Isolation and identification of xylitol-producing yeasts from agricultural residues

Selected yeast strains isolated from corn silage and viticulture residues were screened for their capacities to convert D-xylose into xylitol A conventional TLC was adapted for easy determination of xylose and xylitol in the culture supernatant solutions. This technique is suitable for the first steps of a screening program to select xylitol-producing yeasts from natural environments. Candida tropicalis ASM III (NRRL Y-27290), isolated from corn silage, appears to be a promising strain for xylitol production with a high yield (0.88 g xylitol per g of xylose consumed).     

Lactic acid production from agricultural resources as cheap raw materials

Agricultural resources such as barley, wheat, and corn were hydrolyzed by commercial amylolytic enzymes and fermented into lactic acid by Enterococcus faecalis RKY1. Although no additional nutrients were supplemented to those resources, lactic acid productivities were obtained at >0.8 g/l h from barley and wheat. When 200 g/l of whole wheat flour was hydrolyzed by amylolytic enzymes after the pre-treatment with 0.3% (v/v) sulfuric acid and sterilized by filtration, E. faecalis RKY1 efficiently produced lactic acid with 2.6 g/l h of lactic acid productivity and 5.90 g/l of maximal dry cell weight without additional nutrients. Lactic acid productivity and cell growth could be enhanced to 31% and 12% higher values than those of non-adapted RKY1, by adaptation of E. faecalis RKY1 to CSL-based medium. When the medium contained 200 g/l of whole wheat flour hydrolyzate, 15 g/l of corn steep liquor, and 1.5 g/l of yeast extract, lactic acid productivity and maximal dry cell weight were obtained at 5.36 g/l h and 14.08 g/l, respectively. This result represented an improvement of up to 106% of lactic acid productivity and 138% of maximal dry cell weight in comparison to the fermentation from whole wheat flour hydrolyzate only.     

Bioconversion of Raw Glycerol Generated from the Synthesis of Biodiesel by Different Oleaginous Yeasts: Lipid Content and Fatty Acid Profile of Biomass

In this work, 12 different yeast strains were evaluated to gauge their ability to accumulate lipids using raw glycerol as the main carbon source. Lipomyces lipofer NRRL Y-1155 stood out above the other strains, achieving 9.48 g/l biomass, 57.64 % lipid content and 5.46 g/l lipid production. The fatty acid profile was similar to vegetable oils commonly used in the synthesis of biodiesel, with the predominance of polyunsaturated acids, especially linoleic acid, reaching 68.3 % for Rhodotorula glutinis NRRL YB-252. The occurrence of palmitic acid (39.3 % for Lipomyces starkeyi NRRL Y-11557) was also notable. Thus, yeast biomass with high lipid content can be a sustainable and renewable alternative as a raw material for the biodiesel industry.     

Characterization of a new xylitol-producer Candida tropicalis strain

A xylitol-producer yeast isolated from corn silage and designated as ASM III was selected based on its outstanding biotechnological potential. When cultivated in batch culture mode and keeping the dissolved oxygen at 40% saturation, xylitol production was as high as 130 g l(-1) with a yield of 0.93 g xylitol g(-1) xylose consumed. A preliminary identification of the yeast was performed according to conventional fermentation and assimilation physiological tests. These studies were complemented by using molecular approaches based on PCR amplification, restriction-fragment length polymorphism analysis and sequencing of the rDNA segments: intergenic transcribed spacer (ITS) 1-5.8S rDNA-ITS 2, and D1/D2 domain of the 26S rRNA gene. Results from both the conventional protocols and the molecular characterization, and proper comparisons with the reference strains Candida tropicalis ATCC 20311 and NRRL Y-1367, led to the identification of the isolate as a new strain of C. tropicalis.     

