Kudriavzevii Strains Research Articles

Production and Characterization of Bioethanol Using Native Microbial Isolates from Decomposed Maize Cob via Simultaneous Saccharification and Fermentation

Maize cob a major component of agricultural waste composed mainly of cellulose that can be converted to bioethanol through fermentation as means of waste management. Achieving enhances bioethanol yield without ascertaining proper conversion pathway may be subjective. Therefore, this research aimed at converting maize cob to bioethanol through fermentation using natives microbial isolates from decomposed maize cob via Response Surface Methodology. Microbial strains used were Pichia kudriavzevii strains with accession number KP998095.1 and MN861069.1. A pH value of 5.5 corresponded to bioethanol yield of 52.45 g/L, pH was observed to be sensitive to fermentation hence influence the extent of fermentation process. Optical density (O. D610nm) of 2.2 after eight (8) days of fermentation corresponded to bioethanol yield of 52.45 g/L. The growth pattern observed in most of the samples followed a typical microbial growth pattern depicting lag, log and stationary phases. Reducing soluble sugar of 23.5 mg/L on enzyme hydrolysis corresponded to bioethanol yield of 52.45 g/L, reducing soluble sugar content of the experimental samples were observed to decrease as the fermentation progresses. The infrared spectra of the optimum sample distillate of bioethanol from the fermentation revealed the presence of OH at 3311.7 cm-1 stretching band and C = C at 1636.3 cm−1 stretching band. The elemental compositions of the distillate were oxygen (31.03%), hydrogen (9.74%), carbon (56.85%), nitrogen (1.92%) and sulphur (0.46%) which confirmed the presence of bioethanol. The empirical formula of the best distillate using C, H, and O compositions (%) was found to be C2H5OH, hence revealed the presence of bioethanol. The result of the GC-MS analysis showed that mass spectra comparison of the various peaks revealed the presence of bioethanol as the largest peak with 76.38% ethanol by concentration. Maize cob can be said to be a promising feedstock for bioethanol production using native microbial isolates.

Evaluation of antifungal and apoptotic effects of linalool, citral, and carvacrol separately and in combination with nystatin against clinical isolates of Pichia kudriavzevii

Pichia kudriavzevii (formerly Candida krusei) poses a significant threat to immunocompromised patients due to its inherent resistance to various antifungal drugs. This study explored the anticandidal potential of citral, linalool, and carvacrol in combination with nystatin against P. kudriavzevii strains.Using the microdilution method following CLSI guidelines, Minimum Inhibitory Concentrations (MICs) and fungicidal concentrations (MFCs) were determined. Citral exhibited MIC values ranging from 50 to 100 µg/ml, averaging 70.24 ± 16.99 µg/ml, while carvacrol had MIC values of 50 to 100 µg/ml, averaging 86.90 ± 16.99 µg/ml. Linalool demonstrated weaker antifungal activity, with MIC values between 100 and 200 µg/ml, averaging 150 ± 38.73 µg/ml. The study assessed the synergistic effectsof these phenols with nystatin through fractional inhibitory concentration indices (FICIS). In addition, flow cytometry was employed to assess apoptosis induction in P. kudriavzevii cells.Carvacrol displayed a remarkable synergistic effect in combination with nystatin against all 21 isolates tested. Conversely, linalool showed synergy in 17 isolates, while citral exhibited synergy in only 2 isolates. These findings highlight distinct patterns of synergy between the different compounds and nystatin against P. kudriavzevii. Also, Carvacrol emerged as the most potent inducer of apoptosis across all P. kudriavzevii strains, followed by citral and linalool. This suggests that carvacrol not only possesses a stronger antifungal effect but also has a more pronounced ability to trigger programmed cell death in P. kudriavzevii. In conclusion, the study supports the potential of carvacrol, citral and linalool, as anticandidal agents, suggesting their supplementation with nystatin for treating P. kudriavzevii infections.

In Vitro Characterization and Identification of Potential Probiotic Yeasts Isolated from Zaopocu, a Traditional Fermented Dregs Vinegar from Hainan Island.

