Search results for: S. cerevisiae

Authors: Yetti Marlida, Syukri Arif, Nadirman Haska

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Recently, alcohol fuels have been produced on industrial scales by fermentation of sugars derived from wheat, corn, sugar beets, sugar cane etc. The enzymatic hydrolysis of cellulosic materials to produce fermentable sugars has an enormous potential in meeting global bioenergy demand through the biorefinery concept, since agri-food processes generate millions of tones of waste each year (Xeros and Christakopoulos 2009) such as sugar cane baggase , wheat straw, rice straw, corn cob, and oil palm trunk. In fact oil palm trunk is one of the most abundant lignocellulosic wastes by-products worldwide especially come from Malaysia, Indonesia and Nigeria and provides an alternative substrate to produce useful chemicals such as bioethanol. Usually, from the ages 3 years to 25 years, is the economical life of oil palm and after that, it is cut for replantation. The size of trunk usually is 15-18 meters in length and 46-60 centimeters in diameter. The trunk after cutting is agricultural waste causing problem in elimination but due to the trunk contains about 42% cellulose, 34.4%hemicellulose, 17.1% lignin and 7.3% other compounds,these agricultural wastes could make value added products (Pumiput, 2006).This research was production of bioethanol from oil palm trunk via saccharafication by cocktail carbohydrases enzymes. Enzymatic saccharification of acid treated oil palm trunk was carried out in reaction mixture containing 40 g treated oil palm trunk in 200 ml 0.1 M citrate buffer pH 4.8 with 500 unit/kg amylase for treatment A: Treatment B: Treatment A + 500 unit/kg cellulose; C: treatment B + 500 unit/kgg xylanase: D: treatment D + 500 unit/kg ligninase and E: OPT without treated + 500 unit/kg amylase + 500 unit/kg cellulose + 500 unit/kg xylanase + 500 unit/kg ligninase. The reaction mixture was incubated on a water bath rotary shaker adjusted to 600C and 75 rpm. The samples were withdraw at intervals 12 and 24, 36, 48,60, and 72 hr. For bioethanol production in biofermentor of 5L the hydrolysis product were inoculated a loop of Saccharomyces cerevisiae and then incubated at 34 0C under static conditions. Samples are withdraw after 12, 24, 36, 48 and 72 hr for bioethanol and residual glucose. The results of the enzymatic hidrolysis (Figure1) showed that the treatment B (OPT hydrolyzed with amylase and cellulase) have optimum condition for glucose production, where was both of enzymes can be degraded OPT perfectly. The same results also reported by Primarini et al., (2012) reported the optimum conditions the hydrolysis of OPT was at concentration of 25% (w /v) with 0.3% (w/v) amylase, 0.6% (w /v) glucoamylase and 4% (w/v) cellulase. In the Figure 2 showed that optimum bioethanol produced at 48 hr after incubation,if time increased the biothanol decreased. According Roukas (1996), a decrease in the concentration of ethanol occur at excess glucose as substrate and product inhibition effects. Substrate concentration is too high reduces the amount of dissolved oxygen, although in very small amounts, oxygen is still needed in the fermentation by Saccaromyces cerevisiae to keep life in high cell concentrations (Nowak 2000, Tao et al. 2005). The results of the research can be conluded that the optimum enzymatic hydrolysis occured when the OPT added with amylase and cellulase and optimum bioethanol produced at 48 hr incubation using Saccharomyses cerevicea whereas 18.08 % bioethanol produced from glucose conversion. This work was funded by Directorate General of Higher Education (DGHE), Ministry of Education and Culture, contract no.245/SP2H/DIT.LimtabMas/II/2013

Keywords: oil palm trunk, enzymatic hydrolysis, saccharification

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Authors: Lavinia Ruta, Ileana Farcasanu

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The yeast surface display (YSD) technique enables the expression of proteins on yeast cell surfaces, facilitating the identification and isolation of proteins with targeted binding properties, such as nanobodies. Nanobodies, derived from camelid species, are single-domain antibody fragments renowned for their high affinity and specificity towards target proteins, making them valuable in research and potentially in therapeutics. Their advantages include a compact size (~15 kDa), robust stability, and the ability to target challenging epitopes. The project endeavors to establish and validate a platform for producing Green Fluorescent Protein (GFP) and mCherry nanobodies using the yeast surface display method. mCherry, a prevalent red fluorescent protein sourced from coral species, is commonly utilized as a genetic marker in biological studies due to its vibrant red fluorescence. The GFP-nanobody, a single variable domain of heavy-chain antibodies (VHH), exhibits specific binding to GFP, offering a potent means for isolating and engineering fluorescent protein fusions across various biological research domains. Both GFP and mCherry nanobodies find specific utility in cellular imaging and protein analysis applications.

Keywords: YSD, nanobodies, GFP, Saccharomyces cerevisiae

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Authors: F. A. Ogundolie, F. Okogue, D. V. Adegunloye

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Greywater samples were obtained from three restaurants in the Federal University of Technology; Akure coded SSR, MGR and GGR. Fungi isolates obtained include Rhizopus stolonifer, Aspergillus niger, Mucor mucedo, Aspergillus flavus, Saccharomyces cerevisiae. Of these fungi isolates obtained, R. stolonifer, A. niger and A. flavus showed significant degradation ability on grey water and was used for this research. A simple bioreactor was constructed using biodegradation process in purification of waste water samples. Waste water undergoes primary treatment; secondary treatment involves the introduction of the isolated organisms into the waste water sample and the tertiary treatment which involved the use of filter candle and the sand bed filtration process to achieve the end product without the use of chemicals. A. niger brought about significant reduction in both the bacterial load and the fungi load of the greywater samples of the three respective restaurants with a reduction of (1.29 × 108 to 1.57 × 102 cfu/ml; 1.04 × 108 to 1.12 × 102 cfu/ml and 1.72 × 108 to 1.60 × 102 cfu/ml) for bacterial load in SSR, MGR and GGR respectively. Reduction of 2.01 × 104 to 1.2 × 101; 1.72 × 104 to 1.1 × 101, and 2.50 × 104 to 1.5 × 101 in fungi load from SSR, MGR and GGR respectively. Result of degradation of these selected waste water by the fungi showed that A. niger was probably more potent in the degradation of organic matter and hence, A. niger could be used in the treatment of wastewater.

