Isolation and identification of carotenoid-producing yeast and evaluation of antimalarial activity of the extracted carotenoid(s) against P. falciparum.

Plasmodial resistance to a variety of plant-based antimalarial drugs has led toward the discovery of more effective antimalarial compounds having chemical or biological origin. Since natural compounds are considered as safer drugs, in this study, yeast strains were identified and compared for the production of carotenoids that are well-known antioxidants and this metabolite was tested for its antiparasitic activity. Plasmodium falciparum 3D7 strain was selected as the target parasite for evaluation of antimalarial activity of yeast carotenoids using in vitro studies. Data were analyzed by FACS (fluorescence-activated cell sorter) and counted via gold standard Giemsa-stained smears. The extracted yeast carotenoids showed a profound inhibitory effect at a concentration of 10-3 µg/µl and 10-4 µg/µl when compared to ß- carotene as control. SYBR Green1 fluorescent dye was used to confirm the decrease in parasitaemia at given range of concentration. Egress assay results suggested that treated parasite remained stalled at schizont stage with constricted morphology and were darkly stained. Non-toxicity of carotenoids on erythrocytes and on human liver hepatocellular carcinoma cells (HepG2 cells) was shown at a given concentration. This report provides strong evidence for antimalarial effects of extracted yeast carotenoids, which can be produced via a sustainable and cost-effective strategy and may be scaled up for industrial application.

Strain Improvement of Native Saccharomyces cerevisiae LN ITCC 8246 Strain Through Protoplast Fusion To Enhance Its Xylose Uptake.

Co-utilization of xylose and glucose and subsequent fermentation using Saccharomyces cerevisiae could enhance ethanol productivity. Directed engineering approaches have met with limited success due to interconnectivity of xylose metabolism with other intrinsic, hidden pathways. Therefore, random approaches like protoplast fusion were used to reprogram unidentified mechanisms. Saccharomyces cerevisiae LN, the best hexose fermenter, was fused with xylose fermenting Pichia stipitis NCIM 3498. Protoplasts prepared using glucanex were fused under electric impulse and fusants were selected using 10% ethanol and cycloheximide (50 ppm) markers. Two fusants, 1a.23 and 1a.30 showing fast growth on xylose and tolerance to 10% ethanol, were selected. Higher extracellular protein expression observed in fusants as compared to parents was corroborated by higher number of bands resolved by two-dimensional analysis. Overexpression of XYL1, XYL2, XKS, and XUT4 in fusants as compared to S. cerevisiae LN as observed by RT-PCR analysis was substantiated by higher specific activities of XR, XDH, and XKS enzymes in fusants. During lignocellulosic hydrolysate fermentation, fusants could utilize glucose faster than the parent P. stipitis NCIM 3498 and xylose consumption in fusants was higher than S. cerevisiae LN.

Corrigendum: Augmenting Pentose Utilization and Ethanol Production of Native Saccharomyces Cerevisiae LN Using Medium Engineering and Response Surface Methodology.

[This corrects the article DOI: 10.3389/fbioe.2018.00132.].

Augmenting Pentose Utilization and Ethanol Production of Native Saccharomyces cerevisiae LN Using Medium Engineering and Response Surface Methodology.

Economics of ethanol production from lignocellulosic biomass depends on complete utilization of constituent carbohydrates and efficient fermentation of mixed sugars present in biomass hydrolysates. Saccharomyces cerevisiae, the commercial strain for ethanol production uses only glucose while pentoses remain unused. Recombinant strains capable of utilizing pentoses have been engineered but with limited success. Recently, presence of endogenous pentose assimilation pathway in S. cerevisiae was reported. On the contrary, evolutionary engineering of native xylose assimilating strains is promising approach. In this study, a native strain S. cerevisiae LN, isolated from fruit juice, was found to be capable of xylose assimilation and mixed sugar fermentation. Upon supplementation with yeast extract and peptone, glucose (10%) fermentation efficiency was 78% with ~90% sugar consumption. Medium engineering augmented mixed sugars (5% glucose + 5% xylose) fermentation efficiency to ~50 and 1.6% ethanol yield was obtained with concomitant sugar consumption ~60%. Statistical optimization of input variables Glucose (5.36%), Xylose (3.30%), YE (0.36%), and peptone (0.25%) with Response surface methodology led to improved sugar consumption (74.33%) and 2.36% ethanol within 84 h. Specific activities of Xylose Reductase and Xylitol Dehydrogenase exhibited by S. cerevisiae LN were relatively low. Their ratio indicated metabolism diverted toward ethanol than xylitol and other byproducts. Strain was tolerant to concentrations of HMF, furfural and acetic acid commonly encountered in biomass hydrolysates. Thus, genetic setup for xylose assimilation in S. cerevisiae LN is not merely artifact of xylose metabolizing pathway and can be augmented by adaptive evolution. This strain showed potential for commercial exploitation.