Recently, there has been an increasing interest in researching fermented food-derived yeasts as probiotics because they offer a natural and diverse source of potential strains with unique functional properties and health benefits. In this study, 13 yeast strains isolated from Zaopocu (ZPC), a traditional fermented dregs vinegar on Hainan Island, China, were evaluated for their probiotic characteristics in vitro. Yeast identification was conducted through 5.8S-ITS region sequencing, revealing Kodamaea ohmeri as the predominantly isolated species (ZPC_Y3, Y5, Y6, Y11), followed by Pichia kudriavzevii (ZPC_Y2, Y13, Y14), Rhodotorula mucilaginosa (ZPC_Y9, Y10), Pichia fermentans (ZPC_Y8, Y12), Pichia kluyveri (ZPC_Y4), and Pichia occidentalis (ZPC_Y1). Except for ZPC_Y4, ZPC_Y8, and ZPC_Y12, all isolated yeasts exhibited stable growth at 37°C. The survival rates of all test strains exceeded 60% under challenging conditions at pH = 2 and 0.3% bile salt, along with strong antioxidant activity (> 5 6%), notable autoaggregation (> 70%), and varying levels of cell hydrophobicity with xylene (ranging from 35.32 ± 8.57% to 89.73 ± 4.84%). In addition, all isolates showed resistance to multiple antibiotics, along with antagonistic activity, and were deemed safe as none exhibited hemolytic, gelatinase, or DNase activities. Significantly, two P. kudriavzevii strains (ZPC_Y2, Y14) exhibited the production of catalase, lipase, and β-galactosidase, along with the capacity to synthesize gamma-aminobutyric acid (GABA). In summary, this preliminary study represents the first attempt to identify and characterize potential probiotic yeast strains isolated from Zaopocu, providing a theoretical basis for exploring their application in developing novel therapeutic probiotics.

Differential consumption of malic acid and fructose in apple musts by Pichia kudriavzevii strains.

To assess the capability of Pichia kudriavzevii strains isolated from wine, cider, and natural environments in North Patagonia to produce ciders with reduced malic acid levels. Fermentation kinetics and malic acid consumption were assessed in synthetic media and in regional acidic apple musts. All P. kudriavzevii strains degraded malic acid and grew in synthetic media with malic acid as the sole carbon source. Among these strains, those isolated from cider exhibited higher fermentative capacity, mainly due to increased fructose utilization; however, a low capacity to consume sucrose present in the must was also observed for all strains. The NPCC1651 cider strain stood out for its malic acid consumption ability in high-malic acid Granny Smith apple must. Additionally, this strain produced high levels of glycerol as well as acceptable levels of acetic acid. On the other hand, Saccharomyces cerevisiae ÑIF8 reference strain isolated from Patagonian wine completely consumed reducing sugars and sucrose and showed an important capacity for malic acid consumption in apple must fermentations. Pichia kudriavzevii NPCC1651 strain isolated from cider evidenced interesting features for the consumption of malic acid and fructose in ciders.

Adaptive laboratory evolution under acetic acid stress enhances the multistress tolerance and ethanol production efficiency of Pichia kudriavzevii from lignocellulosic biomass

Second-generation bioethanol production using lignocellulosic biomass as feedstock requires a highly efficient multistress-tolerant yeast. This study aimed to develop a robust yeast strain of P. kudriavzevii via the adaptive laboratory evolution (ALE) technique. The parental strain of P. kudriavzevii was subjected to repetitive long-term cultivation in medium supplemented with a gradually increasing concentration of acetic acid, the major weak acid liberated during the lignocellulosic pretreatment process. Three evolved P. kudriavzevii strains, namely, PkAC-7, PkAC-8, and PkAC-9, obtained in this study exhibited significantly higher resistance toward multiple stressors, including heat, ethanol, osmotic stress, acetic acid, formic acid, furfural, 5-(hydroxymethyl) furfural (5-HMF), and vanillin. The fermentation efficiency of the evolved strains was also improved, yielding a higher ethanol concentration, productivity, and yield than the parental strain, using undetoxified sugarcane bagasse hydrolysate as feedstock. These findings provide evidence that ALE is a practical approach for increasing the multistress tolerance of P. kudriavzevii for stable and efficient second-generation bioethanol production from lignocellulosic biomass.