Keywords: Aspergillus niger, greywater, bacterial, fungi, microbial load, bioreactor, biodegradation, purification, organic matter and filtration

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Authors: Byeong-Uk Lim, Young-Ran Song, Sang-Ho Baik

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This study aimed to develop yeast starters for scientific alcohol production and systematic quality control of whisky. A total of 389 yeast strains were isolated from traditional Korean fermentation starter (nuruk) and rice wine (makgeolli), and ten strains were finally selected for their high alcohol productivities, in which their alcohol productions were above 17.3% (v/v) during 10 days under two steps of glucose feeding condition (30% and then 15%, w/v). By 18s rDNA sequence analysis, all strains were identified as Saccharomyces cerevisiae (SC), and they can grow well under 50% (w/v) glucose and 10% (v/v) ethanol conditions. Furthermore, the capability of ten different SC strains to ferment rice wine for whisky was studied. Rice wine was fermented with only steamed rice, water, and two types of enzymes (glucoamylase and α-amylase) during 14 days at 25 °C, and then their oenological properties have been determined. As the results, the fermented rice wines indicated the final pH range of 4.24-4.38 and acidity range of 0.12-0.18. The highest ethanol production of 20.2% (v/v) was found in the fermentation with a SC-156 strain, whereas SC-92 (16.8%) and SC-119 (16.4%) showed significantly lowest ethanol productions. In addition, the residual sugar contents showed negative correlation with alcohol contents. Moreover, this study focused on nucleotide polymorphisms in the MSN2 and MSN4 genes to investigate the cause of the defective stress responses in yeast. Consequently, it was also confirmed that the deletion of the N termini of Msn4p from identified point mutations in SC-63, SC-95, SC-156, SC-158, and SC-160 strains.

Keywords: yeast, high-alcohol, whisky, rice wine

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Authors: P. Rahnemoon, M. Sarabi Jamab, M. Javanmard Dakheli, A. Bostan

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In recent years, tendency to use of natural antimicrobial agents in food industry has increased. Pomegranate peels containing phenolic compounds and anti-microbial agents, are counted as valuable source for extraction of these compounds. In this study, the extraction of pomegranate peel extract was carried out at different ethanol/water ratios (40:60, 60:40, and 80:20), temperatures (25, 40, and 55 ˚C), and time durations (20, 24, and 28 h). The extraction yield, phenolic compounds, flavonoids, and anthocyanins were measured. ‎Antimicrobial activity of pomegranate peel extracts were determined against some food-borne ‎microorganisms such as Salmonella enteritidis, Escherichia coli, Listeria monocytogenes, ‎‎Staphylococcus aureus, Aspergillus niger, and Saccharomyces cerevisiae by agar diffusion and MIC methods. Results showed that at ethanol/water ratio 60:40, 25 ˚C and 24 h maximum amount of phenolic compounds ‎‏(‎‏‎349.518‎‏ ‏mg gallic acid‏/‏g dried extract), ‎flavonoids (250.124 mg rutin‏/‏g dried extract), anthocyanins (252.047 ‎‏‏mg ‎cyanidin‏‎3‎‏glucoside‏/‏‎100 g dried extract), and the strongest antimicrobial activity were obtained. ‎All extracts’ antimicrobial activities were demonstrated against every tested ‎‎microorganisms‏.‎‏ Staphylococcus aureus showed the highest sensitivity among the tested ‎‎‎microorganisms.

Keywords: antimicrobial agents, phenolic compounds, pomegranate peel, solvent extraction‎

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Authors: Jae-Hyung Jin, Kyung-Tae Lee, Yee-Seul So, Eunji Jeong, Yeonseon Lee, Dongpil Lee, Dong-Gi Lee, Yong-Sun Bahn

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Cryptococcus neoformans causes cryptococcal meningoencephalitis mainly in immunocompromised patients as well as immunocompetent people. But therapeutic options are limited to treat cryptococcosis. Some signaling pathways including cyclic AMP pathway, MAPK pathway, and calcineurin pathway play a central role in the regulation of the growth, differentiation, and virulence of C. neoformans. To understand signaling networks regulating the virulence of C. neoformans, we selected the 114 putative phosphatase genes, one of the major components of signaling networks, in the genome of C. neoformans. We identified putative phosphatases based on annotation in C. neoformans var. grubii genome database provided by the Broad Institute and National Center for Biotechnology Information (NCBI) and performed a BLAST search of phosphatases of Saccharomyces cerevisiae, Aspergillus nidulans, Candida albicans and Fusarium graminearum to Cryptococcus neoformans. We classified putative phosphatases into 14 groups based on InterPro phosphatase domain annotation. Here, we constructed 170 signature-tagged gene-deletion strains through homologous recombination methods for 91 putative phosphatases. We examined their phenotypic traits under 30 different in vitro conditions, including growth, differentiation, stress response, antifungal resistance and virulence-factor production.

Keywords: human fungal pathogen, phosphatase, deletion library, functional genomics

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Authors: Aida Kalantari, Boyang Ji, Tao Chen, Ivan Mijakovic

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3-hydroxypropanoic acid (3-HP) is one of the most important biomass-derivable platform chemicals that can be converted into a number of industrially important compounds. There have been several attempts at production of 3-HP from renewable sources in cell factories, focusing mainly on Escherichia coli, Klebsiella pneumoniae, and Saccharomyces cerevisiae. Despite the significant progress made in this field, commercially exploitable large-scale production of 3-HP in microbial strains has still not been achieved. In this study, we investigated the potential of Bacillus subtilis to be used as a microbial platform for bioconversion of glycerol into 3-HP. Our recombinant B. subtilis strains overexpress the two-step heterologous pathway containing glycerol dehydratase and aldehyde dehydrogenase from various backgrounds. The recombinant strains harboring the codon-optimized synthetic pathway from K. pneumoniae produced low levels of 3-HP. Since the enzymes in the heterologous pathway are sensitive to oxygen, we had to perform our experiments in micro-aerobic conditions. Under these conditions, the cell produces lactate in order to regenerate NAD+, and we found the lactate production to be in competition with the production of 3-HP. Therefore, based on the in silico predictions, we knocked out the glycerol kinase (glpk), which in combination with growth on glucose, resulted in improving the 3-HP titer to 1 g/L and the removal of lactate. Cultivation of the same strain in an enriched medium improved the 3-HP titer up to 7.6 g/L. Our findings provide the first report of successful introduction of the biosynthetic pathway for conversion of glycerol into 3-HP in B. subtilis.

Keywords: bacillus subtilis, glycerol, 3-hydroxypropanoic acid, metabolic engineering

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Authors: Neti Yuliana, Srisetyani, Siti Nurdjanah, Dewi Sartika, Yoan Martiansari, Putri Nabila

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Naturally sweet potato flour usually requires a modification process to improve its inherent property for expanding its application in food system. The study was aimed to modify sweet potato flour (SPF), to increase its utilization for composite noodle production, trough fermentation of sweet potato slices before its flouring process. Fermentation were prepared with five different starters: pickle brine, Lactobacillus plantarum, Leuconostoc mesenteroides, mixed of Lactobacillus plantarum, Leuconostoc mesenteroides , and mixed of Lactobacillus plantarum, Leuconostoc mesenteroides, and Sacharomyces cerevisiae. Samples were withdrawn every 0, 24, 48, 72 and 96 hours. The fermented flours were characterized for swelling power, solubility, paste transmittance, pH, sensory properties (acidic aroma and whiteness), and the amount of broken composite noodle strips. The results indicated that there was no significant effect of different starters on fermented SPF characteristic and on the amount of broken noodle strip, while length of fermentation significantly affected. Longer fermentation, reaching 48-72 h, increased swelling power, pH, acidic aroma and whiteness of flour and reduced solubility, paste transmittance, and the amount of broken noodle strip. The results suggested that fermentation within 48-72 h period of time could provide great composite SPF for noodle.