Joint effect of nitrogen sources and B vitamin supplementation of date juice on lactic acid production by Lactobacillus casei subsp. rhamnosus

The use of date juice as a substrate for lactic acid production was investigated. Various nitrogen sources were compared with yeast extract for efficient lactic acid production by Lactobacillus casei subsp. rhamnosus. Among different nitrogen sources added to date juice (yeast extract, ammonium sulfate, tryptic soy, urea, peptone, and casein hydrolysate), yeast extract was the most efficient. The effect of yeast extract could have been due to its B vitamin content. The addition of five B vitamins at less than 25 mg/l to date juice with any nitrogen source enhanced lactic acid production to some extent, except for date juice with yeast extract or urea or peptone. The most significant increase was obtained with ammonium sulfate. Half of the yeast extract content (10 g/l) in a supplemented date juice could be replaced by a mixture of B vitamins at less than 25 mg/l, and ammonium sulfate at 2.6 g/l with no significant decrease in lactic acid production.     

戊糖乳杆菌发酵培养基氮源条件的优化

对戊糖乳杆菌发酵培养基的氮源条件进行了优化。通过单因素实验及响应面分析优化利用木糖高产乳酸的戊糖乳杆菌发酵培养基的不同氮源组合。优化得到的牛肉膏与柠檬酸氢二铵复合的最佳组成为牛肉膏17.72 g/L,柠檬酸氢二铵1.91 g/L,得到乳酸实际最大产量42.37 g/L。添加玉米浆与酵母粉和无机氮源复合的最佳组成为玉米浆46.54 g/L,酵母粉21.95 g/L,柠檬酸氢二铵9.95 g/L,可得到乳酸最大产量41.06 g/L。通过响应面优化减少了有机氮源的种类。牛肉膏与柠檬酸氢二铵的复合得到了更高的乳酸产量,且减少了有机氮源用量,节约了成本。玉米浆与酵母粉的复合解决了单一玉米浆造成的木糖利用速率过低的问题,同样得到较高浓度的乳酸。     

Ethanol production from fodder beets

Various yeasts such as two strains of Saccharomyces cerevisiae, Saccharomyces diastaticus, and Kluyveromyces marxianus were investigated for their ability to ferment fodder beet juice to alcohol. Juice extracted from fodder beet roots without any additives was used as a fermentation substrate. The fermentation kinetic parameters were determined and compared for each species of yeast tested. The best species for fodder beet juice fermentation was chosen and products obtained by fermentation of one hectare of fodder beet plants are given.     

Domesticating a food spoilage yeast into an organic acid‐tolerant metabolic engineering host: Lactic acid production by engineered Zygosaccharomyces bailii

Lactic acid represents an important class of commodity chemicals, which can be produced by microbial cell factories. However, due to the toxicity of lactic acid at lower pH, microbial production requires the usage of neutralizing agents to maintain neutral pH. Zygosaccharomyces bailii, a food spoilage yeast, can grow under the presence of organic acids used as food preservatives. This unique trait of the yeast might be useful for producing lactic acid. With the goal of domesticating the organic acid‐tolerant yeast as a metabolic engineering host, seven Z. bailii strains were screened in a minimal medium with 10 g/L of acetic, or 60 g/L of lactic acid at pH 3. The Z. bailii NRRL Y7239 strain was selected as the most robust strain to be engineered for lactic acid production. By applying a PAN‐ARS‐based CRISPR‐Cas9 system consisting of a transfer RNA promoter and NAT selection, we demonstrated the targeted deletion of ADE2 and site‐specific integration of Rhizopus oryzae ldhA coding for lactate dehydrogenase into the PDC1 locus. The resulting pdc1::ldhA strain produced 35 g/L of lactic acid without ethanol production. This study demonstrates the feasibility of the CRISPR‐Cas9 system in Z. bailii, which can be applied for a fundamental study of the species.     

Effect of technical threonine sources on homoserine biosynthesis by mutant Brevibacteruim flavum 2T