Towards unlocking the biocontrol potential of Pichia kudriavzevii for plant fungal diseases: in vitro and in vivo assessments with candidate secreted protein prediction

BackgroundPlant fungal pathogens cause substantial economic losses through crop yield reduction and post-harvest storage losses. The utilization of biocontrol agents presents a sustainable strategy to manage plant diseases, reducing the reliance on hazardous chemical. Recently, Pichia kudriavzevii has emerged as a promising biocontrol agent because of its capacity to inhibit fungal growth, offering a potential solution for plant disease management.ResultsTwo novel Pichia kudriavzevii strains, Pk_EgyACGEB_O1 and Pk_EgyACGEB_O2, were isolated from olive brine samples. The microscopic characterization of the strains revealed similar structures. However, there were noticeable differences in their visual morphology. Based on their internal transcribed spacer (ITS) DNA sequences, Pk_EgyACGEB_O1 and Pk_EgyACGEB_O2 strains assigned by GenBank IDs MZ507552.1 and MZ507554.1 shared high sequence similarity (~ 99.8% and 99.5%) with P. kudriavzevii, respectively. Both strains were evaluated in vitro against plant pathogenic fungi. The strains revealed the ability to consistently inhibit fungal growth, with Pk_EgyACGEB_O2 showing higher effectiveness. In addition, both P. kudriavzevii strains effectively controlled grey mold disease caused by B. cinerea in golden delicious apples, suggesting their potential as sustainable and eco-friendly biocontrol agents for post-harvest diseases. Based on a comprehensive bioinformatics pipeline, candidate-secreted proteins responsible for the potent antifungal activity of P. kudriavzevii were identified. A total of 59 proteins were identified as common among the P. kudriavzevii CBS573, SD108, and SD129 strains. Approximately 23% of the secreted proteins in the P. kudriavzevii predicted secretome are hydrolases with various activities, including proteases, lipases, glycosidases, phosphatases, esterases, carboxypeptidases, or peptidases. In addition, a set of cell-wall-related proteins was identified, which might enhance the biocontrol activity of P. kudriavzevii by preserving the structure and integrity of the cell wall. A papain inhibitor was also identified and could potentially offer a supplementary defense against plant pathogens.ConclusionOur results revealed the biocontrol capabilities of P. kudriavzevii against plant pathogenic fungi. The research focused on screening novel strains for their ability to inhibit the growth of common pathogens, both in vitro and in vivo. This study shed light on how P. kudriavzevii interacts with fungal pathogens. The findings can help develop effective strategies for managing plant diseases.

Structure prediction, docking studies and molecular cloning of novel Pichia kudriavzevii YK46 metalloprotease (MetPr) for improvement of feather waste biodegradation

This study addresses the environmental risks associated with the accumulation of keratin waste from poultry, which is resistant to conventional protein degradation methods. To tackle this issue, microbial keratinases have emerged as promising tools for transforming resilient keratin materials into valuable products. We focus on the Metalloprotease (MetPr) gene isolated from novel Pichia kudriavzevii YK46, sequenced, and deposited in the NCBI GenBank database with the accession number OQ511281. The MetPr gene encodes a protein consisting of 557 amino acids and demonstrates a keratinase activity of 164.04 U/ml. The 3D structure of the protein was validated using Ramachandran's plot, revealing that 93% and 97.26% of the 557 residues were situated within the most favoured region for the MetPr proteins of template Pichia kudriavzevii strain 129 and Pichia kudriavzevii YK46, respectively. Computational analyses were employed to determine the binding affinities between the deduced protein and beta keratin. Molecular docking studies elucidated the optimal binding affinities between the metalloprotease (MetPr) and beta-keratin, yielding values of − 260.75 kcal/mol and − 257.02 kcal/mol for the template strains Pichia kudriavzevii strain 129 and Pichia kudriavzevii YK46, respectively. Subsequent molecular cloning and expression of the MetPr gene in E. coli DH5α led to a significantly higher keratinase activity of 281 ± 12.34 U/ml. These findings provide valuable insights into the potential of the MetPr gene and its encoded protein for keratin waste biotransformation, with implications for addressing environmental concerns related to keratinous waste accumulation.

In Vitro Evaluation of the Technological and Probiotic Potential of Pichia kudriavzevii Strains Isolated from Traditional Fermented Foods.