Keywords: starters, fermented flour, sweet potato, composite noodle

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Authors: Hanan Al-Khalaifah, Afaf Al-Nasser

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Probiotics and prebiotics claimed to serve as effective alternatives to antibiotics in the poultry. This study aims to investigate the effect of different probiotics and prebiotics on the economic impact analysis of the use of probiotics and prebiotics in broiler feed. The study involved four broiler cycles, two during winter and two during summer. In the first two cycles (summer and winter), different types of prebiotics and probiotics were used. The probiotics were Bacillus coagulans (1 g/kg dried culture) and Lactobacillus (1 g/kg dried culture of 12 commercial strains), and prebiotics included fructo-oligosaccharides (FOS) (5 g/kg) and mannan-oligosaccharide (MOS) derived from Saccharomyces cerevisiae (5 g/kg). Based on the results obtained, the best treatment was chosen to be FOS, from which different ratios were used in the last two cycles during winter and summer. The levels of FOS chosen were 0.3, 0.5, and 0.7% of the diet. From an economic point of view, it was generally concluded that in all dietary treatments, food was consumed less in cycle 1 than in cycle 2, the total body weight gain was more in cycle 1 than cycle 2, and the average feed efficiency was less in cycle l than cycle 2. This indicates that the weather condition affected better in cycle 1. Also, there were very small differences between the dietary treatments in each cycle. In cycle 1, the best total feed consumption was for the FOS treatment, the highest total body weight gain and average feed efficiency were for B. coagulans. In cycle 2, all performance was better in FOS treatment. FOS significantly reduced the Salmonella sp. counts in the intestine, where the environment was driven towards acidity. FOS was the best on the average taste panel study of the produced meat. Accordingly, FOS prebiotic was chosen to be the best treatment to be used in cycles 3 and 4. The economic impact analysis generally revealed that there were no big differences between the treatments in all of the studied indicators, but there was a difference between the cycles.

Keywords: antibiotic, economic impact, prebiotic, probiotic, broiler

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Authors: Astorga-Trejo Rebeca, Fonseca-Peralta Héctor Manuel, Beltrán-Arredondo Laura Ivonne, Castro-Martínez Claudia

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The use of biomass as feedstock for the production of fuels and other chemicals of interest is an ever-growing accepted option in the way to the development of biorefinery complexes; in the Mexican state of Sinaloa, two million tons of residues from corn crops are produced every year, most of which can be converted to bioethanol and other products through biotechnological conversion using yeast and other microorganisms. Therefore, the objective of this work was to take advantage of corn stover and evaluate its potential as a substrate for the production of second-generation bioethanol (2G), enzymes, and xylitol. To produce bioethanol 2G, an acid-alkaline pretreatment was carried out prior to saccharification and fermentation. The microorganisms used for the production of enzymes, as well as for the production of xylitol, were isolated and characterized in our workgroup. Statistical analysis was performed using Design Expert version 11.0. The results showed that it is possible to obtain 2G bioethanol employing corn stover as a carbon source and Saccharomyces cerevisiae ItVer01 and Candida intermedia CBE002 with yields of 0.42 g and 0.31 g, respectively. It was also shown that C. intermedia has the ability to produce xylitol with a good yield (0.46 g/g). On the other hand, qualitative and quantitative studies showed that the native strains of Fusarium equiseti (0.4 IU/mL - xylanase), Bacillus velezensis (1.2 IU/mL – xylanase and 0.4 UI/mL - amylase) and Penicillium funiculosum (1.5 IU / mL - cellulases) have the capacity to produce xylanases, amylases or cellulases using corn stover as raw material. This study allowed us to demonstrate that it is possible to use corn stover as a carbon source, a low-cost raw material with high availability in our country, to obtain bioproducts of industrial interest, using processes that are more environmentally friendly and sustainable. It is necessary to continue the optimization of each bioprocess.

Keywords: biomass, corn stover, biorefinery, bioethanol 2G, enzymes, xylitol

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Authors: Parul Johri, Mala Trivedi

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Major Intrinsic proteins (MIPs), actively involved in the passive transport of small polar molecules across the membranes of almost all living organisms. MIPs that specifically transport water molecules are named aquaporins (AQPs). The permeability of membranes is actively controlled by the regulation of the amount of different MIPs present but also in some cases by phosphorylation and dephosphorylation of the channel. Based on sequence similarity, MIPs have been classified into many categories. All of the proteins are made up of the 20 amino acids, the only difference is there in their orientations. Again all the 20 amino acids are made up of the basic five elements namely: carbon, hydrogen, oxygen, sulphur and nitrogen. These elements are responsible for giving the amino acids the properties of hydrophilicity/hydrophobicity which play an important role in protein interactions. The hydrophobic amino acids characteristically have greater number of carbon atoms as carbon is the main element which contributes to hydrophobic interactions in proteins. It is observed that the carbon level of proteins in different species is different. In the present work, we have taken a sample set of 150 aquaporins proteins from Uniprot database and a dynamic programming code was written to calculate the carbon percentage for each sequence. This carbon percentage was further used to barcode the aqauporins of animals and plants. The protein taken from Oryza sativa, Zea mays and Arabidopsis thaliana preferred to have carbon percentage of 31.8 to 35, whereas on the other hand sequences taken from Mus musculus, Saccharomyces cerevisiae, Homo sapiens, Bos Taurus, and Rattus norvegicus preferred to have carbon percentage of 31 to 33.7. This clearly demarks the carbon range in the aquaporin proteins from plant and animal origin. Hence the atom level analysis of protein sequences can provide us with better results as compared to the residue level comparison.

Keywords: aquaporins, carbon, dynamic prgramming, MIPs

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Authors: Nuray Yilmaz Baran, Mehmet Saçak

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Schiff base polymers are one class of conjugated polymers, also called as poly(azomethines). They have drawn the attention of researchers in recent years due to their some properties such as, optoelectronic, semiconductive, and photovoltaic, antimicrobial activities and high thermal stability. In this study, Poly(2-[[4-(dimethylamino)benzylidene]amino] phenol) P(2-DBAP), which is a Schiff base polymer, was synthesized by an oxidative polycondensation reaction of -[[4-(dimethylamino)benzylidene]amino]phenol (2-DBAP) with oxidants NaOCl, H₂O₂ and O₂ in various organic medium. At the end of the polymerizations carried out at various temperatures and time, maximum conversion of the monomer to the polymer could be obtained as around 93.7 %. The structures of the monomer and polymer were characterized by UV-Vis, FTIR and ¹HNMR techniques. Thermal analysis of the polymer was identified by TG-DTG and DTA techniques, and the thermal degradation behavior was supported by Thermo-IR spectra recorded in the temperature range of 25-800 °C. The number average molecular weight (Mn), weight average molecular weight (Mw) and polydispersity index (PDI) of the polymer were found to be 26337, 9860 g/mol 2.67, respectively. The change of electrical conductivity value of the P(2-DBAP) doped with iodine vapor at different temperatures and time was investigated its maximum was measured by increasing 10¹⁰ fold as 2 x10⁻⁴ Scm⁻¹ after doping for 48 h at 60 °C. Antibacterial and antifungal activities of P(2-DBAP) Schiff base and its polymer were also investigated against Sarcina lutea, Enterobacter aerogenes, Escherichia coli, Enterococcus Faecalis, Klebsiella pneumoniae, Bacillus subtilis, and Candida albicans, Saccharomyces cerevisiae, respectively.

