Current Biotechnology - Volume 3, Issue 2, 2014

To overcome food borne illness, food quality and safety are major scientific disciplines including food handling, preparation and storage. Detection of contaminants in the food system is the main concern regarding public health. It is necessary to develop an immunoassay system to detect and control food borne pathogens in contaminated foods before they enter into the human food chain and cause infections. The conventional methods including agar plating, chromatography or immunoassay are tedious and time consuming for detection of food contamination, while biosensors are one option to overcome these problems. There is growing interest towards biosensors technology due to high specificity, convenience and quick response. In the field of food science and technology, biosensors technology for the detection of food borne pathogens is more fascinating to the current researchers. In this contest we introduce, quartz crystal microbalance (QCM) a label free and piezoelectric biosensor for the detection of pathogenic microbes and toxins present in the food. The potential significance of the present review is to provide the implementation of QCM based biosensor for the detection of various analytes in food systems including toxic and nontoxic.

Improving the performance of their bioreactors is a continuous distinguished task of process engineers for the entire lifetime of their processes. In this paper new developments which help to improve the performance of bioreactors for recombinant protein formation are reviewed. In order to judge the performance of a cultivation process an important prerequisite is accurately and easily monitoring the key quantities of their processes. Recently, the well-established monitoring techniques, got competition from new, so-called kernel methods, which are more precise as they need a smaller number of free model parameters. In the field of dynamic estimators, “Extended Kalman Filters” are being replaced by “Unscented Kalman Filters” which simplify this sophisticated technique as they do not need Jacobian matrices. They use the full process model instead of linear approximations. Process supervision, optimization and control examples are given from microbial fermentations as well as from animal cell cultures. Optimization the operation of the fermentations, an extremely prominent task, is discussed in case of E. coli cultivations where the product appears in soluble form as well as in the form of inclusion bodies. In order to keep the process on its optimal path at a significant batch-to-batch reproducibility, open loop control along robust trajectories should be used for the initial biomass growth phase. Later, during the product formation phase, closed loop feed forward feedback control is the technique of choice to keep the process on its optimal trajectory. All procedures mentioned can be implemented in modern industrial automation systems. Advances in such bioreactor control systems develop towards virtual plants, which simultaneously allow simulating the fermentation process. They allow for improved controller developments, online process supervision, and can also be used for a more realistic training of the personnel similarly to flight simulators for pilot training.

A bacterial strain producing extracellular alkaline thermostable laccase has been isolated from sawdust effluents and identified as Micrococcus species. The laccase was partially purified and characterized. The isolate showed maximum laccase production after 120 h, at 30ºC with an optimum pH of 9.0. The specific activity was 127.94 U/mg with a purification fold of 5.54 was obtained with DEAE-cellulose anion exchange chromatography. The molecular weight of purified laccase was 23 kDa. A temperature of 40ºC and pH of 9.0 was found to be optimum for laccase activity. Laccase was stable at 50ºC and at a pH of 9.0 after 1 h incubation. Laccase retained 70-80% of its activity in the presence of 5% DMSO, butanol, acetone and isopropanol. The laccase activity was inhibited by 10 mM SDS (90%), mercuric chloride (80%) and p-CMB (80%). However, only 30% of the enzyme activity was inhibited by sodium azide and EDTA. The characteristics of partially purified laccase with respect to inhibitors and metal ions contribute towards the understanding for the use of bacterial laccases for industrial applications.

A purified thermo-tolerant lipase of Streptomyces STL-D8 was immobilized onto the natural fibres namely ‘Bombax’. Hydrolytic activity of this fibre-bound lipase was determined for hydrolysis of p-NPP in a 0.05 M Tris buffer (pH 8.5) at 55°C. This fibre-bound lipase was used to synthesize methyl succinate using methanol and succinic acid as reactants in DMSO at 55°C under shaking. Although a low yield of methyl succinate was observed in reaction mixture at 16 h (14.3 mM) yet the yield of methyl succinate was improved by optimizing molar ratio of reactants to 75 mM: 100 mM (succinic acid: methanol) in DMSO. Molecular sieves added to the reaction system at a concentration of 30 mg/2 mL increased the synthesis of methyl succinate from 30.0 mM to 31.4 mM. The volumetric scale up of the reaction system to 10 and 50 mL produced more or less a similar yield (29.1 mM to 27.9 mM) of methyl succinate after 16 h of reaction. Methyl succinate is a therapeutic molecule that has insulin sensitizing property comparable to that of glucose.

