Sustainable wineries through waste valorisation: A review of grape marc utilisation for value-added products.

Grapes are one of the most cultivated fruits worldwide, with one third of total production used in winemaking. Both red and white winemaking processes result in substantial quantities of solid organic waste, such as grape marc (pomace) and stalks, which requires suitable disposal. Grape marc accounts for approximately 10-30% of the mass of grapes crushed and contains unfermented sugars, alcohol, polyphenols, tannins, pigments, and other valuable products. Being a natural plant product rich in lignocellulosic compounds, grape marc is also a promising feedstock for renewable energy production. However, despite grape marc having such potential, advanced technologies to exploit this have not been widely adopted in wineries and allied industries. This review covers opportunities beyond traditional composting and animal feed, and examines value-added uses via the extraction of useful components from grape marc, as well as thermochemical and biological treatments for energy recovery, fuel or beverage alcohol production, and specialty novel products and applications such as biosurfactants and environmental remediation. New advances in relevant technology for each of these processes are discussed, and future directions proposed at both individual producer and regional facility scales, including advanced processing techniques for integrated ethanol production followed by bioenergy generation from the spent marc.

Purification and characterisation of intracellular alpha-galactosidases from Acinetobacter sp.

Two alpha-galactosidases (Ag-I & Ag-II) were purified from Acinetobacter sp. Both the enzymes were monomeric with pH optima of 7.0 and molecular weight of 65 kDa for Ag-I and 37 kDa for Ag-II. The temperature optima for Ag-I was between 50 and 60 °C and that of Ag-II was 40 °C. Both the enzymes were strongly inhibited by metal ions Ag2+ and Hg+, pCMB and SDS (1 %). The enzymes were found to be active on both natural and synthetic substrates. Artificial substrate, pNPGal, has shown more affinity to enzyme than natural substrate raffinose. The half-life (t 1/2) of Ag-I varied from 1.85 h at 90 °C to 7.6 h at 70 °C.

Role of feed forward neural networks coupled with genetic algorithm in capitalizing of intracellular alpha-galactosidase production by Acinetobacter sp.

Alpha-galactosidase production in submerged fermentation by Acinetobacter sp. was optimized using feed forward neural networks and genetic algorithm (FFNN-GA). Six different parameters, pH, temperature, agitation speed, carbon source (raffinose), nitrogen source (tryptone), and K2HPO4, were chosen and used to construct 6-10-1 topology of feed forward neural network to study interactions between fermentation parameters and enzyme yield. The predicted values were further optimized by genetic algorithm (GA). The predictability of neural networks was further analysed by using mean squared error (MSE), root mean squared error (RMSE), mean absolute error (MAE), mean absolute percentage error (MAPE), and R2-value for training and testing data. Using hybrid neural networks and genetic algorithm, alpha-galactosidase production was improved from 7.5 U/mL to 10.2 U/mL.

Reducing sugars production from corncobs: a comparative study of chemical and biotechnological methods.

Two commonly used chemical pretreatment processes, sulphuric acid, and sodium hydroxide, were tested to provide comparative performance data. A connection between solid to liquid ratio (S/L) and sugars released was observed with an increase in S/L ratio between 0.02 and 0.2. Enzymatic digestibility of 1 M of NaOH-pretreated corncobs were released 210.7 mg ml(-1) of sugars. Further, compared with different concentrations of acid pretreatments at 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, and 0.5 M concentrations, sodium hydroxide pretreatment of corncob substantially increased accessibility and digestibility of cellulose. Another additional observation made was whole-cell and crude enzymatic hydrolysis of different concentrations of acid and NaOH (0.05, 0.1, 0.25 M)-treated materials released lower amount of sugars compared with the sugars released (310.9 mg ml(-1)) with whole-cell hydrolysis of 1 M of NaOH-treated corncobs. NaOH-pretreated corncobs contained higher content of sugars and which is more suitable for production of reducing sugars.

A Proposal for Six Sigma Integration for Large-Scale Production of Penicillin G and Subsequent Conversion to 6-APA.