Notable mixed substrate fermentation by native Kodamaea ohmeri strains isolated from Lagenaria siceraria flowers and ethanol production on paddy straw hydrolysates.

BACKGROUND: Bioethanol obtained by fermenting cellulosic fraction of biomass holds promise for blending in petroleum. Cellulose hydrolysis yields glucose while hemicellulose hydrolysis predominantly yields xylose. Economic feasibility of bioethanol depends on complete utilization of biomass carbohydrates and an efficient co-fermenting organism is a prerequisite. While hexose fermentation capability of Saccharomyces cerevisiae is a boon, however, its inability to ferment pentose is a setback. RESULTS: Two xylose fermenting Kodamaea ohmeri strains were isolated from Lagenaria siceraria flowers through enrichment on xylose. They showed 61% glucose fermentation efficiency in fortified medium. Medium engineering with 0.1% yeast extract and peptone, stimulated co-fermentation potential of both strains yielding maximum ethanol 0.25 g g-1 on mixed sugars with ~ 50% fermentation efficiency. Strains were tolerant to inhibitors like 5-hydroxymethyl furfural, furfural and acetic acid. Both K. ohmeri strains grew well on biologically pretreated rice straw hydrolysates and produced ethanol. CONCLUSIONS: This is the first report of native Kodamaea sp. exhibiting notable mixed substrate utilization and ethanol fermentation. K. ohmeri strains showed relevant traits like utilizing and co-fermenting mixed sugars, exhibiting excellent growth, inhibitor tolerance, and ethanol production on rice straw hydrolysates.

Enhanced biodegradation of PAHs by microbial consortium with different amendment and their fate in in-situ condition.

Microbial degradation is a useful tool to prevent chemical pollution in soil. In the present study, in-situ bioremediation of polyaromatic hydrocarbons (PAHs) by microbial consortium consisting of Serratia marcescens L-11, Streptomyces rochei PAH-13 and Phanerochaete chrysosporium VV-18 has been reported. In preliminary studies, the consortium degraded nearly 60-70% of PAHs in broth within 7 days under controlled conditions. The same consortium was evaluated for its competence under natural conditions by amending the soil with ammonium sulphate, paddy straw and compost. Highest microbial activity in terms of dehydrogenase, FDA hydrolase and aryl esterase was recorded on the 5(th) day. The degradation rate of PAHs significantly increased up to 56-98% within 7 days under in-situ however almost complete dissipation (83.50-100%) was observed on the 30(th) day. Among all the co-substrates evaluated, faster degradation of PAHs was observed in compost amended soil wherein fluorene, anthracene, phenanthrene and pyrene degraded with half-life of 1.71, 4.70, 2.04 and 6.14 days respectively. Different degradation products formed were also identified by GC-MS. Besides traces of parent PAHs eleven non-polar and five polar products were identified by direct and silylation reaction respectively. Various products formed indicated that consortium was capable to degrade PAHs by oxidation to mineralization.

Glycoside hydrolase production by Aspergillus terreus CM20 using mixture design approach for enhanced enzymatic saccharification of alkali pretreated paddy straw.