Keywords: conductive properties, polyazomethines, polycondensation reaction, Schiff base polymers, thermal stability

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Authors: Yazeed Al-Sheikh

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Pregnancy represents a risk factor in the occurrence of vulvovaginal candidiasis. To investigate the prevalence rate of vaginal carriage of Candida species in Saudi pregnant and non-pregnant women, high vaginal swab (HVS) specimens (707) were examined by direct microscopy (10% KOH and Giemsa staining) and parallel cultured on Sabouraud Dextrose Agar (SDA) as well as on “CHROM agar Candida” medium. As expected, Candida-positive cultures were frequently observed in pregnant-test group (24%) than in non-pregnant group (17%). The frequency of culture positive was correlated to pregnancy (P=0.047), parity (P=0.001), use of contraceptive (P=0.146), or antibiotics (P=0.128), and diabetic-patients (P < 0.0001). Out of 707 HVS examined specimens, 157 specimens were yeast-positive culture (22%) on Sabouraud Dextrose Agar or “CHROM agar Candida”. In comparison, the sensitivities of the direct 10% KOH and the Giemsa stain microscopic examination methods were 84% (132/157) and 95% (149/157) respectively but both with 100% specificity. As for the identity of recovered 157 yeast isolates, based on API 20C biotype carbohydrate assimilation, germ tube and chlamydospore formation, C. albicansand C. glabrata constitute 80.3 and 12.7% respectively. Rates of C. tropicalis, C. kefyr, C. famata or C. utilis were 2.6, 1.3, and 0.6% respectively. Sachromyces cerevisiae and Rhodotorula mucilaginosa yeasts were also encountered at a frequency of 1.3 and 0.6% respectively. Finally, among all recovered 157 yeast-isolates, strains resistant to ketoconazole were not detected, whereas 5% of the C. albicans and as high as 55% of the non-albicans yeast isolates (majority C. glabrata) showed resistance to fluconazole. Our findings may prove helpful for continuous determination of the existing vaginal candidiasis causative species during pregnancy, its lab-diagnosis and/or control and possible measures to minimize the incidence of the disease-associated pre-term delivery.

Keywords: vaginal candidiasis, Candida spp., pregnancy, risk factors, API 20C-yeast biotypes, giemsa stain, antifungal agents

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Authors: Irshad Ali Khan, Jian-Ping Lu, Xiao-Hong Liu, Fu-Cheng Lin

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This study reports the functional analysis of a gene MoNUC1 in M. oryzae, which is homologous to the Saccharomyces cerevisiae NUC1 encoding a mitochondrial nuclease protein. The MoNUC1 having a gene locus MGG_05324 is 1002-bp in length and encodes an identical protein of 333 amino acids. We disrupted the gene through gene disruption strategy and isolated two mutants confirmed by southern blotting. The deleted mutants were then used for phenotypic studies and their phenotypes were compared to those of the Guy-11 strain. The mutants were first grown on CM medium to find the effect of MoNUC1 gene disruption on colony growth and the mutants were found to show normal culture colony growth similar to that of the Guy-11 strain. Conidial germination and appressorial formation were also similar in both the mutants and Guy-11 strains showing that this gene plays no significant role in these phenotypes. For pathogenicity, the mutants and Guy-11 mycelium blocks were inoculated on blast susceptible barley seedlings and it was found that both the strains exhibited full pathogenicity showing coalesced and necrotic blast lesions suggesting that this gene is not involved in pathogenicity. Mating of the mutants with 2539 strain formed numerous perithecia showing that MoNUC1 is not essential for sexual reproduction in M. oryzae. However, the mutants were found to form reduced conidia (1.06±8.03B and 1.08±9.80B) than those of the Guy-11 strain (1.46±10.61A) and we conclude that this protein is not required for the blast fungus to cause pathogenicity but plays significant role in conidiation. Proteins of signal transduction pathways that could be disrupted/ intervened genetically or chemically could lead to antifungal products of important fungal cereal diseases and reduce rice yield losses. Tipping the balance toward understanding the whole of pathogenesis, rather than simply conidiation will take some time, but clearly presents the most exciting challenge of all.

Keywords: appressorium formation, conidiation, NUC1, Magnaporthe oryzae, pathogenicity

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Authors: Sonja Djilas, Aleksandra Velićanski, Dragoljub Cvetković, Siniša Markov, Eva Lončar, Vesna Tumbas Šaponjac, Milica Vinčić

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Due to high content of bioactive compounds, sour cherry possesses antioxidant and antimicrobial activity. Additionally, waste material from industrial processing of sour cherry is also a good source of bioactive compounds. The aim of this study was to screen the antimicrobial activity and determine the minimal inhibitory (MIC) and minimal bactericidal concentrations (MBC) of sour cherry pomace extract. Tested strains were Gram-negative bacteria (Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 14028 and wild isolates Escherichia coli and Salmonella sp.), Gram-positive bacteria (Staphylococcus aureus ATCC 11632, Bacillus cereus ATCC 10876 and wild isolates Staphylococcus saprophyticus and Bacillus sp.) and yeasts (Saccharomyces cerevisiae 112, Hefebank Weihenstephan and Candida albicans ATCC 10231). Antimicrobial activity was tested by disc-diffusion method and agar-well diffusion method. MIC and MBC were determined by microdilution method. Screening tests showed that Gram-negative bacteria were resistant to tested extract, with exception of Salmonella typhimurium and Salmonella sp. for which only zones of reduced growth appeared. However, Gram-positive bacteria were more sensitive where the highest clear zones appeared with 100 µl of extract applied. There was no activity against tested yeasts. MIC and MBC values were in the range 3.125-37.5 mg/ml and 6.25-100 mg/ml, respectively. The most susceptible strain was Staphylococcus aureus while the most resistant was Bacillus sp. where MBC was not found in tested concentration range. Sour cherry pomace possesses high antibacterial potential, which indicates that this waste material is a promising source of bioactive compounds and could be used as a functional food ingredient.

Keywords: antimicrobial activity, sour cherry, pomace, bioactive compounds

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Authors: Dharell B. Siano, Wendy C. Mateo, Victorino T. Taylan, Francisco D. Cuaresma

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Biofuel is one of the renewable energy sources adapted by the Philippine government in order to lessen the dependency on foreign fuel and to reduce carbon dioxide emissions. Rain tree pods were seen to be a promising source of bioethanol since it contains significant amount of fermentable sugars. The study was conducted to establish the complete procedure in processing rain tree pods for village level hydrous bioethanol production. Production processes were done for village level hydrous bioethanol production from collection, drying, storage, shredding, dilution, extraction, fermentation, and distillation. The feedstock was sundried, and moisture content was determined at a range of 20% to 26% prior to storage. Dilution ratio was 1:1.25 (1 kg of pods = 1.25 L of water) and after extraction process yielded a sugar concentration of 22 ⁰Bx to 24 ⁰Bx. The dilution period was three hours. After three hours of diluting the samples, the juice was extracted using extractor with a capacity of 64.10 L/hour. 150 L of rain tree pods juice was extracted and subjected to fermentation process using a village level anaerobic bioreactor. Fermentation with yeast (Saccharomyces cerevisiae) can fasten up the process, thus producing more ethanol at a shorter period of time; however, without yeast fermentation, it also produces ethanol at lower volume with slower fermentation process. Distillation of 150 L of fermented broth was done for six hours at 85 °C to 95 °C temperature (feedstock) and 74 °C to 95 °C temperature of the column head (vapor state of ethanol). The highest volume of ethanol recovered was established at with yeast fermentation at five-day duration with a value of 14.89 L and lowest actual ethanol content was found at without yeast fermentation at three-day duration having a value of 11.63 L. In general, the results suggested that rain tree pods had a very good potential as feedstock for bioethanol production. Fermentation of rain tree pods juice can be done with yeast and without yeast.