Six Sigma methodology has been successfully applied to daily operations by several leading global private firms including GE and Motorola, to leverage their net profits. Comparatively, limited studies have been conducted to find out whether this highly successful methodology can be applied to research and development (R&D). In the current study, we have reviewed and proposed a process for a probable integration of Six Sigma methodology to large-scale production of Penicillin G and its subsequent conversion to 6-aminopenicillanic acid (6-APA). It is anticipated that the important aspects of quality control and quality assurance will highly benefit from the integration of Six Sigma methodology in mass production of Penicillin G and/or its conversion to 6-APA.

Significance of biotic factors in mesenchymal stem cell fate in regenerative medicine.

Stem and progenitor cell research is a complex and exciting field which promises curative discoveries in numerous areas including cancer, diabetes, and regenerative medicine. Use of biotic factors or growth factors has played an essential role in the development of stem cell research. These biologically active components have been administered into stem cells either to improve or maintain the stem cell proliferation, or to encourage controlled differentiation into more defined cell types. Small molecules such as 6-Bromoindirubin-3´-oxime (BIO), cardiogenol-C, etc can help stem cell research by controlling or influencing the regulatory changes in a controlled manner and to help understand the mechanisms during stem cell differentiation. Extra cellular matrix (ECM) is another significant biotic factor, which mediates cell and tissue behavior by influencing cell-matrix interactions. Thus, in this review we would like to emphasize significance of various growth factors in stem cell research.

Influence of dilute acid and alkali pretreatment on reducing sugar production from corncobs by crude enzymatic method: a comparative study.

In the present study, two commonly used catalysts in chemical pretreatment, sulfuric acid and sodium hydroxide, were tested to evaluate the effect of solid-to-liquid ratio on pretreatment and enzymatic hydrolysis. Solid to liquid ratio (S/L) was influential on sugars released with an increase in the S/L ratio between 0.03 and 0.2. Enzymatic digestibility of 0.25 M H2SO4 pretreated corncobs were released more sugars (415.12 mg/mL); whereas, corncobs pretreated with NaOH released 350.12 mg/mL of reducing sugars at S/L 0.05. Further, in comparison with NaOH pretreated corncobs, acid treated material substantially increased the accessibility and digestibility of cellulose during crude enzymatic hydrolysis (28.96 FPU) and released 398.95 mg/mL reducing sugars.

Constitutive optimized production of streptokinase in Saccharomyces cerevisiae utilizing glyceraldehyde 3-phosphate dehydrogenase promoter of Pichia pastoris.

A novel expression vector constructed from genes of Pichia pastoris was applied for heterologous gene expression in Saccharomyces cerevisiae. Recombinant streptokinase (SK) was synthesized by cloning the region encoding mature SK under the control of glyceraldehyde 3-phosphate dehydrogenase (GAP) promoter of Pichia pastoris in Saccharomyces cerevisiae. SK was intracellularly expressed constitutively, as evidenced by lyticase-nitroanilide and caseinolytic assays. The functional activity was confirmed by plasminogen activation assay and in vitro clot lysis assay. Stability and absence of toxicity to the host with the recombinant expression vector as evidenced by southern analysis and growth profile indicate the application of this expression system for large-scale production of SK. Two-stage statistical approach, Plackett-Burman (PB) design and response surface methodology (RSM) was used for SK production medium optimization. In the first stage, carbon and organic nitrogen sources were qualitatively screened by PB design and in the second stage there was quantitative optimization of four process variables, yeast extract, dextrose, pH, and temperature, by RSM. PB design resulted in dextrose and peptone as best carbon and nitrogen sources for SK production. RSM method, proved as an efficient technique for optimizing process conditions which resulted in 110% increase in SK production, 2352 IU/mL, than for unoptimized conditions.

Simultaneous pretreatment and sacchariffication of rice husk by Phanerochete chrysosporium for improved production of reducing sugars.

