Microbiology

The overexpression of one single cbh gene making Trichoderma asperellum T-1 a better cellulase producer Abstract Trichoderma asperellum T-1, a traditional bio-control strain, is previously found to be potentially useful in the degradation of natural waste lignocellulose as it can ferment the natural materials without pretreatment. Many problems caused by substrate pretreatment can be therefore avoided. In this study, we intended to engineer a new strain to enhance its lignocellulose degradation ability by modifying the genome of T. asperellum T-1. A genetic transformation system mediated by Agrobacterium tumefaciens AGL-1 (ATMT) was constructed on T. asperellum T-1. On this basis, the overexpressed strain was produced by transforming a recombinant cellobiosidase gene (cbh) under the control of inducible promoter of endo-1, 4-β-xylanase gene, into wild-type T. asperellum T-1. After resistance screening, multiple transmission, growth comparison, and enzyme activity determination, four transformants (M1, M2, M5, and M6) were obtained. Filter paper cellulase activity of these transformants reached, respectively, 36.2%, 30.6%, 32.9%, and 42.7% higher than the wild-type strain. Most importantly, the CMCase, β-glucosidase and xylanase activity were also increased, although only one cbh gene was overexpressed. This work indicated that the enhancement of cellulase production ability of T. asperellumT-1 can be promisingly feasible by genetic modification. And the xylanase gene's promoter can be effectively used in genetic modification to promote T. asperellum T-1 to be more effectively used in lignocellulose degradation.

The effect of pitching rate on the production of higher alcohols by top-fermenting yeast in wheat beer fermentation Abstract Purpose The level of higher alcohols on top-fermentation determines the flavor profile and is one of the most important elements dictating the favorable top-fermented wheat beer (Ale beer) development. The optimization of the pitching rate has been shown to be crucial for industrial beer brewing. This study focused on understanding the effect of the variable inoculum size on the synthesis of higher alcohols. Methods We utilized sequencing to investigate the transcript changes under different inoculum sizes and link the results to fermentation performance. Results Variable cell inoculum density levels were linked with differences in higher alcohol production. Specifically, we observed significantly less higher alcohols produced at lower cell inoculum density during the stationary phase. Importantly, the accumulation of higher alcohols during the exponential growth phase was overall similar between different pitching rates. Moreover, free amino nitrogen (FAN) consumption and yeast cell viability were significantly decreased during stationary phase at the lowest inoculum density. Transcriptomic analysis revealed that amino acid metabolism genes ALD4, ALD6, ARO9, ARO10, and PUT1 were differentially expressed once the cells entered the declining growth phase at the lowest inoculum size. Conclusion The results suggest that the variable accumulation of higher alcohols in the top-fermenting yeast at different inoculum sizes is mostly accounted for in the stationary phase. We discovered that lower pitching rate was associated with a negative effect on amino acid metabolism and synthesis of higher alcohols during the stationary phase, leading to the decrease in higher alcohol concentration at low inoculum densities. Overall, our study provides valuable insights that could benefit wheat beer production.

Isolation and characterization of thermotolerant yeasts for the production of second-generation bioethanol Abstract The purpose of this study was to isolate, identify, and characterize the thermotolerant yeasts for use in high-temperature ethanol fermentation. Thermotolerant yeasts were isolated and screened from soil samples collected from the Mekong Delta, Vietnam, using the enrichment method. Classification and identification of the selected thermotolerant yeasts were performed using matrix-assisted laser desorption ionization/time-of-fight mass spectrometry (MALDI-TOF/MS) and nucleotide sequencing of the D1/D2 domain of the 26S rDNA and the internal transcribed spacer (ITS) 1 and 2 regions. The ethanol production by the selected thermotolerant yeast was carried out using pineapple waste hydrolysate (PWH) as feedstock. A total of 174 yeast isolates were obtained from 80 soil samples collected from 13 provinces in the Mekong Delta, Vietnam. Using MALDI-TOF/MS and nucleotide sequencing of the D1/D2 domain and the ITS 1 and 2 regions, six different yeast species were identified, including Meyerozyma caribbica, Saccharomyces cerevisiae, Candida tropicalis, Torulaspora globosa, Pichia manshurica, and Pichia kudriavzevii. Among the isolated thermotolerant yeasts, P. kudriavzevii CM4.2 displayed great potential for high-temperature ethanol fermentation. The maximum ethanol concentration (36.91 g/L) and volumetric ethanol productivity (4.10 g/L h) produced at 45 °C by P. kudriavzevii CM4.2 were achieved using PWH containing 103.08 g/L of total sugars as a feedstock. These findings clearly demonstrate that the newly isolated thermotolerant yeast P. kudriavzevii CM4.2 has a high potential for second-generation bioethanol production at high temperature.

