Strain Kluyveromyces Marxianus Research Articles

Functionality of Yeast β-Glucan Recovered from Kluyveromyces marxianus by Alkaline and Enzymatic Processes.

β-Glucan (BG), one of the most abundant polysaccharides containing glucose monomers linked by β-glycosidic linkages, is prevalent in yeast biomass that needs to be recovered to obtain this valuable polymer. This study aimed to apply alkaline and enzymatic processes for the recovery of BG from the yeast strain Kluyveromyces marxianus TISTR 5925. For this purpose, the yeast was cultivated to produce the maximum yield of raw material (yeast cells). The effective recovery of BG was then established using either an alkaline or an enzymatic process. BG recovery of 35.45% was obtained by using 1 M NaOH at 90 °C for 1 h, and of 81.15% from 1% (w/v) hydrolytic protease enzyme at 55 °C for 5 h. However, BG recovered by the alkaline process was purer than that obtained by the enzymatic process. Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopy confirmed the purity, the functional groups, and the linkages of BG obtained from different recovery systems and different raw materials. The results of this study suggest that an alkaline process could be an effective approach for the solubilization and recovery of considerable purity of BG from the yeast cells. In addition, the obtained BG had comparable functional properties with commercially available BG. This study reveals the effectiveness of both chemical and biological recovery of BG obtained from yeast as a potential polymeric material.

Optimization of fermentation for γ-aminobutyric acid (GABA) production by yeast Kluyveromyces marxianus C21 in okara (soybean residue).

γ-Aminobutyric acid (GABA) is a non-protein amino acid with a variety of physiological functions. Recently, yeast Kluyveromyces marxianus strains involved in the catabolism and anabolism of GABA can be used as a microbial platform for GABA production. Okara, rich in nutrients, can be used as a low-cost fermentation substrate for the production of functional materials. This study first proved the advantages of the okara medium to produce GABA by K. marxianus C21 when L-glutamate (L-Glu) or monosodium glutamate (MSG) is the substrate. The highest production of GABA was obtained with 4.31g/L at optimization condition of culture temperature 35°C, fermentation time 60h, and initial pH 4.0. Furthermore, adding peptone significantly increased the GABA production while glucose and vitamin B6 had no positive impact on GABA production. This research provided a powerful new strategy of GABA production by K. marxianus C21 fermentation and is expected to be widely utilized in the functional foods industry to increase GABA content for consumers as a daily supplement as suggested.

A Combined In Vitro and In Vivo Assessment of the Safety of the Yeast Strains Kluyveromyces marxianus A4 and A5 Isolated from Korean Kefir.

Kefir is a traditional fermented milk containing beneficial bacteria and yeasts. Despite Kluyveromyces marxianus, isolated from kefir, gaining increasing attention as a potential probiotic yeast owing to its biological function, Saccharomyces boulardii is the only species considered as a probiotic yeast. We evaluated the safety of K. marxianus strains A4 and A5, isolated from Korean kefir, in comparisonwith that of S. boulardii. Virulence attributes were preliminarily assessed in vitro including their ability of gelatin hydrolysis, pseudohyphae formation, and hemolysis. To evaluate in vivo safety, the strains were challenged in a healthy animal model, four-week-old female BALB/c mice. Mice were orally administered 0.2mL of 0.9% sterilized saline (NC_S; n = 6), S. boulardii ATCC MYA-796 (high concentration, S.b_H; low concentration, S.b_L; n = 6 for each), K. marxianus A4 (high concentration, A4_H; low concentration, A4_L; n = 6 for each), or K. marxianus A5 (high concentration, A5_H; low concentration, A5_L; n = 6 for each) for 2weeks. At study end, body weight, spleen and liver weights, and blood parameters were assessed. K. marxianus A4 and A5 were tested negative for gelatinase and hemolysis. Overall, hematological, plasma biochemical, and cytokine (interleukin-1β and tumor necrosis factor-α) parameters were comparable between the experimental and negative control (NC) groups. Notably, the interleukin-6 level of the A5_H group was significantly lower than that of the NC group (p < 0.05), suggesting anti-inflammatory potential of K. marxianus A5.

Development of a Continuous System for 2-Phenylethanol Bioproduction by Yeast on Whey Permeate-Based Medium.