The effect of threonine technical sources on the homoserine biosynthesis by the threonine auxotroph Brevibacterium flavum 2T when cultivated on sucrose and acetic acid containing media was investigated. Various threonine sources (corn extract and fodder yeast, microbial biomass and soybean meal hydrolyzates) prepared by means of different hydrolyzing agents (acids, enzymes, autolysis) were used. The most effective substrate was protein--vitamin concentrate hydrolyzate, particularly combined with corn extract in the ratio 1: 0,25-0.5 (with respect to the dry weight of the initial material). The homoserine yield was 16.2 g/l on the sucrose containing medium and 18.4 g/l on the acetic acid containing medium which was in agreement with controls. The medium containing pure threonine was used as a control. With other threonine sources (corn extract, protein-vitamin concentrate autolyzate and enzymolyzate, fodder yeast and soybean meal hydrolyzates), the homoserine production was significantly lower, i.e. 40-70% of the control. The content of amino acids (threonine, isoleucine, methionine) in the initial material and their suitability for the homoserine biosynthesis were found to be correlated. The substrates with a high content of threonine (over 3.5%) and a low content of methionine (below 0.5%) proved most effective. The use of the material in which the ratio threonine: methionine was less than 6.0 caused the homoserine biosynthesis to be partially replaced with that of lysine.     

Adaptive response of yeasts to furfural and 5-hydroxymethylfurfural and new chemical evidence for HMF conversion to 2,5-bis-hydroxymethylfuran

Renewable lignocellulosic materials are attractive low-cost feedstocks for bioethanol production. Furfural and 5-hydroxymethylfurfural (HMF) are among the most potent inhibitory compounds generated from acid hydrolysis of lignocelluloses to simple sugars for fermentation. In Saccharomyces cerevisiae ATCC 211239 and NRRL Y-12632 and Pichia stipitis NRRL Y-7124, furfural and HMF inhibition were determined to be dose-dependent at concentrations from 10 to 120 mM. The yeast strains were more sensitive to inhibition by furfural than HMF at the same concentration, while combined treatment of furfural and HMF synergistically suppressed cell growth. A metabolite transformed from HMF by strain NRRL Y-12632 was isolated from the culture supernatant, and conclusively identified as 2,5-bis-hydroxymethylfuran, a previously postulated HMF alcohol, with a composition of C₆H₈O₃ and a molecular weight of 128. It is proposed that, in the presence of HMF, the yeast reduces the aldehyde group on the furan ring of HMF into an alcohol, in a similar manner as for furfural. The accumulation of this biotransformed metabolite may be less toxic to yeast cultures than HMF, as evidenced by the rapid yeast fermentation and growth rates associated with HMF conversion. The ability of yeasts to adapt to and transform furfural and HMF offers the potential for in situ detoxification of these inhibitors and suggests a genetic basis for further development of highly tolerant strains for biofuel production.     

Olive-mill wastewaters: a promising substrate for microbial lipase production

The present study investigated the valorization of olive-mill wastewater (OMW) by its use as a possible growth medium for the microbial production of extra-cellular lipase. To this end, strains of Geotrichum candidum (NRRL Y-552 and Y-553), Rhizopus arrhizus (NRRL 2286 and ISRIM 383), Rhizopus oryzae (NRRL 6431), Aspergillus oryzae (NRRL 1988 and 495), Aspergillus niger (NRRL 334), Candida cylindracea (NRRL Y-17506) and Penicillium citrinum (NRRL 1841 and 3754, ISRIM 118) were screened. All strains were able to grow on the undiluted OMW, producing extra-cellular lipase activity. C. cylindracea NRRL Y-17506 showed the highest lipase activity on all the typologies of OMW used. Its lipase production on OMW was markedly affected by the type of nitrogen source and was induced by the addition of olive oil. The highest activity (9.23 IU ml(-1)) of the yeast was obtained on OMW supplemented with NH(4)Cl (2.4 g l(-1)) and olive oil (3.0 g l(-1)).     

Ethanol production from whey permeate by immobilized yeast cells

The ability of two yeast strains to utilize the lactose in whey permeate has been studied. Kluyveromyces marxianus NCYC 179 completely utilized the lactose (9.8%), whereas Saccharomyces cerevisiae NCYC 240 displayed an inability to metabolize whey lactose for ethanol production. Of the two gel matrices tested for immobilizing K. marxianus NCYC 179 cells, sodium alginate at 2% (w/v) concentration proved to be the optimum gel for entrapping the yeast cells effectively. The data on optimization of physiological conditions of fermentation (temperature, pH, ethanol concentration and substrate concentration) showed similar effects on immobilized and free cell suspensions of K. marxianus NCYC 179, in batch fermentation. A maximum yield of 42.6 g ethanol l^?1 (82% of theoretical) was obtained from 98 g lactose l^?1 when fermentation was carried at pH 5.5 and 30°C using 120 g dry weight l^?1 cell load of yeast cells. These results suggest that whey lactose can be metabolized effectively for ethanol production using immobilized K. marxianus NCYC 179 cells.     

