A novel strategy for partial purification of alkane hydroxylase from P. chrysogenum SNP5 through reconstituting its native membrane into liposome.

Integral proteins or enzymes are still challenging to purify into their native state because of their need for an amphipathic environment and cofactors. Alkane hydroxylase (AlkB) is a membrane-bound enzyme that catalyzes the hydroxylation of a range of alkanes that have a broad spectrum of applications. In the current study, a novel approach has been explored for partial purification of alkane hydroxylase (AlkB) in its native state through restructuring the lipid bilayer of Penicillium chrysogenum SNP5 into a liposome to extend the native and protective environment to AlkB enzyme. Three different methods i.e., reverse-phase evaporation method (RPEM), detergent-based method (DBM), and ethanol injection method (EIM) have been used for reconstituting its native membrane into liposome. On characterizing liposomes through fluorescence imaging, AFM, and particle size analysis, the reverse-phase evaporation method gave the best results based on the size distribution (i.e., 100-300 nm), the morphology of liposomes, and maximum AlkB specific activity (i.e., 140.68 U/mg). The maximum reconstitution efficiency of 29.48% was observed in RPEM followed by 17.3% in DBM and 12.3% in EIM. On the characterization of the purified AlkB, the molecular weight was measured of 44.6 KDa and the thermostability of liposomes synthesized with the RPEM method was obtained maximum at 55 °C. This approach may open a new strategy for the purification of integral enzymes/proteins in their native state in the field of protein purification and its applications in diversified industries.

Enhanced ethanol production using hydrophobic resin detoxified Pine forest litter hydrolysate and integrated fermentation process development supplementing molasses.

Globally escalating ethanol demand necessitates the use of hybrid technologies integrating first- and second-generation biofuel feedstocks for achieving the futuristic targets of gasoline replacement with bioethanol. In present study, an optimized two-step sequential pre-treatment (first dilute alkali, then dilute acid) of Pine forest litter (PFL) was developed. Furthermore, the saccharification of pre-treated PFL was optimized through Response Surface Methodology using Box-Behnken Design, wherein 0.558 g/g of reducing sugar was released under the optimized conditions (12.5% w/v of biomass loading, 10 FPU/g of PFL enzyme loading, 0.15% v/v Tween-80 and 48 h incubation time). Moreover, during hydrolysate fermentation using Saccharomyces cerevisiae NCIM 3288 strain, 22.51 ± 1.02 g/L ethanol was produced. Remarkably, hydrophobic resin (XAD-4) treatment of PFL hydrolysate, significantly removed inhibitors (Furfural, 5-hydroxymethylfurfural and phenolics) and increased ethanol production to 27.38 ± 1.18 g/L. Furthermore, during fermentation of molasses supplemented PFL hydrolysate (total initial sugar: 100 ± 3.27 g/L), a maximum of 46.02 ± 2.08 g/L ethanol was produced with 0.482 g/g yield and 1.92 g/l/h productivity. These findings indicated that the integration of molasses to lignocellulosic hydrolysate, would be a promising hybrid technology for industrial ethanol production within existing bio-refinery infrastructure.

Biotransformation of grease waste into fatty acid by Penicillium chrysogenum SNP5 through media engineering and artificial neural network.

Degradation of grease waste remains a challenging task. Current work deals with the biotransformation of grease waste into fatty acids under submerged fermentation using Penicillium chrysogenum SNP5 through media formulation and artificial neural network (ANN). Fermentation media was formulated to ameliorate the uptake of hydrocarbon by enhancing alkane hydroxylase (AlkB) activity, extracellular release of fatty acids and inhibiting beta-oxidation of fatty acid by regulating transketolase. Further, the process parameters of fermentation were optimized through Artificial Neural Network (ANN) using three critical variables viz; inoculum size (spores/ml), pH, and incubation time (days) while media engineering was done with the optimal supplementation of various medium components such as glucose, YPD, MnSO4, tetrahydrobiopterin (THB) and phloretin. The maximum conversion of 66.5% of grease waste into fatty acid was achieved at optimum conditions: inoculums size 3.36 × 107 spores/ml, incubation time 11.5 days, pH 7.2 along with formulated media composed of 1% grease in czapek-dox medium supplemented with 55.5 mM glucose, 0.5% YPD, 16.6 mM hexadecane, 1 mM MnSO4, 1 mM THB, and 1 mM phloretin. The presence of long-chain fatty acids in purified extracts such as oleic acid and octadecanoic acid as end products has valued the evolved process as another source of alternative fuel.