Keywords: fermentation, hydrous bioethanol, fermentation, rain tree pods, village level

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Authors: Elias L. Souza, Noeli Sellin, Cintia Marangoni, Ozair Souza

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Among the different forms of reuse and recovery of agro-residual waste is the production of biofuels. The production of second-generation ethanol has been evaluated and proposed as one of the technically viable alternatives for this purpose. This research work employed the banana pseudostem as biomass. Two different chemical pre-treatment methods (acid hydrolisis with H2SO4 2% w/w and alkaline hydrolysis with NaOH 3% w/w) of dry and milled biomass (70 g/L of dry matter, ms) were assessed, and the corresponding reducing sugars yield, AR, (YAR), after enzymatic saccharification, were determined. The effect on YAR by increasing the dry matter (ms) from 70 to 100 g/L, in dry and milled biomass and also fresh, were analyzed. Changes in cellulose crystallinity and in biomass surface morphology due to the different chemical pre-treatments were analyzed by X-ray diffraction and scanning electron microscopy. The acid pre-treatment resulted in higher YAR values, whether related to the cellulose content under saccharification (RAR = 79,48) or to the biomass concentration employed (YAR/ms = 32,8%). In a comparison between alkaline and acid pre-treatments, the latter led to an increase in the cellulose content of the reaction mixture from 52,8 to 59,8%; also, to a reduction of the cellulose crystallinity index from 51,19 to 33,34% and increases in RAR (43,1%) and YAR/ms (39,5%). The increase of dry matter (ms) bran from 70 to 100 g/L in the acid pre-treatment, resulted in a decrease of average yields in RAR (43,1%) and YAR/ms (18,2%). Using the pseudostem fresh with broth removed, whether for 70 g/L concentration or 100 g/L in dry matter (ms), similarly to the alkaline pre-treatment, has led to lower average values in RAR (67,2% and 42,2%) and in YAR/ms (28,4% e 17,8%), respectively. The acid pre-treated and saccharificated biomass broth was detoxificated with different activated carbon contents (1,2 and 4% w/v), concentrated up to AR = 100 g/L and fermented by Saccharomyces cerevisiae. The yield values (YP/AR) and productivity (QP) in ethanol were determined and compared to those values obtained from the fermentation of non-concentrated/non-detoxificated broth (AR = 18 g/L) and concentrated/non-detoxificated broth (AR = 100 g/L). The highest average value for YP/AR (0,46 g/g) was obtained from the fermentation of non-concentrated broth. This value did not present a significant difference (p<0,05) when compared to the YP/RS related to the broth concentrated and detoxificated by activated carbon 1% w/v (YP/AR = 0,41 g/g). However, a higher ethanol productivity (QP = 1,44 g/L.h) was achieved through broth detoxification. This value was 75% higher than the average QP determined using concentrated and non-detoxificated broth (QP = 0,82 g/L.h), and 22% higher than the QP found in the non-concentrated broth (QP = 1,18 g/L.h).

Keywords: biofuels, biomass, saccharification, bioethanol

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Authors: Dibakar Chandra Deka, Anamika Kalita Deka

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Fermented alcoholic beverage production has its own stand among the tribal communities of North-East India. This biological oxidation method is followed by Ahom, Dimasa, Nishi, Miri, Bodo, Rabha tribes of this region. Bodo tribes among them not only prepare fermented alcoholic beverage but also store it for various time periods like 3 months, 6 months, 9 months, 12 months and 15 months etc. They prepare alcoholic beverage Jou (rice beer) following the fermentation of Oryza sativa with traditional yeast culture Amao. Saccharomyces cerevisiae is the main domain strain present in Amao. Dongphangrakep (Scoparia dulcis), Mwkhna (Clerodendrum viscosum), Thalir (Musa balbisina) and Khantal Bilai (Ananas cosmos) are the main plants used for Amao preparation. The stored Jou is known as Joufinai. They store the fermented mixture (rice and Amao) in anaerobic conditions for the preparation of Joufinai. We observed a successive increase in alcohol content from 3 months of storage period with 11.79 ± 0.010 (%, v/v) to 15.48 ± 0.070 (%, v/v) at 15 months of storage by a simple, reproducible and solution based colorimetric method. A positive linear correlation was also observed between pH and ethanol content with storage having correlation coefficient 0.981. Here, we optimised the detection of change in constituents of Joufinai during storage using reverse phase HPLC method. We found acetone, ethanol, acetic acid, glycerol as main constituents present in Joufinai. A very good correlation was observed from 3 months to 15 months of storage periods with its constituents. Increase in glycerol content was also detected with storage periods and hence Joufinai can be use as a precursor of above stated compounds. We also observed antioxidant activities increase from 0.056 ±2.80 mg/mL for 3 months old to 0.078± 5.33 mg/mL (in ascorbic acid equivalents) for 15 month old beverage by DPPH radical scavenging method. Therefore, we aimed for scientific validation of storage procedure used by Bodos in Joufinai production and to convert the Bodos’ traditional alcoholic beverage to a commercial commodity through our study.

Keywords: Amao, correlation, beverage, joufinai

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Authors: Patarasuda Chaisupa, R. Clay Wright

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The selection of appropriate biomolecular targets is a crucial aspect of biopharmaceutical development. The Auxin-Inducible Degron Degradation (AID) technology has demonstrated remarkable potential in efficiently and rapidly degrading target proteins, thereby enabling the identification and acquisition of drug targets. The AID system also offers a viable method to deplete specific proteins, particularly in cases where the degradation pathway has not been exploited or when the adaptation of proteins, including the cell environment, occurs to compensate for the mutation or gene knockout. In this study, we have engineered an improved AID system tailored to deplete proteins of interest. This AID construct combines the auxin-responsive E3 ubiquitin ligase binding domain, AFB2, and the substrate degron, IAA17, fused to the target genes. Essential genes of fungi with the lowest percent amino acid similarity to human and plant orthologs, according to the Basic Local Alignment Search Tool (BLAST), were cloned into the AID construct in S. cerevisiae (AID-tagged strains) using a modular yeast cloning toolkit for multipart assembly and direct genetic modification. Each E3 ubiquitin ligase and IAA17 degron was fused to a fluorescence protein, allowing for real-time monitoring of protein levels in response to different auxin doses via cytometry. Our AID system exhibited high sensitivity, with an EC50 value of 0.040 µM (SE = 0.016) for AFB2, enabling the specific promotion of IAA17::target protein degradation. Furthermore, we demonstrate how this improved AID system enhances quantitative functional studies of various proteins in fungi. The advancements made in auxin-inducible protein degradation in this study offer a powerful approach to investigating critical target protein viability in fungi, screening protein targets for drugs, and regulating intracellular protein abundance, thus revolutionizing the study of protein function underlying a diverse range of biological processes.