2-Phenylethanol (2-PE) is an alcohol with a rosy scent and antimicrobial activity, and therefore, it is widely used in the food and cosmetic industries as an aroma and preservative. This work was aimed to draw up a technology for 2-PE bioproduction on whey permeate, which is waste produced by the dairy industry, rich in lactase and proteins. Its composition makes it a harmful waste to dispose of; however, with a properly selected microorganism, it could be converted to a value-added product. Herein, two yeast Kluyveromyces marxianus strains and one Kluyveromyces lactis, isolated from dairy products, were tested for 2-PE production, firstly on standard media and then on whey permeate based media in batch cultures. Thereafter, the 2-PE bioproduction in a continuous system in a 4.8 L bioreactor was developed, and subsequently, the final product was recovered from culture broth. The results showed that the yield of 2-PE production increased by 60% in the continuous culture compared to batch culture. Together with a notable reduction of chemical oxygen demand for whey permeate, the present study reports a complete, effective, and environmentally friendly strategy for 2-PE bioproduction with a space-time yield of 57.5 mg L−1 h−1.

Studies on Bioethanol Production with Thermo Tolerant Yeast Isolates and their Co-Cultures using African Wild Cocoyam as Feedstock

In this work different ways of optimally producing bioethanol at various pH with thermotolerant yeasts and their cocultures using a non-human edible starchy food as feedstock was examined. African wild cocoyam, Xanthosoma roseum, sourced from abandoned farmlands in Obukpa, Nsukka, Nigeria was used as the substrate, while strains of Kluyveromyces marxianus and Pichia stipitis were used to ferment them. First the tubers were gelatinized by boiling under pressure above 100oC before hydrolysis with concentrated H2SO4. The hydrolysates were then fermented at 35oC with the thermotolerant yeasts for five days at different pH. Results obtained showed that gelatinized sample of the substrate gave optimum glucose yield when hydrolysed with 1M H2SO4 for 60 minutes. Kluyveromyces marxianus produced more ethanol than Pichia stipitis at all the four fermentation pH values tested. However, optimum ethanol production was obtained when the two yeast strains were used as coculture at pH 4.5. The peak time for ethanol production was 96 hours for the individual yeast cultures while that of their coculture was 72 hours. The results of the study indicated that wild cocoyam is an excellent feedstock for bioethanol production with many advantages including being non-edible, thereby eliminating concerns for food security, and containing high amount of carbohydrate. The study also revealed that fermenting sugar hydrolysates with a coculture of microorganisms during bioethanol production is a more efficient process than using individual cultures.

A biorefinery concept for the production of fuel ethanol, probiotic yeast, and whey protein from a by-product of the cheese industry.

Agroindustrial by-products and residues can be transformed into valuable compounds in biorefineries. Here, we present a new concept: production of fuel ethanol, whey protein, and probiotic yeast from cheese whey. An initial screening under industrially relevant conditions, involving thirty Kluyveromyces marxianus strains, was carried out using spot assays to evaluate their capacity to grow on cheese whey or on whey permeate (100 g lactose/L), under aerobic or anaerobic conditions, in the absence or presence of 5% ethanol, at pH 5.8 or pH 2.5. The four best growing K. marxianus strains were selected and further evaluated in a miniaturized industrial fermentation process using reconstituted whey permeate (100 g lactose/L) with cell recycling (involving sulfuric acid treatment). After five consecutive fermentation cycles, the ethanol yield on sugar reached 90% of the theoretical maximum in the best cases, with 90% cell viability. Cells harvested at this point displayed probiotic properties such as the capacity to survive the passage through the gastrointestinal tract and capacity to modulate the innate immune response of intestinal epithelium, both in vitro. Furthermore, the CIDCA 9121 strain was able to protect against histopathological damage in an animal model of acute colitis. Our findings demonstrate that K. marxianus CIDCA 9121 is capable of efficiently fermenting the lactose present in whey permeate to ethanol and that the remaining yeast biomass has probiotic properties, enabling an integrated process for the obtainment of whey protein (WP), fuel ethanol, and probiotics from cheese whey.Key points• K. marxianus-selected strains ferment whey permeate with 90% ethanol yield.• Industrial fermentation conditions do not affect selected yeast probiotic capacity.• Whey permeate, fuel ethanol, and probiotic biomass can be obtained in a biorefinery.