Microalgae harvesting techniques: updates and recent technological interventions.

Microalgal biomass has garnered attention as a renewable and sustainable resource for producing biodiesel. The harvesting of microalgal biomass is a significant bottleneck being faced by the industries as it is the crucial cost driver in the downstream processing of biomass. Bioharvesting of microalgal biomass mediated by: microbial, animal, and plant-based polymeric flocculants has gained a higher probability of utility in accumulation due to: its higher dewatering potential, less toxicity, and ecofriendly properties. The present review summarizes the key challenges and the technological advancements associated with various such harvesting techniques. The economic and technical aspects of different microalgal harvesting techniques, particularly the cationic polymeric flocculant-based harvesting of microalgal biomass, are also discussed. Furthermore, interactions of flocculants with microalgal biomass and the effects of these interactions on metabolite and lipid extractions are discussed to offer a promising solution for suitability in selecting the most efficient and economical method of microalgal biomass harvesting for cost-effective biodiesel production.

Microbiome systems biology advancements for natural well-being.

Throughout the years all data from epidemiological, physiological and omics have suggested that the microbial communities play a considerable role in modulating human health. The population of microorganisms residing in the human intestine collectively known as microbiota presents a genetic repertoire that is higher in magnitude than the human genome. They play an essential role in host immunity and neuronal signaling. Rapid enhancement of sequence based screening and development of humanized gnotobiotic model has sparked a great deal of interest among scientists to probe the dynamic interactions of the commensal bacteria. This review focuses on systemic analysis of the gut microbiome to decipher the complexity of the host-microbe intercommunication and gives a special emphasis on the evolution of targeted precision medicine through microbiome engineering. In addition, we have also provided a comprehensive description of how interconnection between metabolism and biochemical reactions in a specific organism can be obtained from a metabolic network or a flux balance analysis and combining multiple datasets helps in the identification of a particular metabolite. The review highlights how genetic modification of the critical components and programming the resident microflora can be employed for targeted precision medicine. Inspite of the ongoing debate on the utility of gut microbiome we have explored on the probable new therapeutic avenues like FMT (Fecal microbiota transplant) can be utilized. This review also recapitulates integrating human-relevant 3D cellular models coupled with computational models and the metadata obtained from interventional and epidemiological studies may decipher the complex interactome of diet-microbiota-disease pathophysiology. In addition, it will also open new avenues for the development of therapeutics derived from microbiome or implementation of personalized nutrition. In addition, the identification of biomarkers can also help towards the development of new diagnostic tools and eventually will lead to strategic management of the disease. Inspite of the ongoing debate on the utility of the gut microbiome we have explored how probable new therapeutic avenues like FMT (Fecal microbiota transplant) can be utilized. This review also summarises integrating human-relevant 3D cellular models coupled with computational models and the metadata obtained from interventional and epidemiological studies may decipher the complex interactome of diet- microbiota-disease pathophysiology. In addition, it will also open new avenues for the development of therapeutics derived from the microbiome or implementation of personalized nutrition. In addition, the identification of biomarkers can also help towards the development of new diagnostic tools and eventually will lead to strategic management of disease.

Enhanced production of alkane hydroxylase from Penicillium chrysogenum SNP5 (MTCC13144) through feed-forward neural network and genetic algorithm.