Phanerochete chrysosporium, the white-rot fungus, (a best source for lignolytic enzymes system) was used in the biological pretreatment of rice husk for reducing sugars production. Usually reducing sugar production through biochemical process involves two steps: solid state fermentation (SSF) of fungal pretreatment for delignification, subsequently pretreated biomass subjected to enzymatic hydrolysis. During the fungal pretreatment of rice husk for reducing sugar production along with cellulase and xylanse, the activities of lignin degradation-related enzymes such as lignin peroxidases (LiP), GLOX (glyoxidase), and aryl alcohol oxidases (AAO), were observed. The fungal pretreated rice husk produced highest (895.9 mg/ml/2g of rise husk) reducing sugars on 18th day of fungal treatment. This method may be good alternative to avoid operational costs associated with washing and the removal of inhibitors during the conventional pretreatment methods.

Chemometrics-based process analytical technology (PAT) tools: applications and adaptation in pharmaceutical and biopharmaceutical industries.

Process analytical technology (PAT) is used to monitor and control critical process parameters in raw materials and in-process products to maintain the critical quality attributes and build quality into the product. Process analytical technology can be successfully implemented in pharmaceutical and biopharmaceutical industries not only to impart quality into the products but also to prevent out-of-specifications and improve the productivity. PAT implementation eliminates the drawbacks of traditional methods which involves excessive sampling and facilitates rapid testing through direct sampling without any destruction of sample. However, to successfully adapt PAT tools into pharmaceutical and biopharmaceutical environment, thorough understanding of the process is needed along with mathematical and statistical tools to analyze large multidimensional spectral data generated by PAT tools. Chemometrics is a chemical discipline which incorporates both statistical and mathematical methods to obtain and analyze relevant information from PAT spectral tools. Applications of commonly used PAT tools in combination with appropriate chemometric method along with their advantages and working principle are discussed. Finally, systematic application of PAT tools in biopharmaceutical environment to control critical process parameters for achieving product quality is diagrammatically represented.

Optimisation of batch culture conditions for cell-envelope-associated proteinase production from Lactobacillus delbrueckii subsp. lactis ATCC® 7830™.

Using a combination of conventional sequential techniques, the batch growth conditions for the production of cell-envelope-associated proteinases have for the first time been studied and optimised in Lactobacillus delbrueckii subsp. lactis 313 (ATCC 7830; LDL 313). Concentrations of inoculum (0.1 < X < 10 % vol/vol), agitation speed (0 < S < 200 rpm), varying incubation temperature (30 < T < 50 °C), starting pH (4.5 < pH < 7) and carbon/nitrogen ratio of production medium (0.2 < r < 5) had an individual effect on proteinase yield (p < 0.01). Optimal conditions for proteinase production included an initial pH of 6.0, 45 °C incubation temperature, 2 % (v/v) inoculum size of OD(560) = 1, 150 rpm agitation speed, and growth medium carbon/nitrogen ratio of 1.0. Maximum proteinase activity obtained for whole cells was 0.99 U/ml after 8 h of incubation. The variables studied are very relevant due to their significance in improving the productivity of proteinase synthesis from LDL 313, under process and, likely, economic optimum conditions.

Mixing of acid and base pretreated corncobs for improved production of reducing sugars and reduction in water use during neutralization.

Pretreatment of biomass for bioethanol production makes it necessary to use large amounts of water for removing inhibitors and neutralization. In order to reduce water usage, separate batches of corncobs were hydrolyzed in 1M NaOH and 0.05 M H(2)SO(4), respectively, and the hydrolysis products were mixed to achieve a pH of 7. This approach lowered water usage by 10-fold compared with neutralization by distilled and recycling wash water. Mixing of the pretreated biomasses (121°C, 20 min) increased release of reducing sugars during enzymatic hydrolysis with cellulases (38.49 FPU(IU)) produced by Phanerochaete chrysosporium NCIM 1106 by 2- and 15-fold compared with the sugars released from the unmixed NaOH- and H(2)SO(4)-treated corncobs, respectively. Enzymatic hydrolysis (EH, cell free extract) of the mixed material released 395.15 mg/ml of sugars during 48 h, slightly less than what was achieved by microbial hydrolysis (whole cell hydrolysis), 424.50mg after 120 h.