Simultaneous saccharification and fermentation of sequential dilute acid‐alkali pretreated cotton (Gossypium hirsutum L.) stalk for cellulosic ethanol production

AbstractBACKGROUNDCotton stalk (CS) was deconstructed by sequential dilute acid‐alkali pretreatment (DALP) using sulfuric acid (1%, v/v) and sodium hydroxide (3%, w/v). The enzymatic hydrolysis of DALP‐CS by cellulase was optimized by Taguchi method, employing factors like solid loading, enzyme dose, tween 80, and pH as main process variables. For bioethanol production, pretreated CS was subjected to simultaneous saccharification and fermentation (SSF) using a newly identified yeast strain Kluyveromyces marxianus JKSG‐6.RESULTSSequential pretreatment of CS resulted in 66% and 42% removal of hemicellulose and lignin, respectively. The same was evident from the changes in the fourier transforming infrared (FT‐IR) spectroscopy absorption spectrum. Under optimized conditions, the enzymatic hydrolysis of DALP‐CS released 375.50 ± 0.04 mg g−1 of glucose. Further, by employing K. marxianus JKSG‐6 under SSF at 42 °C, the strain produced an ethanol titer of 26.10 ± 0.05 g L−1 from pretreated CS.CONCLUSIONSequential dilute acid‐alkali pretreatment rendered CS, a highly recalcitrant LCB feedstock for bioethanol, more accessible for hydrolysis by cellulase. SSF of the effectively pretreated CS was successfully carried out for bioethanol production by employing a newly identified yeast K. marxianus JKSG‐6. © 2021 Society of Chemical Industry (SCI).

Valorization of apple pomaces for biofuel production: A biorefinery approach

This study is aimed at assessing the potential of apple pomace (AP) as a substrate for biofuel production following a biorefinery approach. Two different APs, from juice and cider production were evaluated. First, bioethanol generation was performed and its fermentation residues, together with available biobutanol fermentation residues, were studied for biogas production. Moreover, co-digestion of AP and swine manure was investigated following a factorial design. Twelve different bacterial and yeast strains were compared for bioethanol production, obtaining bioethanol concentrations about 50 g L−1 by different strains of Kluyveromyces marxianus, K. lactis, Lachancea thermotolerans and Saccharomyces cerevisiae, with yields of 0.371–0.444 g g−1. Specific methane yields of the fermentation residues of bioethanol and biobutanol production were 463 and 290 mL CH4 g −1VS added, respectively. Methane yield for the co-digestion of AP and swine manure was 596 mL CH4 g−1VS added, with an AP percentage of 14.6% and a substrate concentration of 9.38 g VS L−1.

Purification and characterization of an inulinase produced by a Kluyveromyces marxianus strain isolated from blue agave bagasse

Exo-inulinases are versatile enzymes that have gained attention in recent years due to their ability to hydrolyze linear and branched polyfructose chains found in inulines. Agavin, a branched inulin, is found in Agave plant, the raw matter to produce tequila. Our group has isolated several microbial strains from agave bagasse, an agro-industrial residue from tequila production that increases yearly. Strain ISO3, identified as Kluyveromyces marxianus, showed a remarkable activity towards agavin, and from its fermentation liquor an inulinolytic enzyme (Inu-ISO3) was purified. The isolated enzyme is a glycosylated dimeric protein with a molecular mass of ~256 kDa, as determined by DLS and SEC. The enzyme has an isoelectric pH of 4.6 and has both inulinase and invertase activities with an I/S ratio (ratio of activity with agavin to activity with sucrose) of 1.39. The enzyme has temperature and pH optima of 50 °C and 5.5, respectively, and follows hyperbolic kinetics with agavin (kcat of 339 ± 27 s−1 and KM of 11.8 ± 1.5 mM). The remarkable activity of Inu-ISO3 on linear and branched inulin spotlights this enzyme as a potential player in the treatment of agricultural residua for the generation of added-value products.