The effect of supplementation by different nitrogen sources on the production of lactic acid from date juice by Lactobacillus casei subsp. rhamnosus

Production of lactic acid from date juice by fermentation has been studied using Lactobacillus casei subsp. rhamnosus as the producer organism. The optimum substrate concentration, expressed in its glucose content, was 60 g l(-1). Various nitrogen sources were compared with yeast extract in terms of their efficiency for lactic acid production. None of these nitrogen sources gave lactic acid concentrations as high as that obtained with yeast extract. As yeast extract supplementation was not economically attractive, different proportions of (NH4)2SO4 and yeast extract were used. When the elemental nitrogen ratio of(NH4)2SO4 to yeast extract was 4:1, the substrate use and efficiency of lactic acid production were the same as in date juice supplemented with 20 g l(-1) yeast extract (0:5).     

Green biorefinery: separation of lactic acid from grass silage juice by chromatography using neutral polymeric resin

The aim of this work was to recover lactic acid in undissociated form from grass silage juice. For this aim, chromatographic separation using neutral polymeric resin Amberlite XAD1600 was investigated. Up to now, there is no hint in the literatures about using neutral polymeric resin for lactic acid separation from a mixture. Important factors (flow-rate, concentration of feed and loaded volume) that affect separation performance were firstly investigated with model solutions. The obtained results showed that lactic acid solutions with the purity varying from 93.2% to 99.9% could be obtained at the recovery yields over 99.4%. After that, trials with silage juice were carried out. Due to the complex composition of the feed, the purity of products decreased to 94% at a recovery yield of 97%. Although 99% of inorganic salts and sugars were separated from lactic acid organic acids in general and acetic acid in particular caused a purity problem. It seems that organic acids could not be separated from lactic acid by neutral resin Amberlite XAD1600. Besides the organic acid problem, some amino acids were remained in the products as impurities.     

Interaction effects of lactic acid and acetic acid at different temperatures on ethanol production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in corn mash

The combined effects of lactic acid and acetic acid on ethanol production by S. cerevisiae in corn mash, as influenced by temperature, were examined. Duplicate full factorial experiments (three lactic acid concentrations × three acetic acid concentrations) were performed to evaluate the interaction between lactic and acetic acids on the ethanol production of yeast at each of the three temperatures, 30, 34, and 37°C. Corn mash at 30% dry solids adjusted to pH 4 after lactic and acetic acid addition was used as the substrate. Ethanol production rates and final ethanol concentrations decreased (P<0.001) progressively as the concentration of combined lactic and acetic acids in the corn mash increased and the temperature was raised from 30 to 37°C. At 30°C, essentially no ethanol was produced after 96 h when 0.5% w/v acetic acid was present in the mash (with 0.5, 2, and 4% w/v lactic acid). At 34 and 37°C, the final concentrations of ethanol produced by the yeast were noticeably reduced by the presence of 0.3% w/v acetic acid and ≥2% w/v lactic acid. It can be concluded that, as in previous studies with defined media, lactic acid and acetic acid act synergistically to reduce ethanol production by yeast in corn mash. In addition, the inhibitory effects of combined lactic and acetic acid in corn mash were more apparent at elevated temperatures.     

Production of a New Polysaccharide with Cryptoccus laurentii var. flavescens

Conditions were investigated for the production of a new gum by the yeast Cryptococcus laurentii var. flavescens NRRL Y-1401 in shaken flasks and 20-liter fermentors. The most suitable medium contained 6% commercial glucose, 0.25% autolyzed brewer's yeast, and 0.001% MnSO₄ and had an initial pH of 6.5. Polysaccharide yields, as measured by the dry weight of the alcohol precipitates, were in the range of 30 to 35% based on initial glucose.     

