Current Biotechnology (v.2, #1)

Editorial by Paul A. Dalby (1-1).

Gene Technology: An Effective Tool for Development of Water-Stress Tolerant Crops by Jelili T. Opabode, Oluyemisi A. Akinyemiju (2-9).
Inadequacy of rainfall is one of the negative impacts of climate change globally. Productivity of crops grown in drought or water deficit conditions is greatly reduced which resulted in decline in food production. Conventional breeding has contributed to our knowledge on responses of plants to drought condition. However, limited progress was achieved because response to water-deficit stress by plants is complex and under the control of several genes. Within a short period, researches in Molecular Biology and Biotechnology have greatly increased our understanding of drought resistance in plants. This knowledge is currently being applied to develop water-stress tolerance crop varieties by classical breeding and transgenic approach. This paper documents gene-based techniques and recent discoveries that facilitated development of water stressed-tolerant crops by transgenic approach. Molecular techniques and resources currently being used to increase our knowledge on mechanism of drought tolerant in plants are genomic and complemenary DNA (cDNA) library, polymerase chain reaction (PCR), GenBank database, micro array technology, gene cloning, nucleic acids hybridization and genetic engineering. The conclusion of genome sequencing project on Arabidopsis and rice made possible the discovery of several genes directly or indirectly regulating drought responses in these model plant species. Genes performing similar role in other plants are being discovered in large number using the insights gained from the model species through comparative genomics. Both functional and regulatory genes control biochemical and physiological responses of plants to drought. Through tissue culture and genetic engineering, expression pattern and regulation of drought tolerance genes and their transfer across plant families for development of drought tolerance crop lines were facilitated. Somatic cotyledon, immature embryo, shoot meristem, shoot apex and embryogenic callus are some of the plant tissues being used to incorporate foreign drought resistance gene into cultivated crops. Agrobacterium tumefaciens-mediated method is the most widely used gene transfer technique for developing drought tolerant crops. The paper discussed the need to use biotechnology tools to discover more drought tolerance genes among indigenous plants and incorporate the discovered drought tolerance genes to indigenous cultivated crops for their improvement against water stress. Potential areas of application of the technology and its requirements are also discussed.

Collagen is one of the extremely hard to degrade structural matrix proteins. Collagenases are the enzymes which bring about the degradation of collagen into small peptides. The contribution of collagenases in several human diseases includes hyper production of collagenase while some diseases are originated due to inadequate amounts of collagenase in the body. The management of these diseases includes understanding of collagenase involvement and then developing the therapeutics for the treatment. This review focuses on the studies of different human diseases related to collagenases and discusses the strategies adapted to conquer them.

Isolation and screening of collagen degrading non-pathogenic microorganisms from insects and annelids samples was intended. Production of a novel highly active extracellular collagenase by a non-pathogenic Bacillus tequilensis isolated from earthworm has been reported. The culture was grown on medium containing (g/L) meat extract 10.0, peptone 10.0 and gelatin 1.0 and 2.1-fold increase in enzyme activity was attained on optimizing various fermentation parameters. The cost effective production of collagenase by B. tequilensis was achieved by utilizing wastes of meat industry as the sole carbon and nitrogen source.

Optimization of Process Variables Affecting the Production of Extracellular Protease by Kluyveromyces marxianus by Panagiota-Yiolanda Stergiou, Athanasios Foukis, Leonidas G. Theodorou, Maria Papagianni, Emmanuel M. Papamichael (23-29).
The optimized production of extracellular protease by Kluyveromyces marxianus IF0 0288 is investigated in yeast medium containing glucose as the carbon source in fully aerobic batch fermentation (150 rpm). Four parameters - type of nitrogen source, initial culture pH, temperature and length of fermentation- influencing the growth and protease production, were considered using both the “one variable at a time” approach and response surface methodology (RSM). Experimental and statistical evidence is presented for 36-fold increase in protease volumetric activity during 72 hours of fermentation with conventionally optimized nutrition (0.04% w/v bovine serum albumin) and cultivation (pH 5.5, 25oC) conditions. The optimal initial pH-value 5.46 and temperature 25.9oC were identified by the RMS and further improved on application both the volumetric activity (42.5-fold) and specific protease activity (51-fold). The results provide for the first time the evidence that K. marxianus is potentially an efficient producer of extracellular protease.