Isolation of a Novel Kluyveromyces marxianus Strain QKM-4 and Evidence of Its Volatilome Production and Binding Potentialities in the Biocontrol of Toxigenic Fungi and Their Mycotoxins

To overcome the economic losses associated with fungi and their toxic metabolites, environmentally safe and efficient approaches are needed. To this end, biological control using yeasts and safe bacterial strains and their products are being explored to replace synthetic fungicides. In the present study, the biocontrol effect of a yeast strain of Kluyveromyces marxianus, QKM-4, against the growth and mycotoxin synthesis potential of key toxigenic fungi was evaluated. In vitro assays were performed to find the application of yeast volatile organic compounds (VOCs) against fungal contamination on important agricultural commodities. The removal of ochratoxin A (OTA) and deoxynivalenol (DON) by living and heat-inactivated yeast cells was also explored. VOCs produced by strain QKM-4 were able to significantly limit the fungal growth of 17 fungal species belonging to genera Aspergillus, Penicillium, and Fusarium. Yeast VOCs were able to reduce OTA biosynthesis potential of Penicillium verrucosum and Aspergillus carbonarius by 99.6 and 98.7%, respectively. In vivo application of QKM-4 VOCs against Fusarium oxysporum and A. carbonarius infection on tomatoes and grapes, respectively, determined a complete inhibition of fungal spore germination. GC/MS-based analysis of yeast VOCs identified long-chain alkanes, including nonadecane, eicosane, docosane, heptacosane, hexatriacontane, and tetracosane. In vitro testing of the mycotoxin-binding potential of the living and heat-inactivated QKM-4 cells showed a reduction of OTA and DON up to 58 and 49%, respectively, from artificially contaminated buffers. Our findings clearly demonstrate the strong antifungal potential of K. marxianus QKM-4 and propose this strain as a strong candidate for application in agriculture to safeguard food and feed products.

Batch and Fed-Batch Ethanol Fermentation of Cheese-Whey Powder with Mixed Cultures of Different Yeasts

Eight yeast strains of Lachancea thermotolerans, Kluyveromyces marxianus, and Kluyveromyces waltii have been tested for their ability to ferment lactose into ethanol in mashes containing 10% (w/v) cheese whey powder (CWP). The K. marxianus NCAIM Y00963 achieved 3.5% (v/v) ethanol concentration at 96–120 h of fermentation. The ethanol production by the selected lactose-positive strains and the well-known ethanologenic Saccharomyces cerevisiae (Levuline Fb) in mixed culture was also investigated at different CWP concentrations and inoculation techniques in batch mode. The mixed culture in an equal ratio (1:1) of cell counts of K. marxianus and S. serevisiae showed an increase in lactose conversion rate. The two yeast strains in a ratio of 3:1 (three-quarters of K. marxianus and a quarter of S. cerevisiae in a total of 4.5 × 1010 cells) resulted in 72.33% efficiency of lactose bioconversion and 7.6% (v/v) ethanol production at 17.5% (w/v) of CWP concentration. In the repeated inoculation process, with the addition of three-quarter part of 3:1 ratio of mixed culture (3.3 × 1010 cells of K. marxianus) into 150 mL CWP mash at initiation and the rest quarter part (1.2 × 1010 cells of S. cerevisiae) at 24 h, 8.86% (v/v) ethanol content with 87.5% efficiency of lactose conversion was reached. Both the ethanol concentration and efficiency of bioconversion were increased to 10.34% (v/v) and 92%, respectively, by combination with fed-batch fermentation technology. Our results can serve a very good basis for the development of industrial technology for the utilization of cheese whey.

Short communication: Physicochemical features and microbial community of milk kefir using a potential probiotic Saccharomyces cerevisiae KU200284