Alkane hydroxylase (AlkB), a membrane-bound enzyme has high industrial demand; however, its economical production remains challenging due to its intrinsic nature and co-factor dependency. In the current study, various critical process parameters for optimum production of AlkB have been optimized through feed forward neural network (FFNN) and genetic algorithm (GA) models using Penicillium chrysogenum SNP5 (MTCC13144). AlkB specific activity under preliminary un-optimized conditions i.e., 1% hexadecane, 7.4 pH, 11 days incubation time, 28 °C incubation temperature and 1 ml of inoculum size was 100 U/mg. 'One variable at a time' (OVAT) strategy was used to identify optimum physicochemical parameters and then its output data was fed to develop a model of FFNN with '6-12-1' topology. Outputs of FFNN were further optimized through GA to minimize errors and intensify search level. This has provided superior predictive performances with 0.053 U/mg overall mean absolute percentage error (MAPE), 6.801 U/mg root mean square errors (RMSE), and 0.987 overall correlation coefficient (R). The AlkB specific activity improved by 3.5-fold, i.e., from 100 U/mg under preliminary un-optimized conditions to 351.32 U/mg under optimum physicochemical conditions obtained through FFNN-GA hybrid method, i.e., hexadecane (carbon source): 1.56% v/v, FeSO4: 0.63 mM, incubation temperature: 27.40 °C, pH: 7.38, incubation time: 12.35 days and inoculums size: 1.33 ml. The developed process would be a stepping stone to fulfill the high industrial demands of  Alkane hydroxylase.

Light regulation of light-harvesting antenna size substantially enhances photosynthetic efficiency and biomass yield in green algae†.

One of the major factors limiting biomass productivity in algae is the low thermodynamic efficiency of photosynthesis. The greatest thermodynamic inefficiencies in photosynthesis occur during the conversion of light into chemical energy. At full sunlight the light-harvesting antenna captures photons at a rate nearly 10 times faster than the rate-limiting step in photosynthetic electron transport. Excess captured energy is dissipated by non-productive pathways including the production of reactive oxygen species. Substantial improvements in photosynthetic efficiency have been achieved by reducing the optical cross-section of the light-harvesting antenna by selectively reducing chlorophyll b levels and peripheral light-harvesting complex subunits. Smaller light-harvesting antenna, however, may not exhibit optimal photosynthetic performance in low or fluctuating light environments. We describe a translational control system to dynamically adjust light-harvesting antenna sizes for enhanced photosynthetic performance. By expressing a chlorophyllide a oxygenase (CAO) gene having a 5' mRNA extension encoding a Nab1 translational repressor binding site in a CAO knockout line it was possible to continuously alter chlorophyll b levels and correspondingly light-harvesting antenna sizes by light-activated Nab1 repression of CAO expression as a function of growth light intensity. Significantly, algae having light-regulated antenna sizes had substantially higher photosynthetic rates and two-fold greater biomass productivity than the parental wild-type strains as well as near wild-type ability to carry out state transitions and non-photochemical quenching. These results have broad implications for enhanced algae and plant biomass productivity.

Combined ANN/EVOP Factorial Design Approach for Media Screening for Cost-effective Production of Alkaline Proteases from Rhizopus oryzae (SN5)/NCIM-1447 under SSF.

In order to achieve high yield of fungal protease in a very cost effective way and to meet its increased market demand, current study deals with the screening of various agro-wastes as carbon source for the production of protease from Rhizopus oryzae (SN5)/NCIM-1447 under solid state fermentation. Substrates and culture parameters such as wheat bran, soybean meal, black-gram husk, rice husk, mixture of wheat bran, soybean meal, nitrogen sources, pH, temperature and incubation time were first optimized with one factor at time strategy and then EVOP factorial and yield of alkaline protease was achieved 412.8 U/gds at 28 °C and pH = 6 after 72 h of fermentation taking wheat bran and soybean as a substrate in 4:1 ratio. Further artificial neural networks (ANN), was trained with data of EVOP and yield of protease was enhanced up to 422.6 U/gds with wheat bran: soyabean in ratio of 70:30, pH 6.2 at 30 °C. The evolved process and Rhizopus oryzae (SN5)/NCIM-1447 strain would be promising for protease production at industrial scale at low cost.

Enhanced production of laccase and pectinase using co-culture of Trametes hirsuta and Phanerochaete sp. through EVOP-factorial design technique.