Continuous production of oxytetracycline in fluidized bed bioreactor by immobilized cells of Streptomyces varsoviensis MTCC-1537: effect of dilution and glucose loading rates.

Oxytetracycline (OT) production using glutaraldehyde cross-linked calcium alginate immobilized cells of Streptomyces varsoviensis in continuous fluidized bed reactor (FBR) was investigated. Initially, batch experiments were carried in stirred tank reactor (STR) and FBR using calcium alginate immobilized cells. Higher OT production of 0.45 gm/L was achieved by FBR when compared with 0.33 g/L of OT in STR. All subsequent studies were carried out in continuous mode of operation in FBR. During 21 days of operation, effect of glucose concentration and different dilution rates were studied. A maximum of 0.75 g/L OT was achieved in the medium having 10 g/L of glucose concentration. The highest OT concentration of 0.92 g/L and the highest yield of OT with respect to biomass at 0.1713 g/g were obtained at the dilution rate of 0.25 day(-1).

Multiple responses optimization and modeling of lipase production by Rhodotorula mucilaginosa MTCC-8737 using response surface methodology.

Response surface methodology was employed to optimize culture medium for production of lipase with Rhodotorula sp. MTCC 8737. In the first step, a Plackett-Burman design was used to evaluate the effects of different inducers qualitatively. Of all the seven inducers tested, soybean oil showed significant influence on the lipase production. Further, response surface studies were conducted to quantitatively optimize by considering linear, interactive, and quadratic effects of test variables. A novel approach was proposed to optimize the lipase production system by optimizing the responses in terms of yield kinetics rather than optimizing the direct responses like lipase titer and biomass growth. The coefficient of determination (R(2)) calculated for Y (P/S) (0.769), Y (P/X) (0.799), and Y (X/S) (0.847) indicated that the statistical model could explain 76.9%, 79.99%, and 84.7% of variability in the response.

Constitutive expression and optimization of nutrients for streptokinase production by Pichia pastoris using statistical methods.

The Pichia pastoris clone producing streptokinase (SK) was optimized for its nutritional requirements to improve intracellular expression using statistical experimental designs and response surface methodology. The skc gene was ligated downstream of the native glyceraldehyde 3-phosphate dehydrogenase promoter and cloned in P. pastoris. Toxicity to the host was not observed by SK expression using YPD medium. The transformant producing SK at level of 1,120 IU/ml was selected, and the medium composition was investigated with the aim of achieving high expression levels. The effect of various carbon and nitrogen sources on SK production was tested by using Plackett-Burman statistical design and it was found that dextrose and peptone are the effective carbon and nitrogen sources among all the tested. The optimum conditions of selected production medium parameters were predicted using response surface methodology and the maximum predicted SK production of 2,136.23 IU/ml could be achieved with the production medium conditions of dextrose (x1), 2.90%; peptone (x2), 2.49%; pH, 7.2 (x3), and temperature, 30.4 (x4). Validation studies showed a 95% increase in SK production as compared to that before optimization at 2,089 IU/ml. SK produced by constitutive expression was found to be functionally active by plasminogen activation assay and fibrin clot lysis assay. The current recombinant expression system and medium composition may enable maximum production of recombinant streptokinase at bioreactor level.

Effect of aeration and agitation regimes on lipase production by newly isolated Rhodotorula mucilaginosa-MTCC 8737 in stirred tank reactor using molasses as sole production medium.