The aim of this study was to analyze the β-glucan contents, physicochemical features, and microbial communities in milk kefir prepared using Saccharomyces cerevisiae KU200284 isolated from cucumber jangajji, a fermented vegetable commonly eaten in Korean. Three types of milk kefir were manufactured, with (1) activated kefir grain, (2) activated kefir grain with commercial S. cerevisiae BOF, and (3) activated kefir grain with S. cerevisiae KU200284. β-Glucan contents of milk kefir using kefir grain and kefir grain with S. cerevisiae strains BOF and KU200284 were 8.29, 8.59, and 8.57%, respectively. The pH, titratable acidity, viscosity, Brix level, and alcohol contents of milk kefir using kefir grain with S. cerevisiae strains were acceptable compared with milk kefir using only kefir grain. In milk kefir produced using kefir grains and S. cerevisiae strains, 16S rRNA reads showed representative strains of Lactobacillus kefiranofaciens (>72% relative abundance) and Acetobacter fabarum (>16% relative abundance). In particular, milk kefir using kefir grain with S. cerevisiae KU200284 had the highest relative abundance of L. kefiranofaciens. In addition, the internal transcribed sequence (ITS) rRNA reads in tested milk kefir showed representative strains of Kluyveromyces marxianus (>52% relative abundance) and Saccharomyces cerevisiae (>16% relative abundance). In contrast, milk kefir using S. cerevisiae strains had higher relative abundance of S. cerevisiae (>37%). The β-glucan production, physicochemical properties, and microbial community profiling indicate that S. cerevisiae KU200284 could be used in functional dairy products as a starter culture.

Identification of yeasts present in artisanal yoghurt and traditionally fermented milks consumed in the northern part of Cameroon

Abstract Bacteria generally ferment milk but, sometimes, yeasts are found in fermented milks. The presence of these yeasts in the microbial community of some fermented milks could be intentional or accidental. The Diversity of yeasts in the products was investigated using a molecular technique employing variable regions of 26S rDNA profiles generated by PCR-DGGE (Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis). Two types of samples: artisanal or handcraft yoghurt and traditionally fermented milks were collected in three towns of the three regions of the northern part of Cameroon. Firstly, a comparison was made between the 16 traditionally fermented milks collected in Maroua, Garoua and Ngaoundre each of the 3 regions. Secondly, it was between 26 artisanal fermented milks of each region and finally, between the two types of products. The different PCR-DGGE 26S rDNA profiles obtained were analyzed and DNA sequencing was used to compare yeasts from each method of production. Twelve (12) species of Yeasts were identified as: Malassezia globosa, Hanseniaspora uvarum, Galactomyces candidum, Candida tropicalis, Aureobasidium pullulans, Torulaspora globosa, Saccharomyces cerevisiae/paradoxus, Pichia kluyveri, Candida parapsilosis, Torulaspora delbrueckii, Kluyveromyces marxianus, Candida orthopsilosis and Pseudozyma sp. Yeast diversity was higher for artisanal fermented milks (yoghurt) with at least 10 species, while for traditionally non packed fermented milks only 5 species were identified with a predominance strain of Galactomyces candidum, Candida parapsilosis, Torulaspora delbrueckii, Saccharomyces cerevisiae/paradoxus and Kluyveromyces marxianus. The different species of yeast might be introduced accidentally in artisanal yoghurt; however, for traditionally fermented milks their presence might be associated to the starter.

Characterizing an engineered carotenoid-producing yeast as an anti-stress chassis for building cell factories

BackgroundA microorganism engineered for non-native tasks may suffer stresses it never met before. Therefore, we examined whether a Kluyveromyces marxianus strain engineered with a carotenoid biosynthesis pathway can serve as an anti-stress chassis for building cell factories.ResultsCarotenoids, a family of antioxidants, are valuable natural products with high commercial potential. We showed that the free radical removal ability of carotenoids can confer the engineered host with a higher tolerance to ethanol, so that it can produce more bio-ethanol than the wild type. Moreover, we found that this engineered strain has improved tolerance to other toxic effects including furfurals, heavy metals such as arsenate (biomass contaminant) and isobutanol (end product). Furthermore, the enhanced ethanol tolerance of the host can be applied to bioconversion of a natural medicine that needs to use ethanol as the delivery solvent of hydrophobic precursors. The result suggested that the engineered yeast showed enhanced tolerance to ethanol-dissolved hydrophobic 10-deacetylbaccatin III, which is considered a sustainable precursor for paclitaxel (taxol) bioconversion.ConclusionsThe stress tolerances of the engineered yeast strain showed tolerance to several toxins, so it may serve as a chassis for cell factories to produce target products, and the co-production of carotenoids may make the biorefinary more cost-effective.