Saccharification of Parthenium hysterophorus biomass using cellulase from Streptomyces sp. NAA2 Abstract Parthenium hysterophorus biomass can be used as a non-conventional renewable feedstock for the production of bioethanol. Therefore, the present work was designed to hydrolyze P. hysterophorus biomass using cellulase enzyme produced from an actinomycete, i.e., Streptomyces sp. NAA2 using P. hysterophorus biomass as a substrate. The isolate NAA2 was identified by molecular characterization of 16SrDNA. The enzyme production by strain NAA2 was enhanced by optimization studies conducted under submerged fermentation conditions using P. hysterophorus as a substrate. The crude enzyme produced under optimized conditions was used to hydrolyze alkali-acid pretreated P. hysterophorus biomass. The highest CMCase production was achieved in 4–5 days when steam-pretreated P. hysterophorus biomass was used at 1% (w/v) concentration, using 2 discs (1 disc = 5 × 107 spores/ml) of inoculum, an initial pH 6.5, temperature at 40 °C, an agitation speed of 120–150 rpm, and by supplementing fermentation medium with 1.5% (w/v) carboxymethyl cellulose (CMC) as additional carbon source. Under optimized conditions, the actinomycete strain NAA2 showed production of 0.967 ± 0.016 U/ml CMCase, 0.116 ± 0.08 FPU/ml FPase, and 0.22 ± 0.012 U/ml β-glucosidase enzymes. On utilizing the cellulase enzyme for biomass hydrolysis, maximum 18.2% saccharification yield (of cellulose 0.202 g/g) was achieved in 96 h when enzyme and substrate levels were 30 FPU/100 ml and 2% (w/v) respectively. Parthenium hysterophorus biomass can be hydrolyzed enzymatically yielding considerable amounts of total reducing sugars. It can, therefore, be used as a feedstock for the production of bioethanol. Also, it has the potential to act as a substrate for the production of cellulases. Furthermore, the improved cellulolytic potential of Streptomyces sp. NAA2 can be exploited in various industrial applications.

The Mo- and Fe-nitrogenases of the endophyte Kosakonia sp. UYSO10 are necessary for growth promotion of sugarcane Abstract Aim Sugarcane is a multipurpose crop primarily used to produce sugar, energy and bioethanol. It requires high amounts of N-fertilization for optimal growth, which increases production costs and environmental degradation. The contribution of biological nitrogen fixation to Uruguayan commercial sugarcane cultivars was demonstrated previously, and diazotrophic bacteria that were isolated from the stems were characterized and identified. From this collection, the isolate UYSO10 related to the Kosakonia genus (formerly Enterobacter) was described as a plant growth-promoting endophyte of sugarcane plants. Purpose To evaluate the effect of the inoculation of wild-type and nitrogenase-deficient strains of Kosakonia sp. UYSO10 on sugarcane growth promotion under non-sterile conditions. Methods Kosakonia sp. UYSO10 was inoculated onto sugarcane setts for plant growth promotion greenhouse experiments. Single and double mutants resulting to the nitrogenase-encoding genes (nifH, anfH) were constructed, and the phenotypes were evaluated in vitro and in vivo. Results Kosakonia sp. UYSO10 is able to promote sugarcane growth under non-sterile conditions, that strain UYSO10 harbors two functional nitrogenases and the inactivation of both nitrogenase-encoding genes diminish its capacity of promoting growth on sugarcane. Conclusion All together, the results obtained showed that the biological nitrogen fixation ability of Kosakonia sp. UYSO10 is required for sugarcane growth promotion.