A successful lignocellulosic ethanol production process needs to address the technological impediments such as cost-competitiveness and sustainability of the process. Effective biomass utilization requires a repertoire of enzymes including various accessory enzymes. Developing an enzyme preparation with defined hydrolytic activities can circumvent the need for supplementing cellulases with accessory enzymes for enhanced hydrolysis. With this objective, mixture design approach was used in the present study to enhance glycoside hydrolase production of a fungal isolate, Aspergillus terreus CM20, by determining the proportion of different lignocellulosic components as enzyme inducers in the culture medium. A mixture of paddy straw and wheat straw (1.42:1.58) resulted in improved cellulolytic activities. The precipitated crude enzyme showed higher CMCase (365.03 18 IU g-1), FPase (161.48 IU g-1), avicelase (15.46 IU g-1), ß-glucosidase (920.92 IU g-1) and xylanase (9627.79 IU g-1) activities. The potential of the crude enzyme for saccharification of alkali pretreated paddy straw was also tested. Under optimum conditions, saccharification released 25.0 g L-1 of fermentable sugars. This indicates the superiority of the crude enzyme produced with respect to its hydrolytic enzyme components.

Improvement of antioxidant and defense properties of Tomato (var. Pusa Rohini) by application of bioaugmented compost.

Nutrient management practices play a significant role in improving the nutritional quality of tomato. The present study deals with the evaluation of compost prepared using Effective Microorganisms (EM), on antioxidant and defense enzyme activities of Tomato (Lycopersicon esculentum). A field experiment with five treatments (control, chemical fertilizer and EM compost alone and in combination) was conducted in randomized block design. An increment of 31.83% in tomato yield was recorded with the combined use of EM compost and half recommended dose of chemical fertilizers (N50P30K25 + EM compost at the rate of 5 t ha(-1)). Similarly, fruit quality was improved in terms of lycopene content (35.52%), antioxidant activity (24-63%) and defense enzymes activity (11-54%), in tomatoes in this treatment as compared to the application of recommended dose of fertilizers. Soil microbiological parameters also exhibited an increase of 7-31% in the enzyme activities in this treatment. Significant correlation among fruit quality parameters with soil microbiological activities reveals the positive impact of EM compost which may be adopted as an eco-friendly strategy for production of high quality edible products.

Optimization of Enzymatic Saccharification of Alkali Pretreated Parthenium sp. Using Response Surface Methodology.

Parthenium sp. is a noxious weed which threatens the environment and biodiversity due to its rapid invasion. This lignocellulosic weed was investigated for its potential in biofuel production by subjecting it to mild alkali pretreatment followed by enzymatic saccharification which resulted in significant amount of fermentable sugar yield (76.6%). Optimization of enzymatic hydrolysis variables such as temperature, pH, enzyme, and substrate loading was carried out using central composite design (CCD) in response to surface methodology (RSM) to achieve the maximum saccharification yield. Data obtained from RSM was validated using ANOVA. After the optimization process, a model was proposed with predicted value of 80.08% saccharification yield under optimum conditions which was confirmed by the experimental value of 85.80%. This illustrated a good agreement between predicted and experimental response (saccharification yield). The saccharification yield was enhanced by enzyme loading and reduced by temperature and substrate loading. This study reveals that under optimized condition, sugar yield was significantly increased which was higher than earlier reports and promises the use of Parthenium sp. biomass as a feedstock for bioethanol production.

Comparative efficiency of different pretreatment methods on enzymatic digestibility of Parthenium sp.

The potential of Parthenium sp. as a feedstock for enzymatic saccharification was investigated by using chemical and biological pretreatment methods. Mainly chemical pretreatments (acid and alkali) were compared with biological pretreatment with lignolytic fungi Marasmiellus palmivorus PK-27. Structural and chemical changes as well as crystallinity of cellulose were examined through scanning electron microscopy, fourier transform infra red and X-ray diffraction analysis, respectively after pretreatment. Total reducing sugar released during enzymatic saccharification of pretreated substrates was also evaluated. Among the pretreatment methods, alkali (1% NaOH) treated substrate showed high recovery of acid perceptible polymerised lignin (7.53 ± 0.5 mg/g) and significantly higher amount of reducing sugar (513.1 ± 41.0 mg/gds) compared to uninoculated Parthenium (163.4 ± 21.2) after 48 h of hydrolysis. This is the first report of lignolytic enzyme production from M. palmivorus, prevalent in oil palm plantations in Malaysia and its application in biological delignification of Parthenium sp. Alkali (1% NaOH) treatment proves to be the suitable method of pretreatment for lignin recovery and enhanced yield of reducing sugar which may be used for bioethanol production from Parthenium sp.