Potential production of 2-phenylethanol and 2-phenylethylacetate by non-Saccharomyces yeasts from Agave durangensis

The participation of non-Saccharomyces yeasts in fermentation processes is of great importance due to their participation in the formation of esters and superior alcohols, which confer characteristic aromas to beverages such as wine and mescal. The aim of this study was identify and evaluate the potential aroma production of yeast native of Agave fermentation by the mescal production in Durango, Mexico. Isolated yeasts were molecularly identified by 5.8s ribosomal gene; the potential production of aromas was carried out in fermentations with the addition of l-phenylalanine and evaluated after 24 h of fermentation. Extraction and quantification of aromatic compounds by headspace solid-phase micro-extraction (HS-SPME) and gas chromatograph mass spectrometry (GC-MS). The isolated non-Saccharomyces yeasts could be classified into six different genera Saccharomyces cerevisiae, Clavispora lusitaniae, Torulaspora delbrueckii, Kluyveromyces dobzhanskii, Kluyveromyces marxianus, and Kluyveromyces sp. All probed strains presented a potential aroma production (ethyl acetate, isoamyl acetate, isoamyl alcohol, benzaldehyde, 2-phenylethyl butyrate, and phenylethyl propionate), particularly 2-phenylethanol and 2-phenylethylacetate; the levels found in the Kluyveromyces marxianus ITD0211 yeast have the highest 2-phenylethylacetate production at 203 mg/L and Kluyveromyces marxianus ITD0090 with a production of 2-phenylethanol at 1024 mg/L. Non-Saccharomyces yeasts were isolated from the mescal fermentation in Durango; the Kluyveromyces genus is the most predominant. For the production of aromas, highlighting two strains of Kluyveromyces marxianus produces competitive quantities of compounds of great biotechnological interest such as 2-phenylethanol and 2-phenylethylacetate, without resorting to the genetic modification of yeasts or the optimization of the culture medium.

Effect of Temperature on Production Intracellular and Extracellular Invertase by Potential Indigenous Strain Kluyveromyces Marxianus Using Sugarcane Molasses

This study is focused on enzymolgy regarding enzyme purification technique and application of thermotolerant specie Kluyveromyces marxianus yeast in bio reactions for the research and optimization of fermentation temperature for intracellular and extracellular enzyme production. Fermentation studies were carried out in shake flask level to optimize intracellular and extracellular enzyme production by changing process conditions like temperature range from (30 to 55 0 C), the pH (5.5), speed (350). The substrate type sugar cane molasses 15% added as a carbon and ammonium sulphate (0.75%) as nitrogen source. The optimized fermentation temperature was found 45 o C, at pH 5.5, and rpm was 350. The production of intracellular invertase was (890µmoles/min/g) while the extracellular (120µmoles/min/g) Kluyveromyces marxianus was greater efficiency as compared to other specie because of its metabolic activity, which express more heat stability and Invertase activty upto 65 0 C. Keywords: Yeast, Fermentation, K.marxianus DOI : 10.7176/JNSR/9-9-02 Publication date :May 31 st 2019

Fermentative capabilities of native yeast strains grown on juices from different Agave species used for tequila and mezcal production.

The Asparagaceae family is endemic from America, being the Agave genus the most important. The Agave species possess economic relevance and are use as raw material to produce several distilled alcoholic beverages, as bacanora, tequila, and mezcal. The fermentation process has been carry out either spontaneously or by adding a selected yeast strain. The latter is generally responsible for the production of ethanol and volatile compounds. This study comprised five Agave species (A. angustifolia, A. cupreata, A. durangensis, A. salmiana, and A. tequilana) and eight endogenous yeast strains: five of them were non-Saccharomyces (Torulaspora delbrueckii, Zygosaccharomyces bisporus, Candida ethanolica, and two Kluyveromyces marxianus) and three Saccharomyces cerevisiae strains. The results showed that the S. cerevisiae strains were not able to grow on A. durangensis and A. salmiana juices. The Kluyveromyces marxianus strains grew and fermented all the agave juices and displayed high ethanol production (48-52gL-1) and volatile compounds. The ethanol production was higher on A. angustifolia juice (1.1-2.8-fold), whereas the volatile compound was dependent on both yeast strain and the Agave species. The use of endogenous non-Saccharomyces yeast strains is feasible, as they may outperform S. cerevisiae regarding the production of fermented beverages from agave plants with a high content of ethanol and aromatic compounds. Graphical abstract.