The isolation of endogenous yeast strains from traditionally fermented food products to use as functional starter cultures has become more popular for improved food safety, quality, and beneficial health effects. In this study, 107 Pichia kudriavzevii strains were isolated from sourdough, shalgam, tarhana, artisanal Tulum cheese, and yogurt. The strains were identified by DNA fingerprinting using iPBS-PCR method before technological and probiotic characterization. The multivariate statistical approach revealed that five strains were most promising in terms of technological characterization, including different harsh growth conditions. These strains were also examined in terms of probiotic properties with a commercial S. cerevisiae var boulardii MYA-796 strain. The multivariate statistical analyses indicated that P. kudriavzevii 5S5 were most promising in in vitro probiotic properties such as surviving in human GI conditions, adhering to intestinal cell lines, and exhibiting high hydrophobicity. Therefore, it seems to be a great starter candidate for the production of functional fermented food products.

Deoxynivalenol Detoxification by a Novel Strain of Pichia kudriavzevii via Enzymatic Degradation and Cell Wall Adsorption.

Deoxynivalenol (DON) is a mycotoxin that significantly threatens the food and feed industry. Corn steep liquor (CSL) is an acidic byproduct of the corn starch industry, and DON is concentrated in CSL once the material is contaminated. In this work, a Pichia kudriavzevii strain that could remove DON from CSL was isolated and characterized. The strain P. kudriavzevii E4-205 showed detoxifying activity in a pH range of 4.0~7.0 and temperature of 25~42 °C, and 39.4% DON was reduced by incubating this strain in CSL supernatant diluted by 2-fold (5 μg/mL DON) for 48 h at pH 5.0 and 30 °C. Further mechanism studies showed that P. kudriavzevii E4-205 could adsorb DON by the cell wall and degrade DON by intracellular enzymes with NADH as a cofactor. The degradation product was identified as 3,7,8,15-tetrahydroxyscirpene by liquid chromatography-tandem mass spectrometry. DON adsorption by inactivated cells was characterized, and the adsorption followed pseudo first-order kinetics. This study revealed a novel mechanism by which microbes degrade DON and might serve as a guide for the development of DON biological detoxification methods.

Saccharification of starchy food waste through thermochemical and enzymatic pretreatment, towards enhanced bioethanol production via newly isolated non-conventional yeast strains

A starchy food waste containing mainly cooked wasted rice (WR) was exploited for bioethanol production using novel yeast strains was investigated. Different pretreatment schemes of the waste at solids loading 10%–30% TS WR (w/v) i.e. enzymatic, thermochemical and combined thermochemical/enzymatic pretreatment, were evaluated aiming to the maximum liberation of fermentable carbohydrates and their subsequent bioconversion to ethanol. Fermentation tests of the whole pretreated slurries were initially performed with the yeasts strains that were identified as Kluyveromyces marxianus isolate V3-19,Pichia kudriavzevii strain YF1702 and K. marxianus strain TTG-428, and their fermentation efficiencies (FE) were comparatively assessed. It was shown that the combined pretreatment led to the maximum saccharification, whereas FEs were higher for K. marxianus, V3-19, exceeding 90% of the theoretical maximum. In the case of the highest organic loading of WR, though, up to 25% of soluble carbohydrates remained unexploitable after 72 h of fermentation, indicating that kinetic restrictions occurred in the process. Further experiments with the hydrolysates that were recovered after combined pretreatment, revealed that the removal of solids enhances the consumption of sugars and leads to complete uptake for the loading 20% TS WR (w/v).

A thermotolerant yeast from cow's rumen utilize lignocellulosic biomass from wheat straw for xylanase production and fermentation to ethanol