The effects of selected yeast extract (YE) compounds on 2,3-butanediol production by Paenibacillus polymyxa DSM 365 were studied. The intention was to substitute complex sources of nutrients by defined compounds thus reducing the high demand of YE as well as lowering the costs of fermentation media. For this purpose the depletion of different inorganic compounds during cultivations was analyzed. Then, their impact on product synthesis (acetoin and 2,3butanediol) was investigated in shake flasks by systematic replenishing with inorganic compounds to a basal medium containing 5 g L^-1YE. Calcium was found to be a key element to enhance product synthesis. In a similar way, studies on mixtures of 8 vitamins and of 12 amino acids, resp., were done. The best results were established for the combination of inorganics (calcium, zinc and permanganate salts) and vitamins. With the enriched medium the product formation could be enhanced by 67% after 48h compared to the product formation using basal medium. Furthermore, a synthetic medium for 2,3-butanediol production was developed as a basis for further studies.

The problem of uneven and faulty dyeing in the finished quality of fabrics is usually tackled through a chemical stripping process to remove the loose dyes from the surface of dyed cotton fabrics. This research work was undertaken to investigate the color stripping of cotton fabric dyed with C.I. reactive black 5 in an environmentally-friendly way using enzymes instead of applying chemicals. Such bio-degradable and economical enzymes were prepared by growing white rot fungus Ganoderma lucidum IBL-05 under the optimized conditions of solid state fermentation (SSF). The biological color stripping process using SSF-enzyme extract was optimized for necessary parameters for effective color strippingsuch as SSF-enzyme concentration, pH, temperature and the duration of the enzyme reaction. The color stripping efficiency of this enzymatic reaction process was evaluated by measuring the K/S values of dyed fabric before and after the color stripping. In the present study 70.81% color stripping of dyed fabric was achieved using a cheaper preparation of crude enzyme extract consisting of three significant enzymes lignin peroxidase (EC 1.11.1.14) (530U/mL), manganese peroxidase (EC 1.11.1.13) (395U/mL) and laccase (EC 1.10.3.2) (68U/mL) under the optimized reaction conditions of stripping medium pH 4.5, at 30°Creaction temperature, and 10 days period of stripping. This biological approach is environmentally friendly as compared to chemical methods used for color stripping in textile industry.

The aim of this study was to evaluate the utility of isolated fungus in biodegradation of non-sterile molasses spent wash (MSW). The non-sterile MSW was used to check the biodegradation capabilities of isolated fungus in the presence of other contaminants. The irrigation site for culture isolation was selected to verify the presence of microbes that are suitable for MSW biodegradation. Three different fungi were isolated from the soil where MSW has been used for irrigation purpose. Higher decolorization was obtained with green color fungus, which was identified as Aspergillus nidulans Var. nidulans. The role of different carbon, nitrogen, pH, and MSW concentration was studied in biodegradation of non-sterile MSW. Higher decrease in chemical oxygen demand (COD) was obtained with sucrose, whereas galactose showed higher decolorization. Similarly, higher decrease in COD and color was obtained with sodium nitrate and ammonium sulphate respectively. However, higher decrease in COD and color was observed with yeast extract. Interestingly, significant increase in color was observed in the presence of urea. The pH optimization study has shown higher decrease in COD and color at pH 6.0, 8.0, and 10.0. Whereas, MSW concentration optimization study has shown higher reduction in COD (62 ± 1.8%) and color (79.6 ± 2.5%) at 12.5% concentration on 7 day. However, 74.0 ± 2.2% average decolorization was obtained for 12.5%, 25.0% and 37.5% MSW concentration. Results strongly suggest the suitability of isolated fungus in non-sterile MSW biodegradation at higher concentrations. Our findings may encourage the use of anaerobically digested MSW for irrigation.