Production of bakers' yeast in cheese whey ultrafiltrate

A process for the production of bakers' yeast in whey ultrafiltrate (WU) is described. Lactose in WU was converted to lactic acid and galactose by fermentation. Streptococcus thermophilus was selected for this purpose. Preculturing of S. thermophilus in skim milk considerably reduced its lag. Lactic fermentation in 2.3x-concentrated WU was delayed compared with that in unconcentrated whey, and fermentation could not be completed within 60 h. The growth rate of bakers' yeast in fermented WU differed among strains. The rate of galactose utilization was similar for all strains, but differences in lactic acid utilization occurred. Optimal pH ranges for galactose and lactic acid utilization were 5.5 to 6.0 and 5.0 to 5.5, respectively. The addition of 4 g of corn steep liquor per liter to fermented WU increased cell yields. Two sources of nitrogen were available for growth of Saccharomyces cerevisiae: amino acids (corn steep liquor) and ammonium (added during the lactic acid fermentation). Ammonium was mostly assimilated during growth on lactic acid. This process could permit the substitution of molasses by WU for the industrial production of bakers' yeast.     

Statistical screening of medium components by Plackett-Burman design for lactic acid production by Lactobacillus sp. KCP01 using date juice

Statistical screening of media components for production of lactic acid by Lactobacillus sp. KCP01 using date juice as a sugar source was carried out by Plackett-Burman design. Date juice at 5% sugar concentration when used alone showed 2.6 g/l of lactic acid production. Increase in lactic acid production (15.1 g/l) was observed with supplementation of salts and organic nitrogen sources of MRS medium and after optimization of pH and temperature using date juice as a C-source. Plackett-Burman design showed peptone, K2HPO4, sodium acetate and date juice as significant components influencing the lactic acid production.     

Production of poly(β-l-malic acid) (PMA) from agricultural biomass substrates by Aureobasidium pullulans

For the first time the production of poly(β-l -malic acid) (PMA) has been achieved using agricultural biomass substrates by the yeast-like fungus Aureobasidium pullulans. Strains NRRL Y-2311-1, NRRL 50382, NRRL 50383, and NRRL 50384, representing diverse isolation sources and phylogenetic clades, produced PMA from alkaline H₂O₂-pretreated corn fiber and wheat straw as sole carbon sources. Pretreated wheat straw was better than pretreated corn fiber, and strain NRRL 50383 gave the highest overall yields of PMA. The addition of CaCO₃ plus supplementary hydrolytic enzymes enhanced PMA production. Four basal media were compared for PMA production, and the best was found to be a N-limited pullulan production medium (PM). In this medium, PMA production took place during growth limitation. Under optimal conditions, strain NRRL 50383 produced more than 20 g PMA/l from 5 % (w/v) pretreated wheat straw in PM with 3 % (w/v) CaCO₃ and supplementary enzymes.     

New finding and optimal production of a novel extracellular alkaline lipase from Yarrowia lipolytica NRRL Y-2178

Lipases are industrially useful versatile enzymes that catalyze numerous different reactions including hydrolysis of triglycerides, transesterification, and chiral synthesis of esters under natural conditions. Although lipases from various sources have been widely used in industrial applications, such as in food, chemical, pharmaceutical, and detergent industries, there are still substantial current interests in developing new microbial lipases, specifically those functioning in abnormal conditions. We screened 17 lipase-producing yeast strains, which were prescreened for substrate specificity of lipase from more than 500 yeast strains from the Agricultural Research Service Culture Collection (Peoria, IL, U.S.A.), and selected Yarrowia lipolytica NRRL Y-2178 as a best lipase producer. This report presents new finding and optimal production of a novel extracellular alkaline lipase from Y. lipolytica NRRL Y-2178. Optimal c ulture conditions f orlipase production by Y. lipolytica NRRL Y-2178 were 72 h incubation time, 27.5 degrees C, pH 9.0. Glycerol and glucose were efficiently used as the most efficient carbon sources, and a combination of yeast extract and peptone was a good nitrogen source for lipase production by Y. lipolytica NRRL Y-2178. These results suggested that Y. lipolytica NRRL Y-2178 showsgood industrial potential as a new alkaline lipase producer.