For recycling agricultural wastes into the production of biofuel, fermentation technology is currently gaining more attention. Bioethanol is a widely used biofuel, however in industries, the fermentation of hemicellulosic feedstock is challenging since fermenting yeasts consume fewer xylose. To enhance the availability of renewable biofuels, Pioneers are keen to develop industrial strains that assimilate xylose along with glucose. In attempt to eliminate the obstacle, xylanolytic yeast with enhanced xylanase production were investigated. A thermotolerant yeast species, Pichia kudriavzevii strain SVMS2019, isolated from the cow's rumen was studied for xylanase production under solid-state fermentation with simultaneous degradation and saccharification of alkali-treated wheat straw biomass for the liberation of xylose sugars and then conversion into bioethanol. After statistical optimization through response surface methodology, the xylanase enzyme yield was enhanced up to 2.23 fold; the optimum conditions were 30 °C, pH 3.5, 72 h of incubation, and 1.5% of pretreated wheat straw to produce 273.02 IU/ml xylanase activity. The maximum of crude xylanase activity was shown at 50 °C and pH 6. The fermentation of xylose to ethanol by Pichia kudriavzevii SVMS2019, bioethanol was obtained at a maximum of about 3.18% at 42 °C in 72 h intervals. Through the novel source, we have obtained the strain that has a significant of hemicellulosic enzymatic saccharification with 2.23 fold increase in xylanase production yield through statistical optimization and produce bioethanol. While xylanolytic activity of yeast has been optimized, potential benefit of cellulosic hydrolytic activity also need to be studied to achieve the co-utilization of glucose and xylose.

Isolation, Identification, and Characterization of an Acid-Tolerant Pichia kudriavzevii and Exploration of Its Acetic Acid Tolerance Mechanism

Pichia kudriavzevii is one of the major non-Saccharomyces cerevisiae yeasts in Chinese baijiu brewing, which has shown a substantially higher tolerance to acid, heat, and ethanol. Exploring the mechanism of P. kudriavzevii could have a positive effect on the artificially controlled production of baijiu. In this study, an efficient acetic-acid-tolerant P. kudriavzevii strain, Y2, was isolated from the yellow water of strong-flavored baijiu brewing waste, and its molecular mechanism of acetic acid tolerance was investigated through a comparative transcriptomic analysis. The strain Y2 could tolerate 12 g/L of acetic acid. According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, differentially expressed genes (DEGs) were mainly enriched in oxidative phosphorylation, the citrate cycle, glycolysis/gluconeogenesis, and carbon metabolism under low (AL group) and high (AH group) concentrations of acetic acid. However, the DEG enrichment was more profound in the AH group when compared to the control. Compared with the AL group, the expression of genes related to oxidative phosphorylation was more significantly upregulated, while in terms of the TCA cycle, phosphoenolpyruvate carboxykinase was significantly upregulated in both the AH and AL groups and was positively correlated with tolerance to acetic acid. This was followed by citrate synthase, isocitrate dehydrogenase, malate dehydrogenase, and succinate dehydrogenase. These results illustrated a possible mechanism of acid tolerance by regulating the metabolism-related pathways in P. kudriavzevii and provided a basis for the further investigation of the acid tolerance mechanism.

Characterization of Yeast Isolated from Berkeep, a Traditional Fermented Milk Products of Darfur State, Western Sudan

Berkeep is a naturally fermented yoghurt-like milk product consumed as common food in parts of the Darfur State, Western Sudan. Its production and consumption derive much food security and economic benefits to the rural people in the region. The objective of this study was to isolate and characterize yeasts of Berkeep by both conventional and molecular-based methods. A total of 220 yeast isolates, were obtained from 75 samples of Berkeep from three production sites namely, Elkoumah, Manwashy and Kass in Darfur state, were purified then characterized to species level by molecular tools. Based on ITS-PCR using the forward primer Cy5-Y-ITS1 and reverse primer Y-ITS4, the isolates were screened into five groups. Representative of each group was selected for sequence analysis of ITS rRNA gene. The gene sequence was subjected to nucleotide BLAST. The yeast microbiota of Berkeep was dominated by Kluyveromyces marxianus (YG4, 85 isolates) which showed 100% homology towards Kluyveromyces marxianus isolate QKM4. 14.54% (32 isolates), Sixty-eight (Group YG5) identified as Saccharomyces cerevisiae showed 97.22% homology towards Saccharomyces cerevisiae isolate JKSP39. Group YG3 revealed 100% homology towards Pichia kudriavzevii isolate Sugarcane. Eleven isolates, (YG2) showed 99.37% homology towards Pichia kudriavzevii strain YS179. The twenty-four isolates of group, (YG1) recorded 99.70% similarity towards Candida intermedia isolate B-NC-12-f 16i. Very minute variation was observed with in the three production sites concerning the yeast count and type of yeast species.