To facilitate the use of biotechnological techniques for conservation, germplasm exchange and improvement of Tetrapleura tetraptera, the study investigated in vitro plant regeneration via somatic embryogenesis from isolated axillary meristem of the plant. Indirect primary somatic embryos were induced on MS basal medium supplemented with nine different concentrations of 2,4-D. Pre-embryogenic calli were made to mature on MS basal medium containing BAP alone or with four concentrations of NAA. Secondary somatic embryos were induced on MS basal medium supplemented with nine different concentrations of 2,4-D. Maturation of the secondary pre-embryogenic calli took place on MS medium fortified with eight concentrations of BAP. Medium fortified with 6.0 mg/l 2, 4-D had the highest frequency of primary embryogenic callus (16.4%) and average number of primary immature embryo per callus (2.3). Culture media supplemented with 0.6 mg/l BAP and 0.1 mg/l NAA recorded the highest primary embryogenesis (24.5%) and average number of matured primary embryo per explant (2.8 embryos/explants). Medium fortified with 1.2 mg/l BAP gave the highest secondary embryogenesis (72.4%) and average number of matured embryo per callus (3.1 embryos per callus). Explants in medium receiving 1.0 mg/l BAP and 0.7 mg/l IBA recorded largest number of shoot-bud (27.6%) and average number of shoot-bud per explant (2.4/explant) with vigorous and green appearance. Shoot elongation was greatest in medium fortified with 1.0 mg/l BAP and 15.0 mg/l IBA with vigorous appearance. The largest number of roots (4.3/plantlet) were formed by plantlets grown in medium supplemented with 1.0 mg/l BAP and 15.0 mg/l IBA with normal appearance. The study concluded that indirect somatic embryos obtained from isolated axillary meristem with 2,4-D, BAP, NAA were capable of regeneration into normal vigorous plants.

Endo-glucanase (CMCase) is widely used in detergents, textile and biofuel industry as color caring agent, smooth surfacing agent of cotton fabrics and biofuels production from plant biomass. Extracellular CMCase of Scytalidium thermophilum grown on 2.0% rice polish medium (pH 5.5) for 96 h at 45o C was purified to homogeneity level by sequential application of ammonium sulphate precipitation, ion exchange and gel filtration chromatography. CMCase was monomeric with 49 kDa as molecular mass. Temperature- and pH-dependent kinetic parameters for CMC hydrolysis were determined. Enzyme exhibited stability up to 85o C with t1/2 of 102 min and over a pH range of 3.0-11.0 with maximum t1/2of 521 min at pH 6.5. Vmax for CMC hydrolysis was 250.0 ± 24 U mg^-1 and Km of 5 mg ml^-1 at 60 °C. The turnover number (Kcat) was 50 sec-1 at 60 °C. The pKa1 and pKa2 and heat of ionization of ionizable groups of active site residues controlling Vmax indicated the involvement of Asp or Glu on acidic limb and imidazole group on basic limb. Thermodynamic parameters (ΔH* = 43.26 kJ mol-1, ΔS* = -161.46 J mol^-1.K-1) of irreversible inactivation in a temperature range of 50-85 °C were also determined.

The goal of this study was to develop a fermentation process for the production of β-glucosidase, an important enzyme in the hydrolysis of lignocellulose and has many applications in food and flavor industries, using low-cost agricultural residues as substrates. Based on statistical experimental design, high-titer production of β-glucosidase on wheat bran and glycerol by Aspergillus niger in a submerged culture was achieved. A 2-level Plackett-Burman design was first used to screen the bioprocess parameters affecting β-glucosidase production. Among the tested parameters, the concentrations of wheat bran, glycerol, corn steep liquor and KCl showed significant effects on β-glucosidase production. These four medium components were further optimized using a 3-level Box-Behnken design, and their optimal levels were found to be: wheat bran, 3.5 g/L; glycerol, 5 g/L; KCl, 0.1 g/L and corn steep liquor, 7.5 g/L, giving a high β- glucosidase titer of 9.37 IU/ml, 1.6-fold of the maximum level obtained in the screening experiment. With the optimized medium, 5.41 IU/ml of β-glucosidase was produced in a stirred-tank bioreactor inoculated with a spore suspension. When the reactor was inoculated with precultured cell pellets and operated at optimized agitation (450 rpm) and aeration rates (2 vvm), β-glucosidase production increased to 9.33 IU/ml, which was comparable to the level obtained in shake-flasks. This study demonstrated a scalable fermentation process for the production of β-glucosidase on low-cost wheat bran and glycerol, which should have important industrial applications.