The influence of media and process parameters (aeration and agitation) on fermentation broth rheology and biomass formation has been studied in 1.5-l stirred tank reactor for lipase production using Rhodotorula mucilaginosa MTCC 8737. Molasses, as sole production medium, is used for lipase production by varying aeration (1, 2, and 3 vvm) and agitation speeds (100, 200, and 300 rpm). Maximum lipase activity of 72 U/ml was obtained during 96 h of fermentation at 2 vvm, 200 rpm, pH 7, and 25 +/- 2 degrees C temperature. Lipase production kinetics with respect to dry cell weight of biomass showed Y (P/S) of 25.71 U/mg, specific product formation of 10.9 U/mg DC, and Y (X/S) 2.35 mg/mg. Maximum lipase activity (MC 2) of 56 U/ml was observed at 1% molasses, and a further increase in the molasses concentration of (%) 1.5 and 2 inhibited the product formation of lipase with 15 and 8.5 U/ml, respectively. The production kinetics of molasses media showed Y (P/X) was 14 U/mg DC, Y (P/S) 16 U/mg, and Y (X/S) 1.14 mg/mg during 96 h of bioreactor operation. The k(L)a values for all batches (MC 1-MC 4) at 96 h of fermentation were 32, 28, 21, and 19/h, and the |oxygen transfer rate were 54.4, 56, 35.7, and 17.29 mg/l h, respectively. Increase in molasses concentration resulted in decreased lipase activity by increase in viscosity of the fermentation broth.

Biological treatment of toxic petroleum spent caustic in fluidized bed bioreactor using immobilized cells of Thiobacillus RAI01.

In the present studies, newly isolated Thiobacillus sp was used for the treatment of synthetic spent sulfide caustic in a laboratory-scale fluidized bed bioreactor. The sulfide oxidation was tested using Ca-alginate immobilized Thiobacillus sp. Initially, response surface methodology was applied for the optimization of four parameters to check the sulfide oxidation efficiency in batch mode. Further, reactor was operated in continuous mode for 51 days at different sulfide loading rates and retention times to test the sulfide oxidation and sulfate and thiosulfate formation. Sulfide conversions in the range of 90-98% were obtained at almost all sulfide loading rates and hydraulic retention times. However, increased loading rates resulted in lower sulfide oxidation capacity. All the experiments were conducted at constant pH of around 6 and temperature of 30 +/- 5 degrees C.

Gentamicin production by Micromonospora echinospora (Me- 22) in stirred tank reactor: effect of various parameters.

Effect of production medium components, initial starch and soyabean meal concentrations, for the enhanced production of gentamicin by Micromonospora echinospora (Me- 22) was studied in a lab scale stirred tank reactor. Also effect of different aeration (0.5, 1, 2, and 4 vvm) and agitation rates (100, 200, 300 and 400 rpm) in a stirred tank reactor was examined. A maximum gentamicin concentration of 2.68 g l(-1) was achieved in the medium having low concentrations of initial starch (7.5 g l(-1)) and high concentrations of initial soyabean meal (4 g l(-1)). Both aeration and agitation significantly affected gentamicin concentration, productivity and biomass formation. The maximum gentamicin concentration of 4.12 g l(-1) and the highest yield of gentamicin on substrate 0.967 g g(-1) were obtained at impeller speed of 200 rpm and aeration rate of 2 vvm. Under optimal culture conditions in STR the production of gentamicin could be increased 3 fold when compared with shake flask.

Evaluation of various parameters of calcium-alginate immobilization method for enhanced alkaline protease production by Bacillus licheniformis NCIM-2042 using statistical methods.

Calcium-alginate immobilization method for the production of alkaline protease by Bacillus licheniformis NCIM-2042 was optimized statistically. Four variables, such as sodium-alginate concentration, calcium chloride concentration, inoculum size and agitation speed were optimized by 2(4) full factorial central composite design and subsequent analysis and model validation by a second-order regression equation. Eleven carbon, 11 organic nitrogen and seven inorganic nitrogen sources were screened by two-level Plackett-Burman design for maximum alkaline protease production by using optimized immobilized conditions. The levels of four variables, such as Na-alginate 2.78%; CaCl(2), 2.15%; inoculum size, 8.10% and agitation, 139 rpm were found to be optimum for maximal production of protease. Glucose, soybean meal and ammonium sulfate were resulted in maximum protease production at 644 U/ml, 720 U/ml, and 806 U/ml when screened for carbon, organic nitrogen and inorganic nitrogen sources, respectively, using optimized immobilization conditions. Repeated fed batch mode of operation, using optimized immobilized conditions, resulted in continuous operation for 12 cycles without disintegration of beads. Cross-sectional scanning electron microscope images have shown the growth pattern of B. licheniformis in Ca-alginate immobilized beads.