Cell Wall Surface Properties of Kluyveromyces marxianus Strains From Dairy-Products.

Thirty-three Kluyveromyces marxianus strains were tested for the ability to form biofilm and mat structures in YPD and whey and for cell surface hydrophobicity. To identify genes potentially involved in adhesion properties, a RT-qPCR analysis was performed. Eight strains were able to adhere on polystyrene plates in both media and formed a mature mat structure. These strains showed a different level of hydrophobicity ranging from 55 to 66% in YPD and from 69 to 81% in whey. Four K. marxianus orthologs genes (FLO11, STE12, TPK3, and WSC4), known from studies in other yeast to be involved in biofilm formation, have been studied. FLO11 and STE12 showed the highest fold changes in all conditions tested and especially in whey: 15.05 and 11.21, respectively. TPK3 was upregulated only in a strain, and WSC4 in 3 strains. In YPD, fold changes were lower than in whey with STE12 and FLO11 genes showing the highest fold changes. In mat structures FLO11 and STE12 fold changes ranged from 3.6–1.3 to 2–1.17, respectively. Further studies are necessary to better understand the role of these genes in K. marxianus adhesion ability.

The Influence of pH, NaCl, and the Deacidifying Yeasts Debaryomyces hansenii and Kluyveromyces marxianus on the Production of Pigments by the Cheese-Ripening Bacteria Arthrobacter arilaitensis.

Arthrobacter arilaitensis is a food-related bacterial species under investigation for its involvement in the coloration of surface-ripened cheeses. Presently, information about this species in association with the development of appropriate cheese coloration is still lacking. This study was performed in order to investigate—with the use of spectrocolorimetry—the influence of pH, NaCl, and deacidifying yeasts on the pigmentation of Arthrobacter arilaitensis biofilms. Three types of cheese-based (curd) solid media were prepared by using different deacidification methods: (i) chemical deacidification by NaOH (CMNaOH); (ii) biological deacidification by the yeast strain Debaryomyces hansenii 304 (CMDh304); and (iii) biological deacidification by the yeast strain Kluyveromyces marxianus 44 (CMKm44). Each medium was prepared with initial pH values of 5.8, 7.0, and 7.5. After pasteurization, agar was incorporated and NaCl was added in varying concentrations (0%, 2%, 4%, and 8% (w/v)). A. arilaitensis Po102 was then inoculated on the so prepared “solid-curd” media, and incubated at 12 °C under light conditions for 28 days. According to the data obtained by spectrocolorimetry in the Compagnie Internationale de l’Eclairage (CIE) L*a*b* color system, all controlled factors appeared to affect the pigments produced by the A. arilaitensis strain. NaCl content in the media showed distinct inhibitory effects on the development of color by this strain when the initial pH was at 5.8. By contrast, when the initial pH of the media was higher (7.0, 7.5), only the highest concentration of NaCl (8%) had this effect, while the coloring capacity of this bacterial species was always higher when D. hansenii 304 was used for deacidification compared to K. marxianus 44.

Efficient l-lactic acid production from corncob residue using metabolically engineered thermo-tolerant yeast

Lactic acid is an important industrial product and the production from inexpensive and renewable lignocellulose can reduce the cost and environmental pollution. In this study, a Kluyveromyces marxianus strain which produced lactic acid efficiently from corncob was constructed. Firstly, two of six different lactate dehydrogenases, which from Plasmodium falciparum and Bacillus subtilis, respectively, were proved to be effective for l-lactic acid production. Then, five single genetic modifications were conducted. The overexpression of Saccharomyces cerevisiae proton-coupled monocarboxylate transporter, K. marxianus 6-phosphofructokinase, or disruption of K. marxianus putative d-lactate dehydrogenase enhanced the l-lactic acid accumulation. Finally, the strain YKX071, obtained via combination of above effective genetic engineering, produced 103.00 g/L l-lactic acid at 42 °C with optical purity of 99.5% from corncob residue via simultaneous saccharification and co-fermentation. This study first developed an effective platform for high optical purity l-lactic acid production from lignocellulose using yeast with inexpensive nitrogen sources.