The present study deals with the coproduction of laccase and pectinase enzymes through solid state fermentation using mixed fungal culture of Trametes hirsuta and Phanerochaete sp., to minimize the cost and time of the process. Substrates selected for the enzyme production were wheat bran, pulse husk and mustard peel. To get optimum yield of laccase and pectinase in a single fermenter, EVOP factorial design technique with factors like pH, incubation temperature and substrates ratio have been explored. At search level of EVOP, outcomes of the one factor at a time has been considered as incubation temperature 30 °C for 7 days at pH 5 with wheat bran: pulse husk: mustard peel ratio of 2:2:1 (w/w/w) and yield of laccase and pectinase was found as 78.1 U/gds and 105.0 U/gds respectively. In first setup of EVOP factorial both laccase and pectinase activities were not found optimum in same set of experiments; therefore, on the basis of its decision making steps, second set of experiment was performed by taking decisions of first set of EVOP as search level. Optimum yield of laccase and pectinase was achieved about 250 U/gds and 247 U/gds respectively at 34 °C, pH 4.5 with 1.75:1.75:1.5 (w/w/w) wheatbran: pulse husk: mustard peel as substrate, which was 2-3 times higher than the outcomes of one factor at a time method.

Engineering PGPMOs through Gene Editing and Systems Biology: A Solution for Phytoremediation?

In light of extensive urbanization and deforestation, toxic wastes are being released into the atmosphere, causing increased air and soil pollution. Conventional methods of soil remediation are time consuming and labor and cost intensive, rendering them uneconomical to maintain sustainable agriculture. One solution is to use natural resources like plants and microbes for phytoremediation. A thorough systemic knowledge of plant-microbe interactions will allow the use of gene editing and gene manipulation techniques to increase the efficiency of plants in phytoremediation. This Opinion article focuses on gene editing techniques used in plants and microbes for phytoremediation and also emphasizes their effectiveness, advancement, and future implications for sustainable and environmentally friendly agriculture.

Improvements in algal lipid production: a systems biology and gene editing approach.

BACKGROUND: In the wake of rising energy demands, microalgae have emerged as potential sources of sustainable and renewable carbon-neutral fuels, such as bio-hydrogen and bio-oil. PURPOSE: For rational metabolic engineering, the elucidation of metabolic pathways in fine detail and their manipulation according to requirements is the key to exploiting the use of microalgae. Emergence of site-specific nucleases have revolutionized applied research leading to biotechnological gains. Genome engineering as well as modulation of the endogenous genome with high precision using CRISPR systems is being gradually employed in microalgal research. Further, to optimize and produce better algal platforms, use of systems biology network analysis and integration of omics data is required. This review discusses two important approaches: systems biology and gene editing strategies used on microalgal systems with a focus on biofuel production and sustainable solutions. It also emphasizes that the integration of such systems would contribute and compliment applied research on microalgae. CONCLUSIONS: Recent advances in microalgae are discussed, including systems biology, gene editing approaches in lipid bio-synthesis, and antenna engineering. Lastly, it has been attempted here to showcase how CRISPR/Cas systems are a better editing tool than existing techniques that can be utilized for gene modulation and engineering during biofuel production.

Biological and analytical techniques used for detection of polyaromatic hydrocarbons.

Polycyclic aromatic hydrocarbons contain two or more fused benzene rings that are considered as cosmo-pollutants ubiquitously found in the environment. The identification and monitoring of polycyclic aromatic hydrocarbons (PAHs) are of great interests for rapid and on-site detection. Therefore, many analytical and biological techniques have been proposed for the qualitative and quantitative assessments of PAHs. Non-biological analytical techniques such as infrared, Raman, and fluorescence spectroscopies are commonly exploited as non-destructive techniques while gas chromatography (GC) and high-performance liquid chromatography (HPLC) with multiple detectors are extensively employed for the separation and detection of an analyte. Even though spectroscopy and chromatography are more accurate, convenient, and feasible techniques, often, these methods are expensive and sophisticated which require high maintenance cost. On the other hand, biological approaches, i.e., immunoassay, PCR, and microarray, offer comprehensive high-throughput specificity and sensitivity for a similar analyte. Biosensor- and immunoassay-mediated detections of PAHs have opened up new avenues in terms of low cost, rapid determination, and higher sensitivity. In this review, we have discussed the strengths and limitations of biological and analytical techniques that were explored for precise evaluation and were trusted at both the legislation and research levels.

Biodegradation of waste grease by Penicillium chrysogenum for production of fatty acid.