Aspartase (L-aspartate ammonia-lyase; EC 4.3.1.1) catalyzes the reversible amination of fumaric acid to produce L-aspartic acid. L-aspartic acid is an essential ingredient of artificial sweetner aspartame widely used in nutraceuticals and pharmaceuticals. The optimum production of aspartase by Aeromonas media NFB-5 at shake-flask level has been investigated using response surface methodology (RSM). Six central composite rotatable design (CCRD) parameters with varying concentrations were explored to find the most suitable combination for optimal production of aspartase and biomass yield. Aspartase production was the desired response and biomass yield was considered as the second response. Six variables optimized were glycerol (0.04-0.66%, v/v), peptone (0.11-0.89%, w/v), yeast extract (0.11- 0.89%, w/v), sodium L-aspartate (0.09-0.71%, w/v), KH2PO4 (0.09-0.71%, w/v) and K2HPO4 (0.14-0.46%, w/v), while NaCl (0.2%, w/v), MgSO4 (0.01%, w/v) and pH (7.0±0.2) were kept constant during shake-flask fermentation. Optimal levels of glycerol (0.50%), peptone (0.68%), yeast extract (0.39%), sodium L-aspartate (0.58%), KH2PO4 (0.36%) and K2HPO4 (0.39%) were determined. Aspartase activity (136.57 U/g wet wt.) and biomass yield (0.969 OD600) obtained by using the optimal concentrations of media constituents were in good agreement with the predicted values. This is the first report on employment of RSM for production of aspartase from Aeromonas media NFB-5 and can be very useful for scale-up studies.

Dextrans are a class of polysaccharides of varying structure with contiguous α(1-6) glycosidic linkages in the main chain and varying percentage of α(1-2), α(1-3), and α(1-4) linkages. Recently Weissella sp. has drawn attention for its high dextran production capacity and linearity in its dextran. In our earlier report Weissella confusa Cab3, isolated from fermented cabbage, was explored for its dextransucrase and dextran production capacity. Sequential optimization strategy based on statistical experimental designs was employed to enhance dextran production by Weissella confusa Cab3. A two-level Plackett–Burman design was employed, where six variables were studied for their influence on dextran production. Sucrose, K2HPO4 and Tween 80 were significant variables for dextran production. The combined effect of these nutrients on dextran production were studied using a 23 full-factorial central composite design. A second- order polynomial was established to identify the relationship between the dextran production and the three medium components. The optimal concentrations of variables for maximum dextran production were 6.06% (w/v) sucrose and 1.63% (w/v) K2HPO4 and 0.34% (v/v) Tween 80. The maximum concentration of dextran obtained by predicted model was 29.7 mg/mL. The dextran production medium was validated at shake flask level which was in good agreement with the experimental determined value (28.0 mg/mL). This value of dextran concentration was 3.3 fold higher as compared to unoptimized medium that gave 9.0 mg/mL of dextran. The dextran production from Weissella confusa was scaled up in a 3L bioreactor. At bioreactor level, Weissella confusa efficiently utilized the medium sucrose and produced 29.0 mg/mL of dextran.

Coated-Wall Mini Reactor for Inulin Hydrolysis by Elisabete Marques Ribeiro, Pedro Fernandes (47-52).
Miniaturized reactors and enzyme immobilization are tools that can contribute significantly for the implementation of efficient and cost effective bioprocesses. The present work fits within such scope, and focuses on the development of a continuous flow, low-cost mini reactor, aimed to provide a suitable platform for enzymatic bioconversion systems. In this particular case, inulin hydrolysis to fructose promoted by inulinase, was used as model system. Thus, the inner walls of a silicone tube were coated with amino groups and inulinase was subsequently immobilized through glutaraldehyde cross-linking. As an outcome of immobilization, the optimal temperature increased by 10ºC, and allowed operation up to 80ºC, at which temperature the free enzyme is totally inactive. Moreover, the activity of immobilized enzyme was enhanced at pH 4, as compared to the free form. Enzyme-substrate affinity was not affected by immobilization. A product yield of 80% was obtained during 5 days of continuous operation.

Production and Characterization of a Novel Bioflocculant from Klebsiella sp. by L. Muthulakshmi, H. Nellaiah, Siddhardha Busi (53-58).
A bioflocculant - producing bacterium was isolated from water samples collected from the Vaigai river and was identified as Klebsiella sp. by morphological and biochemical characteristics. Chemical analysis of the purified bioflocculant indicated that it was a sugar-protein derivative, composed of sugar (76.83%, w/w) and protein (23.17%, w/w). Infrared spectrophotometry revealed that the bioflocculant contains carboxyl, hydroxyl and amino groups as structural components. Scanning electron microscopy (SEM) of the purified bioflocculant showed that it has an irregular, coarse-grained structure connected in netted textures. The concentration of bioflocculant produced after 48 h of cultivation was 0.73 g/L. At the optimal concentration, the bioflocculant caused 91.28% more settling of kaolin than the control in 5 min.