Characterization and antioxidant ability of potential probiotic lactic acid bacteria in ogi liquor and lemon juice- ogi liquor Abstract Purpose Ogi is an indigenous edible fermented cereal slurry but the steep liquor is usually wasted or administered as therapeutic to suppress certain illnesses. The combination of lemon juice and ogi steep liquor (OSL) is known to possess bioactive metabolites. Method This study evaluated potential probiotic lactic acid bacteria (LAB) in different OSL (Zea mays, Sorghum bicolor, and Pennisetum glaucum L.) and lemon juice-ogi steep liquor (LJOSL) based on low pH, bile and lysozyme tolerances, hydrophobicity and auto-aggregation, antibiotic, cholesterol removal, exopolysaccharide production, β-galactosidase, and antimicrobial and hemolytic activities using standard methods. Presumptive LAB were sequenced and assayed for radical scavenging using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and lipid peroxidation inhibitory (LPI) tests. Results Presumptive LAB counts were higher in maize OSL (0 h:5.09 log CFU/ml) and combined cereal OSL (24–48 h:7.65 and 7.72 log CFU/ml) but decreased in all steep liquors at 72 h, except in millet OSL (7.72 log CFU/ml). A total of 120 LAB isolates were randomly selected. Based on pH and bile tolerances, 14 isolates were comparable to reference strains. All these isolates demonstrated probiotics properties except for three that did not show γ-hemolysis. Sequenced LAB isolates were identified as Lactobacillus plantarum, Lactobacillus fermentum, Pediococcus pentosaceus, and Weissella cibara. DPPH activities of LAB gradually increased during fermentation with the highest activity of DPPH (58.77%) and LPI (57.94%) activity in L. plantarum. Strong correlations were found between DPPH and LPI in all the selected isolates. Conclusion The antioxidant property of probiotic LAB in OSL and LJOSL could contribute to its therapeutic nature.

Non-pathogenic Staphylococcus strains augmented the maize growth through oxidative stress management and nutrient supply under induced salt stress Abstract Purpose The present study was conducted to elucidate the role of phytobeneficial bacteria to control the cellular oxidative damage in maize (Zea mays L.) plants caused by salinity. Methods Bacteria were isolated from the rhizosphere of kallar grass (Leptochloa fusca L.) through serial dilution method and taxonomically identified on the basis of their 16S ribosomal RNA gene sequencing. In vitro phosphate solubilization, indole-3-acetic acid (IAA) synthesis, and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity were evaluated by solubilization index measurement, colorimetric method, and turbidity assay, respectively. In the pot experiment, the impact of single and mixed inoculation of these strains at four levels (0, 50, 100, and 200 mM) of salt stress was evaluated in terms of growth and physiological response of maize plants to salinity. Results The bacterial strains (STN-1, STN-5, and STN-14) were taxonomically classified as Staphylococcus spp. At 5% NaCl level, the strains demonstrated substantial potential for phosphate solubilization, ACC deaminase activity, and IAA production both with and without tryptophan. The inoculation of strains STN-1, STN-5, and mixed inoculation resulted in substantial growth improvement of maize plants along with increased antioxidant enzyme activity and decreased levels of reactive oxygen species. In addition, single inoculation of STN-1 and STN-5 along with mixed inoculation augmented the uptake of N, P, K, and Ca+2 and reduced Na+ uptake. Conclusion Current results demonstrated that the strains STN-1 and STN-5 modulated stress-responsive mechanisms and regulated ion balance in induced salinity to promote maize growth.

Genomic annotation and validation of bacterial consortium NDMC-1 for enhanced degradation of sugarcane bagasse Abstract Purpose This study aims at designing a consortium using rumen bacterial isolates for enhancing the hydrolysis of sugarcane bagasse (SB) for efficient biofuel formation. Methods The microbial population was screened through biochemical and molecular tools along with enzymatic activity to obtain potential isolates for diverse cellulolytic and hemicellulolytic carbohydrate active enzyme (CAZyme).Five strains (Paenibacillus, Bacillus, Enterobacter, and Microbacterium) were selected for designing the consortium NDMC-1. The hydrolytic efficiency of NDMC-1 was determined based on cellulase production with simultaneous rise in monosaccharides, oligosaccharides, and soluble chemical oxygen demand (sCOD) concentration. Cellulolytic machinery of these isolates was further explored using genome sequencing. Result The isolates selected for consortia NDMC-1 interacted synergistically leading to enhanced cellulase production. Maximal endoglucanase (1.67 μmol ml−1 min−1), exoglucanase (0.69 μmol ml−1 min−1), and β-glucosidase (2.03 μmol ml−1 min−1) activity were achieved with SB as a sole carbon source after 48 h of incubation. Enhancement in SB hydrolysis employing NDMC-1 was evident by the increase in sCOD from 609 to 2589 mg/l and release of 1295 mg/l reducing sugar, comprising 59.8%, 8.23%, and 6.16% of glucose, cellobiose, and cellotriose, respectively, which resulted in 5.5-fold rise in biogas production. On genome annotation, total 472 contigs from glycoside hydrolase family: 84 from Microbacterium arborescens ND21, 72 from Enterobacter cloacae ND22, 61 from Bacillus subtilis ND23, 116 from Paenibacillus polymyxa ND24, and 140 from Paenibacillus polymyxa ND25 were identified. On further analysis, total 33 cellulases, 59 hemicellulases, and 48 esterases were annotated in the reported genomes. Conclusion This work proposes the application of consortia-based bioprocessing systems over the conventionally favorable single organism approach for efficient hydrolysis of cellulosic substrates to fermentable sugars.