Keywords: synthetic biology, bioengineering, molecular biology, biotechnology

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Authors: Wighens Ngoie Ilunga, Pamela J. Welz, Olewaseun O. Oyekola, Daniel Ikhu-Omoregbe

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Currently, most sludge from the wastewater treatment plants of edible oil factories is disposed to landfills, but landfill sites are finite and potential sources of environmental pollution. Production of biodiesel from wastewater sludge can contribute to energy production and waste minimization. However, conventional biodiesel production is energy and waste intensive. Generally, biodiesel is produced from the transesterification reaction of oils with alcohol (i.e., Methanol, ethanol) in the presence of a catalyst. Homogeneously catalysed transesterification is the conventional approach for large-scale production of biodiesel as reaction times are relatively short. Nevertheless, homogenous catalysis presents several challenges such as high probability of soap. The current study aimed to reuse wastewater sludge from the edible oil industry as a novel feedstock for both monounsaturated fats and bioethanol for the production of biodiesel. Preliminary results have shown that the fatty acid profile of the oilseed wastewater sludge is favourable for biodiesel production with 48% (w/w) monounsaturated fats and that the residue left after the extraction of fats from the sludge contains sufficient fermentable sugars after steam explosion followed by an enzymatic hydrolysis for the successful production of bioethanol [29% (w/w)] using a commercial strain of Saccharomyces cerevisiae. A novel nano-magnetic catalyst was synthesised from mineral processing alkaline tailings, mainly containing dolomite originating from cupriferous ores using a modified sol-gel. The catalyst elemental chemical compositions and structural properties were characterised by X-ray diffraction (XRD), scanning electron microscopy (SEM), Fourier transform infra-red (FTIR) and the BET for the surface area with 14.3 m²/g and 34.1 nm average pore diameter. The mass magnetization of the nano-magnetic catalyst was 170 emu/g. Both the catalytic properties and reusability of the catalyst were investigated. A maximum biodiesel yield of 78% was obtained, which dropped to 52% after the fourth transesterification reaction cycle. The proposed approach has the potential to reduce material costs, energy consumption and water usage associated with conventional biodiesel production technologies. It may also mitigate the impact of conventional biodiesel production on food and land security, while simultaneously reducing waste.

Keywords: biodiesel, bioethanol, edible oil wastewater sludge, nano-magnetism

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Authors: Lethukuthula Ngobese, Abin Gupthar, Patrick Govender

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The ability of yeast cell wall-derived mannoproteins (glycoproteins) to positively contribute to oenological properties has been a key factor that stimulates research initiatives into these industrially important glycoproteins. In addition, and from a fundamental research perspective, yeast cell wall glycoproteins are involved in a wide range of biological interactions. To date, and to the best of our knowledge, our understanding of the fine molecular structure of these mannoproteins is fairly limited. Generally, the amino acid sequences of their protein moieties have been established from structural and functional analysis of the genomic sequence of these yeasts whilst far less information is available on the glycosyl moieties of these mannoproteins. A novel strategy was devised in this study that entails the genetic engineering of yeast strains that over-express and release cell wall-associated glycoproteins into the liquid growth medium. To this end, the Flo1p mannoprotein was overexpressed in Saccharomyces cerevisiae laboratory strains bearing a specific deletion in KNR4 and GPI7 genes involved in cell wall biosynthesis that have been previously shown to extracellularly hyper-secrete cell wall-associated glycoproteins. A polymerase chain reaction (PCR) -based cloning strategy was employed to generate transgenic yeast strains in which the native cell wall FLO1 glycoprotein-encoding gene is brought under transcriptional control of the constitutive PGK1 promoter. The modified Helm’s flocculation assay was employed to assess flocculation intensities of a Flo1p over-expressing wild type and deletion mutant as an indirect measure of their abilities to release the desired mannoprotein. The flocculation intensities of the transformed strains were assessed and all the strains showed similar intensities (>98% flocculation). To assess if mannoproteins were released into the growth medium, the supernatant of each strain was subjected to the BCA protein assay and the transformed Δknr4 strain showed a considerable increase in protein levels. This study has the potential to produce mannoproteins in sufficient quantities that may be employed in future investigations to understand their molecular structures and mechanisms of interaction to the benefit of both fundamental and industrial applications.

Keywords: glycoproteins, genetic engineering, flocculation, over-expression

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Authors: Marija Milašinović-Šeremešić, Valentina Semenčenko, Milica Radosavljević, Dušanka Terzić, Ljiljana Mojović, Ljubica Dokić

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Maize (Zea mays L.) is one of the most important cereal crops, and as such, one of the most significant naturally renewable carbohydrate raw materials for the production of energy and multitude of different products. The main goal of the present study was to investigate a suitability of selected maize hybrids of different genetic background produced in Maize Research Institute ‘Zemun Polje’, Belgrade, Serbia, for starch, bioethanol and animal feed production. All the hybrids are commercial and their detailed characterization is important for the expansion of their different uses. The starches were isolated by using a 100-g laboratory maize wet-milling procedure. Hydrolysis experiments were done in two steps (liquefaction with Termamyl SC, and saccharification with SAN Extra L). Starch hydrolysates obtained by the two-step hydrolysis of the corn flour starch were subjected to fermentation by S. cerevisiae var. ellipsoideus under semi-anaerobic conditions. The digestibility based on enzymatic solubility was performed by the Aufréré method. All investigated ZP maize hybrids had very different physical characteristics and chemical composition which could allow various possibilities of their use. The amount of hard (vitreous) and soft (floury) endosperm in kernel is considered one of the most important parameters that can influence the starch and bioethanol yields. Hybrids with a lower test weight and density and a greater proportion of soft endosperm fraction had a higher yield, recovery and purity of starch. Among the chemical composition parameters only starch content significantly affected the starch yield. Starch yields of studied maize hybrids ranged from 58.8% in ZP 633 to 69.0% in ZP 808. The lowest bioethanol yield of 7.25% w/w was obtained for hybrid ZP 611k and the highest by hybrid ZP 434 (8.96% w/w). A very significant correlation was determined between kernel starch content and the bioethanol yield, as well as volumetric productivity (48h) (r=0.66). Obtained results showed that the NDF, ADF and ADL contents in the whole maize plant of the observed ZP maize hybrids varied from 40.0% to 60.1%, 18.6% to 32.1%, and 1.4% to 3.1%, respectively. The difference in the digestibility of the dry matter of the whole plant among hybrids (ZP 735 and ZP 560) amounted to 18.1%. Moreover, the differences in the contents of the lignocelluloses fraction affected the differences in dry matter digestibility. From the results it can be concluded that genetic background of the selected maize hybrids plays an important part in estimation of the technological value of maize hybrids for various purposes. Obtained results are of an exceptional importance for the breeding programs and selection of potentially most suitable maize hybrids for starch, bioethanol and animal feed production.