Waste Combustion Gases and Algae by Henry R. Bungay (59-63).
The poor match between continuous combustion and algal farming that has diurnal discontinuities and seasonal variations poses almost impossible problems for joining them for efficient and economical removal of carbon dioxide from waste gas. Nevertheless, the waste heat and high concentrations of carbon dioxide can benefit a factory that makes products such as biodiesel using algae. Keys to low initial investment, reasonable labor costs, and efficient operations are very inexpensive photobioreactors, self-actuation of gassing and addition of nutrient medium with sun valves, and provisions for weather emergencies and climatic changes. A major recommendation is mounting the photobioreactors on land that need not be leveled or graded. This reduces cost of land acquisition and site preparation to a minimum. Thin, transparent, very wide plastic bags can be fabricated to have a gassing system and an overflow for harvesting of the algal suspension that are integral to the bag thus eliminating much of the accessory piping. Both the flow rate of gas and nutrient medium may depend on sun valves that respond to the intensity of sunlight and that eliminate the need for human operators to stop flows at night and to adjust them during the day.

There has been a quantum increase in the use of nanoparticles (NPs) in all spheres of life. Their increased presence in the environment necessitates a basic understanding about their potential impact on the environment and interactions with biological systems. Toxic effects of NPs, dubbed as “nanotoxicity” are being increasingly evidenced. Studies on animals and cell culture have amply demonstrated loss of cell viability, tissue damage and inflammatory reactions. A bigger threat is however perceived for the microbial community owing to the accumulation of NPs. Study of nanotoxicity in microbial systems holds importance because (i) the discharge of NPs in water and soil might affect the microbial diversity (ii) antimicrobial activity of NPs could be usefully exploited for application in medical science (iii) their interactions with membrane proteins, DNA and various biomolecules inside the cells need to be understood. The review focuses on microbial nanoparticle interactions with a view to analyze their effects on microbes and environment. Interactive effects of NPs on bacterial cells have been emphasized as unicellular model system. The article comprehensively encompasses the current level of understanding of the emerging domain of nanotoxicology.

Bacillus amyloliquefaciens PFB-01 produced extracellular alkaline protease (385.25 U/ml) under shake flask conditions. Effects of process parameters such as cultivation period, pH, temperature, carbon and nitrogen sources on protease production were investigated for optimization. Production of protease was optimized with 5.0% inoculum size in culture medium (pH 8.0) incubated for 48 h at 37°C and 200 rpm. A combination of gelatin (0.75%) as nitrogen source and glucose (0.5%) as carbon source gave maximum yield of protease (525.82U/mL) which was 138% increase over enzyme yield in basal media. Soybean meal (0.75%) and glucose (0.5%) supported protease yield of 510.5 U/ml and 134% increase over basal media. The protease had optimum pH and temperature of 9.0 and 60 °C. Remarkably, it showed 60% residual activity after 60 min of incubation at 60 ºC in the absence of CaCl2. Protease activity was enhanced in the presence of most organic solvents studied and most stable in the presence of 25% DMSO with residual activity of 93.7%. The enzyme was compatible with the commercial detergents tested. Production of this protease in optimized media with inexpensive and readily available soybean meal as alternative nitrogen source indicates that its production can be cost effective for industrial applications. The physicochemical properties exhibited by the protease from B. amyloliquefaciens PFB-01 make this enzyme commercially viable for exploitation in detergent industry and ester synthesis.

Pseudomonas aeruginosa MTCC 7925: Producer of a Novel SCL-LCLPHA Co-Polymer by Akhilesh Kumar Singh, Ranjana Bhati, Shilalipi Samantaray, Nirupama Mallick (81-88).
Pseudomonas aeruginosa MTCC 7925, a sludge isolate, was found to synthesize a novel short-chain-lengthlong- chain-length (SCL-LCL) co-polymer with 3-hydroxybutyric acid (3HB), 3-hydroxyvaleric acid (3HV), 3- hydroxyhexadecanoic acid (3HHD) and 3-hydroxyoctadecanoic acid (3HOD) as constituents. Under batch mode study, cells harvested at the stationary phase of growth depicted maximum PHAs accumulation, i.e. 24% of dry cell weight (dcw) at 48 h of incubation. The co-polymer accumulation was raised to 49% (dcw) under 2% ethanol-supplemented condition. A further, rise up to 78% (dcw) was recorded by optimizing the critical variables by Response Surface Methodology (RSM). When palm oil and its cakes were used as carbon sources, yield of 5.9 g/l was achieved, which was almost 85-fold higher against control. The thermal and mechanical properties of the polymer are comparable with polypropylene and polyethylene, thus opening new possibilities for various industrial applications.