Biological delignification of paddy straw and Parthenium sp. using a novel micromycete Myrothecium roridum LG7 for enhanced saccharification.

A new lignolytic micromycete fungus Myrothecium roridum LG7 was isolated and selected for biological delignification of agro residue-paddy straw and herbaceous weed Parthenium sp. Physical and chemical modifications in the biomass following pretreatment with M. roridum LG7 for 7 days in term of structural modification and lignin removal, changes in lignin skeleton, and alteration of cellulose crystallinity was observed through SEM-EDXA, FTIR and XRD analysis, respectively. Colonization of the fungus led to high amount of lignin removal (5.8-6.98mg/gds) from pretreated biomass which could be recovered as a value added product. Enzymatic hydrolysis of M. roridum LG7 pretreated biomass released significantly higher amount of reducing sugars (455.81-509.65 mg/gds) as compared to respective raw biomass within 24h. This study illustrates the promise of M. roridum LG7 for biological pretreatment through structural and chemical alteration of biomass beside creation of alkaline environment which prevent the growth of other contaminants.

Streptomyces griseorubens mediated delignification of paddy straw for improved enzymatic saccharification yields.

Biological pretreatment of paddy straw was carried out using an actinomycete isolate, identified as Streptomyces griseorubens ssr38, for delignification under solid state fermentation and enhanced sugar recovery by enzymatic saccharification. After 10 days incubation, the inoculated paddy straw was extracted with mild alkali and high absorbance at 205 nm was shown by the extracts indicating the ability of S. griseorubens ssr38 to depolymerize/solubilize lignin to a high extent. Also, almost 25% of depolymerized lignin could be recovered as value-added acid-precipitable polymeric lignin (APPL) as compared to controls. Enrichment in carbohydrate content of inoculated paddy straw following delignification led to a high saccharification efficiency of 97.8% upon enzymatic hydrolysis with Accelerase®1500. The study, therefore, proves the potential of actinomycetes, besides the conventionally used white-rot fungi, for biological pretreatment, in the biomass to bioethanol process, with respect to the high extent of delignification, lignin recovery, cellulose enrichment and very high saccharification efficiency.

Deciphering the traits associated with PAH degradation by a novel Serratia marcesencs L-11 strain.

Polycyclic aromatic hydrocarbons (PAHs) are wide spread industrial pollutants that are released into the environment from burning of coal, distillation of wood, operation of gas works, oil refineries, vehicular emission, and combustion process. In this study a lipolytic bacterium was isolated from mixed stover compost of Saccharum munja and Brassica campestris. This strain was identified by both classical and 16S ribosomal DNA sequencing method and designated as Serratia marcesencs L-11. HPLC-based quantitation revealed 39- 100% degradation of PAH compounds within seven days. Further its ability to produce catechol 1, 2-dioxygenase (1.118 µM mL(-1) h(-1)) and biosurfactants (0.88 g L(-1)) during growth in PAH containing medium may be responsible for its PAH-degradation potential. This novel bacterium with an ability to produce lipases, biosurfactant and ring cleavage enzyme can prove to be useful for in-situ degradation of PAH compounds.

Pretreatment of paddy straw with Trametes hirsuta for improved enzymatic saccharification.