Keywords: bioethanol, biomass quality, maize, starch

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Authors: Peter. M. Kusen, Georg Wandrey, Christopher Probst, Dietrich Kohlheyer, Jochen Buchs, Jorg Pietruszkau

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Light as a stimulus provides the capability to develop regulation techniques for customizable gene expression. A great advantage is the extremely flexible and accurate dosing that can be performed in a non invasive and sterile manner even for high throughput technologies. Therefore, light regulation in a multiwell microbioreactor system was realized providing the opportunity to control gene expression with outstanding complexity. A light-regulated gene expression system in Saccharomyces cerevisiae was designed applying the strategy of caged compounds. These compounds are photo-labile protected and therefore biologically inactive regulator molecules which can be reactivated by irradiation with certain light conditions. The “caging” of a repressor molecule which is consumed after deprotection was essential to create a flexible expression system. Thereby, gene expression could be temporally repressed by irradiation and subsequent release of the active repressor molecule. Afterwards, the repressor molecule is consumed by the yeast cells leading to reactivation of gene expression. A yeast strain harboring a construct with the corresponding repressible promoter in combination with a fluorescent marker protein was applied in a Photo-BioLector platform which allows individual irradiation as well as online fluorescence and growth detection. This device was used to precisely control the repression duration by adjusting the amount of released repressor via different irradiation times. With the presented screening platform the regulation of complex expression procedures was achieved by combination of several repression/derepression intervals. In particular, a stepwise increase of temporally-constant expression levels was demonstrated which could be used to study concentration dependent effects on cell functions. Also linear expression rates with variable slopes could be shown representing a possible solution for challenging protein productions, whereby excessive production rates lead to misfolding or intoxication. Finally, the very flexible regulation enabled accurate control over the expression induction, although we used a repressible promoter. Summing up, the continuous online regulation of gene expression has the potential to synchronize gene expression levels to optimize metabolic flux, artificial enzyme cascades, growth rates for co cultivations and many other applications addicted to complex expression regulation. The developed light-regulated expression platform represents an innovative screening approach to find optimization potential for production processes.

Keywords: caged-compounds, gene expression regulation, optogenetics, photo-labile protecting group

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Authors: Peter G. Hollis, Kim G. Clarke

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Accurate quantification of the overall volumetric oxygen transfer coefficient (KLa) is ubiquitously measured in bioprocesses by analysing the response of dissolved oxygen (DO) to a step change in the oxygen partial pressure in the sparge gas using a DO probe. Typically, the response lag (τ) of the probe has been ignored in the calculation of KLa when τ is less than the reciprocal KLa, failing which a constant τ has invariably been assumed. These conventions have now been reassessed in the context of multiphase bioprocesses, such as a hydrocarbon-based system. Here, significant variation of τ in response to changes in process conditions has been documented. Experiments were conducted in a 5 L baffled stirred tank bioreactor (New Brunswick) in a simulated hydrocarbon-based bioprocess comprising a C14-20 alkane-aqueous dispersion with suspended non-viable Saccharomyces cerevisiae solids. DO was measured with a polarographic DO probe fitted with a Teflon membrane (Mettler Toledo). The DO concentration response to a step change in the sparge gas oxygen partial pressure was recorded, from which KLa was calculated using a first order model (without incorporation of τ) and a second order model (incorporating τ). τ was determined as the time taken to reach 63.2% of the saturation DO after the probe was transferred from a nitrogen saturated vessel to an oxygen saturated bioreactor and is represented as the inverse of the probe constant (KP). The relative effects of the process parameters on KP were quantified using a central composite design with factor levels typical of hydrocarbon bioprocesses, namely 1-10 g/L yeast, 2-20 vol% alkane and 450-1000 rpm. A response surface was fitted to the empirical data, while ANOVA was used to determine the significance of the effects with a 95% confidence interval. KP varied with changes in the system parameters with the impact of solid loading statistically significant at the 95% confidence level. Increased solid loading reduced KP consistently, an effect which was magnified at high alkane concentrations, with a minimum KP of 0.024 s-1 observed at the highest solids loading of 10 g/L. This KP was 2.8 fold lower that the maximum of 0.0661 s-1 recorded at 1 g/L solids, demonstrating a substantial increase in τ from 15.1 s to 41.6 s as a result of differing process conditions. Importantly, exclusion of KP in the calculation of KLa was shown to under-predict KLa for all process conditions, with an error up to 50% at the highest KLa values. Accurate quantification of KLa, and therefore KP, has far-reaching impact on industrial bioprocesses to ensure these systems are not transport limited during scale-up and operation. This study has shown the incorporation of τ to be essential to ensure KLa measurement accuracy in multiphase bioprocesses. Moreover, since τ has been conclusively shown to vary significantly with process conditions, it has also been shown that it is essential for τ to be determined individually for each set of process conditions.

Keywords: effect of process conditions, measuring oxygen transfer coefficients, multiphase bioprocesses, oxygen probe response lag

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Authors: Andre Pastori D'Aurea, Lauriston Bertelli Feranades, Luis Eduardo Ferreira, Leandro Dias Pinto, Fabiana Ayumi Shiozaki

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The aim of this study was to improve the production of beef cattle on tropical pasture without harming this environment. On tropical pastures, cattle's live weight gain is lower than feedlot, and forage production is seasonable, changing from season to season. Thus, concerned with sustainable livestock production, the Premix Company has developed strategies to improve the production of beef cattle on tropical pasture to ensure sustainability of welfare and production. There are two important principles in this productivity system: 1) increase individual gains with use of better supplementation and 2) increase the productivity units with better forage quality like corn silage or other forms of forage conservations, actually used only in winter, and adding natural additives in the diet. This production system was applied from June 2017 to May 2018 in the Research Center of Premix Company, Patrocínio Paulista, São Paulo State, Brazil. The area used had 9 hectares of pasture of Brachiaria brizantha. 36 steers Nellore were evaluated for one year. The initial weight was 253 kg. The parameters used were daily average gain and gain per area. This indicated the corrections to be made and helped design future fertilization. In this case, we fertilized the pasture with 30 kg of nitrogen per animal divided into two parts. The diet was pasture and protein-energy supplements (0.4% of live weight). The supplement used was added with natural additive Fator P® – Premix Company). Fator P® is an additive composed by amino acids (lysine, methionine and tyrosine, 16400, 2980 and 3000 mg.kg-1 respectively), minerals, probiotics (Saccharomyces cerevisiae, 7 x 10E8 CFU.kg-1) and essential fatty acids (linoleic and oleic acids, 108.9 and 99g.kg-1 respectively). Due to seasonal changes, in the winter we supplemented the diet by increasing the offer of forage, supplementing with maize silage. It was offered 1% of live weight in silage corn and 0.4% of the live weight in protein-energetic supplements with additive Fator P ®. At the end of the period, the productivity was calculated by summing the individual gains for the area used. The average daily gain of the animals were 693 grams per day and was produced 1.005 kg /hectare/year. This production is about 8 times higher than the average of Brazilian meat national production. To succeed in this project, it is necessary to increase the gains per area, so it is necessary to increase the capacity per area. Pasture management is very important to the project's success because the dietary decisions were taken from the quantity and quality of the forage. We, therefore, recommend the use of animals in the growth phase because the response to supplementation is greater in that phase and we can allocate more animals per area. This system's carbon footprint reduces emissions by 61.2 percent compared to the Brazilian average. This beef cattle production system can be efficient and environmentally friendly to the natural. Another point is that bovines will benefit from their natural environment without competing or having an impact on human food production.

