacteria ferment a broad range of carbohydrates into ethanol, and some of them display potential for ethanologenic fermentation at high yield. Thirdly, the establishment of the genetic tools for thermophilic bacteria has allowed metabolic engineering, in particular with emphasis on improving ethanol yield, and this facilitates their employment for ethanol production. Finally, different processes for second-generation ethanol production based on thermophilic bacteria have been proposed with the aim to achieve cost-competitive processes. However, thermophilic bacteria exhibit an inherent low tolerance to ethanol and inhibitors in the pretreated biomass, and this is at present the greatest barrier to their industrial application. Further improvement of the properties of thermophilic bacteria, together with the optimization production processes, is equally important for achieving a realistic industrial ethanol production.     

Towards valorization of pectin-rich agro-industrial residues: Engineering of Saccharomyces cerevisiae for co-fermentation of d-galacturonic acid and glycerol

Pectin-rich plant biomass residues represent underutilized feedstocks for industrial biotechnology. The conversion of the oxidized monomer d-galacturonic acid (d-GalUA) to highly reduced fermentation products such as alcohols is impossible due to the lack of electrons. The reduced compound glycerol has therefore been considered an optimal co-substrate, and a cell factory able to efficiently co-ferment these two carbon sources is in demand. Here, we inserted the fungal d-GalUA pathway in a strain of the yeast S. cerevisiae previously equipped with an NAD-dependent glycerol catabolic pathway. The constructed strain was able to consume d-GalUA with the highest reported maximum specific rate of 0.23 g g_CDW⁻¹ h⁻¹ in synthetic minimal medium when glycerol was added. By means of a ¹³C isotope-labelling analysis, carbon from both substrates was shown to end up in pyruvate. The study delivers the proof of concept for a co-fermentation of the two ‘respiratory’ carbon sources to ethanol and demonstrates a fast and complete consumption of d-GalUA in crude sugar beet pulp hydrolysate under aerobic conditions. The future challenge will be to achieve co-fermentation under industrial, quasi-anaerobic conditions.     

High value added lipids produced by microorganisms: a potential use of sugarcane vinasse

This review aims to present an innovative concept of high value added lipids produced by heterotrophic microorganisms, bacteria and fungi, using carbon sources, such as sugars, acids and alcohols that could come from sugarcane vinasse, which is the main byproduct from ethanol production that is released in the distillation step. Vinasse is a rich carbon source and low-cost feedstock produced in large amounts from ethanol production. In 2019, the Brazilian Ministry of Agriculture, Livestock and Food Supply estimates that growth of ethanol domestic consumption will be 58.8 billion liters, more than double the amount in 2008. This represents the annual production of more than 588 billion liters of vinasse, which is currently used as a fertilizer in the sugarcane crop, due to its high concentration of minerals, mainly potassium. However, studies indicate some disadvantages such as the generation of Greenhouse Gas emission during vinasse distribution in the crop, as well as the possibility of contaminating the groundwater and soil. Therefore, the development of programs for sustainable use of vinasse is a priority. One profitable alternative is the fermentation of vinasse, followed by an anaerobic digester, in order to obtain biomaterials such as lipids, other byproducts, and methane. Promising high value added lipids, for instance carotenoids and polyunsaturated fatty acids (PUFAS), with a predicted market of millions of US$, could be produced using vinasse as carbon source, to guide an innovative concept for sustainable production. Example of lipids obtained from the fermentation of compounds present in vinasse are vitamin D, which comes from yeast sucrose fermentation and Omega 3, which can be obtained by bacteria and fungi fermentation. Additionally, several other compounds present in vinasse can be used for this purpose, including sucrose, ethanol, lactate, pyruvate, acetate and other carbon sources. Finally, this paper illustrates the potential market and microbial processes, using microorganisms, for lipid production.     

Effect of sodium chloride on bakers' yeast growing in gelatin

In recent years, industrial fermentation researchers have shifted their attention from liquid to solid and semisolid culture conditions. We converted liquid cultures to the semisolid mode by adding high levels of gelatin. Previous studies on liquid cultures have revealed the inhibitory activity of mineral salts, such as NaCl, on the fermentation of sugars by yeasts. We made a kinetic study of the effects of 1 to 5% (wt/vol) NaCl on the alcoholic fermentations of glucose by Saccharomyces cerevisiae in a growth medium containing 16% gelatin. Our results showed that the effect of high salt content on semisolid culture is essentially the same as the effect on liquid culture; i.e., as the salt content increased, the following occurred: (i) the growth of yeasts decreased, (ii) the lag period of the yeast biomass curve lengthened, (iii) the sugar intake was lowered, (iv) the yield of ethanol was reduced, and (v) the production of glycerol was increased. We observed a new relationship correlating the area of kinetic hysteresis with ethanol production rate, acetaldehyde concentration, and the initial NaCl concentration.