The aim of present work was to effectively remediate grease waste by Penicillium chrysogenum. For efficient degradation, grease waste was pre-treated using various lipases, among them lipolase was the best. The pretreated grease was used as a substrate by P. chrysogenum resulting into the production of fatty acids. Process was optimized by response surface methodology (RSM) using four variables viz; FeCl2 (mM), spore concentration (spores/ml), time period (days) and amount of grease (g). The optimized conditions viz; FeCl2 1.25mM, culture amount 5×1011spores/ml and time period 16days led to the production of 6.6mg/g fatty acid from 10.0g of pre-treated grease mixed with 5.0g wheat bran in 10.0ml czapek-dox medium under solid state fermentation. The fermented media was extracted with hexane and subjected to GCMS analysis, which showed the presence of higher amount of palmitic acid. It was purified by crystallization method and 2.8g of palmitic acid was recovered from 1.0kg grease waste in tray fermentation.

Biosurfactant production through Bacillus sp. MTCC 5877 and its multifarious applications in food industry.

In this study Bacillus sp. MTCC5877 was explored for the production of biosurfactant (BSs) and various carbon sources 1% (w/v), 0.5% (w/v) nitrogen sources were tested at different pH, and temperature. Yield was measured in terms of Emulsification index (EI), Oil Displacement Area (ODA) and Drop Collapse Area (DCA) and maximum emulsification activities of BSs were found (E24) 50%, 76% and 46%, respectively, and maximum ODA of 5.0, 6.2 and 4.7cm, were shown respectively. The BS was able to reduce the surface tension of water from 72 to 30mN/m and 72 to 32mN/m. Structural compositions of BS were confirmed by FTIR, GC-MS and NMR. Anti-adhesive property of BS was determined and found effective against biofilm formation. It could remove 73% Cd from vegetable which confirms its application in food industry.

Impact of surfactant assisted acid and alkali pretreatment on lignocellulosic structure of pine foliage and optimization of its saccharification parameters using response surface methodology.

In present study, two hybrid methods such as surfactant assisted acid pretreatment (SAAP) and surfactant assisted base pretreatment (SABP) of pine foliage (PF) were found efficient for removal of 59.53 ± 0.76% and 73.47 ± 1.03% lignin, respectively. Assessment of the impact of pretreatment over the structure of PF were studied by scanning electron microscopy, Fourier transform infrared and X-ray diffraction analysis. Parameters for saccharification of SAAP and SABP biomass were optimized by Box-Behnken design method and 0.588 g/g and 0.477 g/g of reducing sugars were obtained, respectively. The ethanol fermentation efficiency of Saccharomyces cerevisiae (NCIM 3288) of hydrolysates was increased by 16.1% and 6.01% in SAAP-PFF and SABP-PFF after detoxification with XAD-4 resin. The mass balance analysis of the process showed that 67.7% and 70.12% cellulose were utilized during SAAP and SABP, respectively. These results indicated that SAAP would be more economic for bioethanol production.

Transformation of waste cooking oil into C-18 fatty acids using a novel lipase produced by Penicillium chrysogenum through solid state fermentation.

The prime aim of the current work was to illustrate the components existing in repeatedly used cooking oil and to develop an economical process for the production of fatty acids from low cost feedstock waste. The waste cooking oil was characterized by the occurrence of high molecular weight hydrocarbons and polymerized derivative of esters. Triacontanoic acid methyl ester, 2,3,5,8-Tetramethyldecane, 3,3 dimethyl heptane, and 2,2,3,3-teramethyl pentane were detected as thermal and oxidative contaminants that adversely affect the quality of cooking oil. Fundamentally, waste cooking oil comprises ester bonds of long chain fatty acids. The extracellular lipase produced from P. chrysogenum was explored for the hydrolysis of waste cooking oil. The incorporation of lipase to waste cooking oil in 1:1 proportion released 17 % oleic acid and 5 % stearic acid.

An overview of key pretreatment processes for biological conversion of lignocellulosic biomass to bioethanol.