Saccharomyces cerevisiae wine strains show a wide range of competitive abilities and differential nutrient uptake behavior in co-culture with S. kudriavzevii

The wine industry has implemented complex starters with multiple yeast species as an efficient method to improve certain wine properties. Strains’ competitive fitness becomes essential for its use in such cases. In the present work, we studied this trait in 60 S. cerevisiae strains from different origins, co-inoculated with a S. kudriavzevii strain, and confirmed it to be associated with the strains' origin. To gather deeper knowledge about the characteristics of strains with highly competitive ability versus the rest, microfermentations using representative strains from each group were performed and the carbon and nitrogen sources uptake was analysed. Our results demonstrate that despite wine strains being the subclade with the highest competitive ability, they present a wide range of behaviors as well as nutrient uptake dynamics, which points to a heterogeneous nature of domestication processes. An interesting strategy was observed in the highly competitive strains (GRE and QA23), the nitrogen sources uptake in the competition was accelerated and the sugar fermentation was slowing despite the fermentation finish at the same time. Therefore, this competition study, using particular combinations of strains, expands the knowledge in the field of the usage of mixed starters in wine manufactured products.

Lactic acid bacteria and yeast strains isolated from fermented fish (Budu) identified as candidate ruminant probiotics based on in vitro rumen fermentation characteristics.

Probiotic supplementation can assist with manipulating the rumen microbial ecosystem. Lactic acid bacteria and yeast from fermented fish (Budu) as the indigenous food from West Sumatra, Indonesia, are potential probiotics for livestock. This study aims to select the best candidate lactic acid bacteria and yeast strains from fermented fish as ruminant probiotics and evaluate the effect of their supplementation on the characteristics of rumen fermentation, feed digestion, and total gas production in vitro. This study used nine treatments, performed in triplicate, in a completely randomized design. The substrate ratio comprised of 70% Pennisetum purpureum forage and 30% concentrate. Five lactic acid bacteria and three yeast isolates were used in this study. Treatments were as follows: T0: control (basal diet); T1: T0 + Lactobacillus parabuchneri strain 3347; T2: T0 + Lactobacillus buchneri strain 5296; T3: T0 + Lactobacillus harbinensis JCM 16178; T4: T0 + Schleiferilactobacillus harbinensis strain LH991; T5: T0 + L. parabuchneri strain 6902; T6: T0 + Pichia kudriavzevii strain B-5P; T7: T0 + P. kudriavzevii strain CBS 5147; and T8: T0 + commercial yeast (Saccharomyces cerevisiae). The lactic acid bacteria inoculum contained 1.02 × 1011 colony-forming unit (CFU)/mL, while the yeast inoculum contained 1.5 × 1010 CFU/mL. The results showed that four lactic acid bacteria and three yeast produced a higher total gas yield (104-183.33 mL) compared to the control (103 mL). Supplementation with lactic acid bacteria in the rumen fermentation in vitro showed dry matter digestibility of 63%-70% and organic matter digestibility (OMD) of 64%-71%. We observed that total volatile fatty acid (VFA) production in all treatments was significantly higher (86-121 mM) compared to the control (81 mM). The concentration of NH3 production was higher in all treatments (12.33-16.83 mM) than in the control (12.25 mM). Meanwhile, the probiotic supplementation did not cause a significant change in the rumen pH (6.86-7.12). Supplementation with the lactic acid bacteria S. harbinensis strain LH991 consistently demonstrated the best results from the parameters of dry and OMD (70.29% and 71.16%, respectively), total VFA (121.67 mM), NH3 (16.83 mM), and total gas production (149.17 mL). The best results were observed from the yeast candidate P. kudriavzevii strain B-5P, where the results were dry and OMD (67.64% and 69.55% respectively), total VFA (96.67 mM), NH3 (13.42 mM), and total gas production (183.33 mL). Based on the obtained results, lactic acid bacteria S. harbinensis strain LH991 and yeast P. kudriavzevii strain B-5P are attractive candidates to be utilized as probiotics for ruminants based on their potential to improve rumen fermentation in vitro. This probiotic supplementation can increase the digestibility of feed ingredients, production of total VFA and NH3, and total gas produced.

Adaptation of Saccharomyces Species to High-Iron Conditions.