Assessment of the probiotic potential of lactic acid bacteria isolated from kefir grains: evaluation of adhesion and antiproliferative properties in in vitro experimental systems Abstract The main objective of this study was to isolate lactic acid bacteria from kefir grains and investigate their probiotic potential. In this study, 48 bacterial strains were isolated from kefir grains, whereas 39 strains were categorized to the genus Lactobacillus. Evaluation of the probiotic potential of the isolated stains was performed, including resistance to low pH, tolerance to pepsin, pancreatin and bile salts, and antibiotic resistance. In addition, evaluation of adhesion and antiproliferative properties in in vitro experimental systems was also conducted. Strains SP2 and SP5 that displayed the best performance in the conducted in vitro tests were selected for further studies. Firstly, genotypic identification of the two strains was performed by partial 16S rRNA gene sequencing, BLAST analysis, and species-specific multiplex PCR assay. The two strains were confirmed to be Pediococcus pentosaceus SP2 and Lactobacillus paracasei SP5. Then, the adhesion properties of the two strains were examined in vitro. Both strains displayed substantial adherence capacity to HT-29 human colon cancer cells. Moreover, a significant decrease of HT-29 cell growth after treatment with viable P. pentosaceus SP2 or L. paracasei SP5 was recorded. In addition, downregulation of anti-apoptotic genes and over-expression of cell cycle–related genes was recorded by real-time PCR analysis. Treatment with conditioned media of the two strains also caused significant reduction of cancer cell proliferation in a time- and concentration-dependent manner. P. pentosaceus SP2 and L. paracasei SP5 displayed the best probiotic properties that exerted substantial adherence on human colon cancer cells as well as significant anti-proliferative properties.

Methicillin resistance and clonal diversity of Staphylococcus aureus isolated from nasal samples of healthy horses in Iran Abstract Purpose The aims of the current study were to investigate the frequency and genetic diversity of Staphylococcus aureus from healthy horses, including both methicillin-resistant (MRSA) and -susceptible S. aureus (MSSA). Methods Three hundred-one nasal swabs were collected from healthy horses in three provinces, Iran. Sixty-one of the 301 tested samples contained S. aureus (20.3%), among which five were MRSA. Isolates were typed by spaPCR-RFLP and agr typing, followed by sequence-based spa typing and MLST on representative strains from each restriction pattern and SCCmec typing for MRSA strains. The presence of Panton-Valentine Leukocidin (PVL) encoding genes was also tested using PCR. Results Eight distinct RFLP patterns (designated as N1-N8) were observed, with N2 (23/61; 37.7%) and N4 (18/61; 29.5%) the most common. On sequencing, N1-N8 patterns were found to be of clonal types ST15-t084, ST2151-t2484, ST291-t937, ST1-t127, and ST1-t1383, ST700-t11926, ST133-t1166, and ST1278-t12595, respectively. No PVL-positive S. aureus were detected. Five MRSA were identified as ST2151-t2484-SCCmecIVa (2 isolates), ST15-t084-SCCmecIVa, ST1-t1383-SCCmecIVa, and t12595-SCCmecIVa (one isolate each). Majority of S. aureus isolates were ascribed to agr types III (n = 30; 49.2%) and IV (n = 28; 45.9%), followed by types II (n = 2, 3.3%) and I (n = 1, 1.6%). The carriage of S. aureus was found to be associated with geographic locations. Conclusions This study for the first time describes the circulation of diverse clones of MSSA and MRSA among the Iranian horse population. This may pose a public health risk, which supports the need for their epidemiological monitoring.

Alexandros Sfakianakis
Anapafseos 5 . Agios Nikolaos
Crete.Greece.72100
2841026182

6948891480

alsfakia@gmail.com