Delignification of paddy straw with the white-rot fungus, Trametes hirsuta under solid state fermentation, for enhanced sugar recovery by enzymatic saccharification was studied. T. hirsuta MTCC136 showed high ligninase and low cellulase activities. Solid state fermentation of paddy straw with T. hirsuta enhanced carbohydrate content by 11.1% within 10 days of incubation. Alkali extracts of Trametes pretreated paddy straw showed high absorbance at 205 nm indicating high lignin break down. The amount of value-added lignin recovered from the Trametes pretreated paddy straw was much higher than controls. Enzymatic hydrolysis of the Trametes pretreated paddy straw yielded much higher sugars than controls and yields increased till 120 h of incubation. Saccharification efficiency of the biologically pretreated paddy straw with Accelerase®1500 was 52.69% within 72 h and was higher than controls. Thus, the study brings out the delignification potential of T. hirsuta for pretreatment of lignocellulosic substrate and facilitating efficient enzymatic digestibility of cellulose.

Biological pretreatment of lignocellulosic substrates for enhanced delignification and enzymatic digestibility.

Sheer enormity of lignocellulosics makes them potential feedstock for biofuel production but, their conversion into fermentable sugars is a major hurdle. They have to be pretreated physically, chemically, or biologically to be used by fermenting organisms for production of ethanol. Each lignocellulosic substrate is a complex mix of cellulose, hemicellulose and lignin, bound in a matrix. While cellulose and hemicellulose yield fermentable sugars, lignin is the most recalcitrant polymer, consisting of phenyl-propanoid units. Many microorganisms in nature are able to attack and degrade lignin, thus making access to cellulose easy. Such organisms are abundantly found in forest leaf litter/composts and especially include the wood rotting fungi, actinomycetes and bacteria. These microorganisms possess enzyme systems to attack, depolymerize and degrade the polymers in lignocellulosic substrates. Current pretreatment research is targeted towards developing processes which are mild, economical and environment friendly facilitating subsequent saccharification of cellulose and its fermentation to ethanol. Besides being the critical step, pretreatment is also cost intensive. Biological treatments with white rot fungi and Streptomyces have been studied for delignification of pulp, increasing digestibility of lignocellulosics for animal feed and for bioremediation of paper mill effluents. Such lignocellulolytic organisms can prove extremely useful in production of bioethanol when used for removal of lignin from lignocellulosic substrate and also for cellulase production. Our studies on treatment of hardwood and softwood residues with Streptomyces griseus isolated from leaf litter showed that it enhanced the mild alkaline solubilisation of lignins and also produced high levels of the cellulase complex when growing on wood substrates. Lignin loss (Klason lignin) observed was 10.5 and 23.5% in case of soft wood and hard wood, respectively. Thus, biological pretreatment process for lignocellulosic substrate using lignolytic organisms such as actinomycetes and white rot fungi can be developed for facilitating efficient enzymatic digestibility of cellulose.

Spontaneous splenic infarction associated with sumatriptan use.

Triptans are specific agonists of the serotonergic 5-HT(1B/1D) receptors that have increasingly been used in the treatment of migraine and cluster headaches. Though they are generally considered safe, there have been a few reports of myocardial infarction and stroke associated with triptan use. We report a patient who developed spontaneous splenic infarction after the use of sumatriptan for the treatment of migraine headache.

Optimization of a biological process for treating potato chips industry wastewater using a mixed culture of Aspergillus foetidus and Aspergillus niger.

Potato chips industry wastewater was collected and analyzed for biochemical oxygen demand (BOD), chemical oxygen demand (COD), total suspended solids (TSS) and total carbohydrates. Two Aspergillus species, A. foetidus and A. niger, were evaluated for their ability to grow and produce biomass and reduce the organic load of the wastewater. A. foetidus MTCC 508 and A. niger ITCC 2012 were able to reduce COD by about 60% and produce biomass 2.4 and 2.85 gl(-1), respectively. Co-inoculation of both Aspergillus strains resulted in increased fungal biomass production and higher COD reduction than in individual culture at different culture pH. pH 6 was optimum for biomass production and COD reduction. Amendment of the wastewater with different N and P sources, increased the biomass production and COD reduction substantially. Under standardized conditions of pH 6 and amendment of wastewater with 0.1% KH2PO4 and 0.1% (NH4)2 SO4, a mixed culture gave 90% reduction in COD within 60 h of incubation.