Keywords: cattle production, environment, pasture, sustainability

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Authors: Chang-Hui Shen, Michelle Esposito, Andrew J. Shen, Michael Adejokun, Diana Laterman

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The packaging of DNA into nucleosomes is critical to genomic compaction, yet it can leave gene promoters inaccessible to activator proteins or transcription machinery and thus prevents transcriptional initiation. Both chromatin remodelers and histone acetylases (HATs) are the two main transcription co-activators that can reconfigure chromatin structure for transcriptional activation. Ino80p is the core component of the INO80 remodeling complex. Recently, it was shown that Ino80p dissociates from the yeast INO1 promoter after induction. However, when certain HATs were deleted or mutated, Ino80p accumulated at the promoters during gene activation. This suggests a link between HATs’ presence and Ino80p’s dissociation. However, it has yet to be demonstrated that Ino80p can be acetylated. To determine if Ino80p can be acetylated, wild-type Saccharomyces cerevisiae cells carrying Ino80p engineered with a double FLAG tag (MATa INO80-FLAG his3∆200 leu2∆0 met15∆0 trp1∆63 ura3∆0) were grown to mid log phase, as were non-tagged wild type (WT) (MATa his3∆200 leu2∆0 met15∆0 trp1∆63 ura3∆0) and ino80∆ (MATa ino80∆::TRP1 his3∆200 leu2∆0 met15∆0 trp1∆63 ura3∆0) cells as controls. Cells were harvested, and the cell lysates were subjected to immunoprecipitation (IP) with α-FLAG resin to isolate Ino80p. These eluted IP samples were subjected to SDS-PAGE and Western blot analysis. Subsequently, the blots were probed with the α-FLAG and α-acetyl lysine antibodies, respectively. For the blot probed with α-FLAG, one prominent band was shown in the INO80-FLAG cells, but no band was detected in the IP samples from the WT and ino80∆ cells. For the blot probed with the α-acetyl lysine antibody, we detected acetylated Ino80p in the INO80-FLAG strain while no bands were observed in the control strains. As such, our results showed that Ino80p can be acetylated. This acetylation can explain the co-activator’s recruitment patterns observed in current gene activation models. In yeast INO1, it has been shown that Ino80p is recruited to the promoter during repression, and then dissociates from the promoter once de-repression begins. Histone acetylases, on the other hand, have the opposite pattern of recruitment, as they have an increased presence at the promoter as INO1 de-repression commences. This Ino80p recruitment pattern significantly changes when HAT mutant strains are studied. It was observed that instead of dissociating, Ino80p accumulates at the promoter in the absence of functional HATs, such as Gcn5p or Esa1p, under de-repressing processes. As such, Ino80p acetylation may be required for its proper dissociation from the promoters. The remodelers’ dissociation mechanism may also have a wide range of implications with respect to transcriptional initiation, elongation, or even repression as it allows for increased spatial access to the promoter for the various transcription factors and regulators that need to bind in that region. Our findings here suggest a previously uncharacterized interaction between Ino80p and other co-activators recruited to promoters. As such, further analysis of Ino80p acetylation not only will provide insight into the role of epigenetic modifications in transcriptional activation, but also gives insight into the interactions occurring between co-activators at gene promoters during gene regulation.

Keywords: acetylation, chromatin remodeler, epigenetic modification, Ino80p

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Authors: Denis V. Axenov-Gribanov, Irina V. Voytsekhovskaya, Evgenii S. Protasov, Maxim A. Timofeyev

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Isolated ecosystems existing under specific environmental conditions have been shown to be promising sources of new strains of actinobacteria. The taiga forest of Baikal Siberia has not been well studied, and its actinobacterial population remains uncharacterized. The proximity between the huge water mass of Lake Baikal and high mountain ranges influences the structure and diversity of the plant world in Siberia. Here, we report the isolation of eighteen actinobacterial strains from male cones of Pinus sylvestris trees growing on the shore of the ancient Lake Baikal in Siberia. The actinobacterial strains were isolated on solid nutrient MS media and Czapek agar supplemented with cycloheximide and phosphomycin. Identification of actinobacteria was carried out by 16S rRNA gene sequencing and further analysis of the evolutionary history. Four different liquid and solid media (NL19, DNPM, SG and ISP) were tested for metabolite production. The metabolite extracts produced by the isolated strains were tested for antibacterial and antifungal activities. Also, antiradical activity of crude extracts was carried out. Strain Streptomyces sp. IB 2014 I 74-3 that active against Gram-negative bacteria was selected for dereplication analysis with using the high-yield liquid chromatography with mass-spectrometry. Mass detection was performed in both positive and negative modes, with the detection range set to 160–2500 m/z. Data were collected and analyzed using Bruker Compass Data Analysis software, version 4.1. Dereplication was performed using the Dictionary of Natural Products (DNP) database version 6.1 with the following search parameters: accurate molecular mass, absorption spectra and source of compound isolation. Thus, in addition to more common representative strains of Streptomyces, several species belonging to the genera Rhodococcus, Amycolatopsis, and Micromonospora were isolated. Several of the selected strains were deposited in the Russian Collection of Agricultural Microorganisms (RCAM), St. Petersburg, Russia. All isolated strains exhibited antibacterial and antifungal activities. We identified several strains that inhibited the growth of the pathogen Candida albicans but did not hinder the growth of Saccharomyces cerevisiae. Several isolates were active against Gram-positive and Gram-negative bacteria. Moreover, extracts of several strains demonstrated high antioxidant activity. The high proportion of biologically active strains producing antibacterial and specific antifungal compounds may reflect their role in protecting pollen against phytopathogens. Dereplication of the secondary metabolites of the strain Streptomyces sp. IB 2014 I 74-3 was resulted in the fact that a total of 59 major compounds were detected in the culture liquid extract of strain cultivated in ISP medium. Eight compounds were preliminarily identified based on characteristics described in the Dictionary of Natural Products database, using the search parameters Streptomyces sp. IB 2014 I 74-3 was found to produce saframycin A, Y3 and S; 2-amino-3-oxo-3H-phenoxazine-1,8-dicarboxylic acid; galtamycinone; platencin A4-13R and A4-4S; ganefromycin d1; the antibiotic SS 8201B; and streptothricin D, 40-decarbamoyl, 60-carbamoyl. Moreover, forty-nine of the 59 compounds detected in the extract examined in the present study did not result in any positive hits when searching within the DNP database and could not be identified based on available mass-spec data. Thus, these compounds might represent new findings.

Keywords: actinobacteria, Baikal Lake, biodiversity, male cones, Pinus sylvestris

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