Iron is an indispensable element that participates as an essential cofactor in multiple biological processes. However, when present in excess, iron can engage in redox reactions that generate reactive oxygen species that damage cells at multiple levels. In this report, we characterized the response of budding yeast species from the Saccharomyces genus to elevated environmental iron concentrations. We have observed that S. cerevisiae strains are more resistant to high-iron concentrations than Saccharomyces non-cerevisiae species. Liquid growth assays showed that species evolutionarily closer to S. cerevisiae, such as S. paradoxus, S. jurei, S. mikatae, and S. arboricola, were more resistant to high-iron levels than the more distant species S. eubayanus and S. uvarum. Remarkably, S. kudriavzevii strains were especially iron sensitive. Growth assays in solid media suggested that S. cerevisiae and S. paradoxus were more resistant to the oxidative stress caused by elevated iron concentrations. When comparing iron accumulation and sensitivity, different patterns were observed. As previously described for S. cerevisiae, S. uvarum and particular strains of S. kudriavzevii and S. paradoxus became more sensitive to iron while accumulating more intracellular iron levels. However, no remarkable changes in intracellular iron accumulation were observed for the remainder of species. These results indicate that different mechanisms of response to elevated iron concentrations exist in the different species of the genus Saccharomyces.

Endogenous Ethanol and Triglyceride Production by Gut Pichia kudriavzevii, Candida albicans and Candida glabrata Yeasts in Non-Alcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) increases with fructose consumption and metabolic syndrome and has been recently linked with endogenous ethanol production, notably by high alcohol-producing Klebsiella pneumoniae (HiAlc Kpn). Candida yeasts are the main causes of auto-brewery syndromes but have been neglected in NASH. Here, the fecal ethanol and microbial content of 10 cases and 10 controls were compared. Ethanol was measured by gas chromatography-mass spectrometry. Species identification was performed by MALDI-TOF MS, and triglyceride production was assessed by a colorimetric enzymatic assay. The fecal ethanol concentration was four times higher in patients with NASH (median [interquartile range]: 0.13 [0.05-1.43] vs. 0.034 [0.008-0.57], p = 0.037). Yeasts were isolated from almost all cases but not from controls (9/10 vs. 0/10, p = 0.0001). Pichia kudriavzevii was the most frequent (four patients), while Candida glabrata, Candida albicans, and Galactomyces geotrichum were identified in two cases each. The concentration of ethanol produced by yeasts was 10 times higher than that produced by bacteria (median, 3.36 [0.49-5.60] vs. 0.32 [0.009-0.43], p = 0.0029). Using a 10% D-fructose restricted medium, we showed that NASH-associated yeasts transformed fructose in ethanol. Unexpectedly, yeasts isolated from NASH patients produced a substantial amount of triglycerides. Pichia kudriavzevii strains produced the maximal ethanol and triglyceride levels in vitro. Our preliminary human descriptive and in vitro experimental results suggest that yeasts have been neglected. In addition to K. pneumoniae, gut Pichia and Candida yeasts could be linked with NASH pathophysiology in a species- and strain-specific manner through fructose-dependent endogenous alcohol and triglyceride production.

The ability of Pichia kudriavzevii to tolerate multiple stresses makes it promising for developing improved bioethanol production processes.

Thermotolerant ethanol fermenting yeasts have been extensively used in industrial bioethanol production. However, little is known about yeast physiology under stress during bioethanol processing. This study investigated the physiological characteristics of the thermotolerant yeast Pichia kudriavzevii, strains NUNS-4, NUNS-5 and NUNS-6, under the multiple stresses of heat, ethanol and sodium chloride. Results showed that NUNS-4, NUNS-5 and NUNS-6 displayed higher growth rates under each stress condition than the reference strain, Saccharomyces cerevisiae TISTR5606. Maximum specific growth rates under stresses of heat (45°C), 15% v/v ethanol and 1·0 M sodium chloride were 0·23 ± 0·04 (NUNS-4), 0·11 ± 0·01 (NUNS-5) and 0·15 ± 0·01 h-1 (NUNS-5), respectively. Morphological features of all yeast studied changed distinctly with the production of granules and vacuoles when exposed to ethanol, and cells were elongated under increased sodium chloride concentration. This study suggests that the three P. kudriavzevii strains are potential candidates to use in industrial-scale fermentation due to a high specific growth rate under multiple stress conditions. Multiple stress-tolerant P. kudriavzevii NUNS strains have received much attention not only for improving large-scale fuel ethanol production, but also for utilizing these strains in other biotechnological industries.