Second-generation bioethanol can be produced from various lignocellulosic biomasses such as wood, agricultural or forest residues. Lignocellulosic biomass is inexpensive, renewable and abundant source for bioethanol production. The conversion of lignocellulosic biomass to bioethanol could be a promising technology though the process has several challenges and limitations such as biomass transport and handling, and efficient pretreatment methods for total delignification of lignocellulosics. Proper pretreatment methods can increase concentrations of fermentable sugars after enzymatic saccharification, thereby improving the efficiency of the whole process. Conversion of glucose as well as xylose to bioethanol needs some new fermentation technologies to make the whole process inexpensive. The main goal of pretreatment is to increase the digestibility of maximum available sugars. Each pretreatment process has a specific effect on the cellulose, hemicellulose and lignin fraction; thus, different pretreatment methods and conditions should be chosen according to the process configuration selected for the subsequent hydrolysis and fermentation steps. The cost of ethanol production from lignocellulosic biomass in current technologies is relatively high. Additionally, low yield still remains as one of the main challenges. This paper reviews the various technologies for maximum conversion of cellulose and hemicelluloses fraction to ethanol, and it point outs several key properties that should be targeted for low cost and maximum yield.

Development of bioprocess for the production of laccase by Pleurotus ostreatus MTCC 1802 using evolutionary optimization technique.

For cost effective production of laccase enzyme (benzenediol: oxygen oxidoreductase) from P. ostreatus MTCC 1802 through solid sate fermentation, physico-chemical parameters such as temperature (20-35 degrees C), incubation period (9-17 days) and substrate (Neem bark and wheat bran, in various ratios, w/w) were optimized first by one parameter at time approach and then obtained optimum conditions were considered as zero level in evolutionary optimization factorial design technique. At statistically optimized conditions yield of laccase was found 303.59 + 16.8) U/gds after 13 days of incubation at 25 degrees C taking wheat bran and neem bark as substrate at a ratio of 3:2 (w/w). The results obtained could be a base line for industrial scale production of laccase.

Synthesis of well-dispersed silver nanorods of different aspect ratios and their antimicrobial properties against Gram positive and negative bacterial strains.

In the present contribution, we describe the synthesis of highly dispersed silver nanorods (NRs) of different aspect ratios using a chemical route. The shape and size of the synthesized NRs were characterized by Transmission Electron Microscopy (TEM) and UV-visible spectroscopy. Longitudinal and transverse absorptions bands confirm the rod type structure. The experimentally recorded UV-visible spectra of NRs solutions were fitted by using an expression of the extinction coefficient for rod like nano structures under the dipole approximation. Simulated and experimentally observed UV-visible spectra were compared to determine the aspect ratios (R) of NRs. The average values of R for NR1, NR2 and NR3 solutions are estimated to be 3.0 ± 0.1, 1.8 ± 0.1 and 1.2 ± 0.1, respectively. These values are in good agreement with those obtained by TEM micrographs. The silver NRs of known aspect ratios are used to study antimicrobial activities against B. subtilis (gram positive) and E. coli (gram negative) microbes. We observed that the NRs of intermediate aspect ratio (R = 1.8) have greater antimicrobial effect against both, B. subtilis (gram positive) and E. coli (gram negative). The NRs of aspect ratio, R = 3.0 has better antimicrobial activities against gram positive than on the gram negative.

Experimental investigation of microbiologically influenced corrosion of selected steels in sugarcane juice environment.

In the current study, ferritic stainless grades AISI 439 and AISI 444 were investigated as possible construction materials for machinery and equipment in the cane-sugar industry. Their performance in corrosive cane-sugar juice environment was compared with the presently used low carbon steel AISI 1010 and austenitic stainless steel AISI 304. The Tafel plot electrochemical technique was used to evaluate general corrosion performance. Microbiologically influenced corrosion (MIC) behaviour in sugarcane juice environment was studied. Four microbial colonies were isolated from the biofilms on the metal coupon surfaces on the basis of their different morphology. These were characterized as Brevibacillus parabrevis, Bacillus azotoformans, Paenibacillus lautus and Micrococcus sp. The results of SEM micrographs showed that AISI 439 and AISI 304 grades had suffered maximum localized corrosion. MIC investigations revealed that AISI 444 steel had the best corrosion resistance among the tested materials. However from the Tafel plots it was evident that AISI 1010 had the least corrosion resistance and AISI 439 the best corrosion resistance.