Identifikasi Jamur Pada Pupuk Organik Cair (POC) Limbah Dapur Di Desa Sanur Kauh

Identification of Fungi in Kitchen Waste Liquid Organic Fertilizer (LOF) in Sanur Kauh Village. Decomposed kitchen waste contains certain microorganisms, one of which is fungus. Fungi are one of the microorganisms that are widespread in soil and water and have the potential in the process of decomposition of organic matter. The research objective was to determine the composition of kitchen waste and obtain fungi species from LOF of kitchen waste in Sanur Kauh Village. The research was conducted from September to December 2020. Sampling was conducted in Sanur Kauh Village, South Denpasar District. Furthermore, LOF making and analysis was carried out at the Laboratory of Soil and Environmental Sciences, Faculty of Agriculture, Udayana University and at the Indonesian Genetika Science Laboratory, Tangerang. The method used is a descriptive analysis experiment, which consists of field exploration, laboratory analysis and molecular identification. The results showed that the most dominant percentage of LOF from kitchen waste composition was fruit at 46.69% followed by vegetables at 24.17%, rice at 18.92%, egg shells, bones and meat at 5.16% and the remaining side dishes of 5.06%. The results of molecular identification of fungal species on LOF fermentation of kitchen waste isolates LDA 2 and LDB 2 were similar to Pichia kudriavzevii strain CBS5174 chromosome 2 (99.71%) and Pichia kudriavzevii culture CBS: 5147 (99.56%).

ASSESSMENT OF PAINT-PIGMENT DEGRADING MICROORGANISMS FROM PAINT INDUSTRIES EFFLUENT-CONTAMINATED SITES IN ABA, SOUTH-EAST NIGERIA

The potential of microorganisms isolated from paint industry effluent-polluted soils to degrade the constituents of the effluents and contaminant removal efficiency after the remediation period were evaluated. Samples were collected from six different paint industries in Aba. Samples from the various paint industries reveals sample F showed the highest bacterial plate counts of 6.21±0.20 x 104cfu/g while samples B and D had the least bacterial counts of 1.30±0.20 x 104cfu/g and 1.30±0.31 x 104cfu/g respectively. The fungal counts of the polluted soils indicates sample D as having the highest count of 2.30±0.24 x 104cfu/g while sample A had the least count of 1.00±0.30 x104cfu/g. Total Paint Utilizing Fungal (TPUF) counts ranges from 1.23±0.02 x 104cfu/g to 3.45±0.10 x 104cfu/g and showed a significant difference between sample C and the control (p<0.05). The bacterial isolates from the contaminated soil include Pseudomonas spp., Escherichia coli, Citrobacter spp., Klebsiella spp., Proteus spp., Enterobacter spp., Providencia rettgeri, Shigella flexneri, Staphylococcus spp., Streptococcus spp. and Enterococcus spp. while the fungal isolates include Saccharomyces cerevisiae, Rhodotorula species, Rhizopus species, Aspergillus niger, Aspergillus flavus and Penicillum notatum. The counts of actual paint utilizing fungi were relatively lower than the heterotrophic groups. The paint degrading microorganisms isolated from the paint industry effluent polluted soils include paint effluent bacterial degraders: Pseudomonas spp. (33.3%), Escherichia coli(33.3%),, Citrobacter spp. (25%), Klebsiella spp.(25%), Proteus spp.(25%), Enterobacter spp.(12.5%). and Providencia rettgeri(12.5%), While the fungal paint effluent degraders include Geotrichum candidum(33.3%), Pichia kudriavzevii(33.3%), Fusarium sp.(25%), Aspergillus niger(25%), Penicillium funiculosum(25%) and Mucor sp.(12.5%). Geotrichum candidum strain GAD1 (MN63874.1) and Pichia kudriavzevii strain (MK811096.1) showed highest degrees of growth indicated by high turbidities during the screening test. This study showed that majority of the Indigenous organisms has the ability to utilize paints as their sole carbon source hence are the major bioremediating agents in the sampling areas.