Absract Archive June 2008 Research article Extractive Fermentation of Ethanol in an Aqueous Two-Phase system by Saccharomyces cerevisiae NCIM 3288 Abstract Product purification and separation is always the most challenging area in the field of biochemical engineering and the application efficiency of which determines 50-90 % of the product cost. Another major problem that very frequently arises during fermentation is product inhibition and product reutilization. The only answer to all such problems is extractive fermentation, which removes the product from fermentation beer during the process itself. The aqueous two-phase systems that are well established for product isolation and purification and are known to be bio-friendly to the organisms as well as to the biomolecules tried here as the means for extractive fermentation of ethanol using Sacchromyces Cerevisiae NCIM 3288. Aqueous two-phase can be obtained by mixing two incompatible polymers e.g. Polyethylene glycol (PEG) and dextran in water above critical concentrations and the composition of which can be so tailored that the product goes to the top phase while cells remain in the bottom. A phase system that was used in the present study contained 8.7 % w/w dextran 40000 and 9.0 % w/w PEG 6000 that gave a volume ratio of 1.61 and tie-line of 22.84 % w/w. The corresponding top and bottom phase compositions were 1.38 % w/w dextran/13.13 % PEG/85.49 % w/w water and 20.83 % w/w dextarn/2.26 % w/w PEG/76.91 % w/w water respectively. Initially, the effect of PEG 6000 and dextran 40000 were studied with 5 %, 10 % and 15 % concentrations in each case. It was found that 15 % showed better results than 5 and 10 %. The effect of 10 % PEG 4000 were also studied and compared with 10 % PEG 6000. It was observed that PEG 4000 was toxic to the cells. So, PEG 6000 was used in our further studies as phase system. The results with aqueous two-phase system using 6 % v/v 24 hr fresh inoculum was encouraging showing higher product formation efficiency and the ethanol was stripped off (partition coefficient was 2.57) to the top phase giving a total yield of 86 % in the cell free system with very rapid glucose uptake and cell growth. Key words: Alcoholic fermentation, aqueous two-phase systems, and Polyethylene glycol, Dextran, Sacchromyces Cerevisiae Introduction Ethanol also known as ethyl alcohol is not only the key product in the production of alcoholic beverages but also traditionally employed as a chemical feed stock for a variety of industrial chemicals such as acetaldehyde, acetic acid, ethyl acetate and butadiene etc. Pharmaceutical industry is one of the major users of ethanol for various drug formations. Traditionally ethanol has been produced by batch fermentation; employing Saccharomyces cerevesiae the name first applied by Meyen (1838) to distinguish beer yeasts from those isolated from other alcoholic beverages is still a major concern of all the researchers in the field. Yeasts are the only organisms currently used for large scale industrial ethanol production. However; the inherent disadvantages such as low productivity, low cell density, longer fermentation and prolonged downtimes have necessitated to develop an alternative high productivity system. Many of these newly developed bioprocesses for ethanol are mainly aimed at to enhance the productivity by employing high cell densities in the fermentor. It has been well recognized that alcoholic fermentation are limited due to the inhibitory effects of both substrate and product. The only answer to such problems is extractive fermentation. These processes have been developed to remove alcohol as it is produced, Cysewski et al. (1977) and Ramalingham et al. (1977) there by allowing high carbohydrate concentrations to be fermented without product inhibition. Author:Anuradha Karunanidhi. Short Communication Genetics of Recurrent Spontaneous Abortion Abstract Recurrent Spontaneous Abortion (RSA) is defined as the loss of 3 or more consecutive pregnancies before the 24th week of gestation. RSA is a complex multifactorial problem associated with endocrine dysfunction, autoimmune disorders, advanced maternal and paternal age, infectious processes, environmental toxins and congenital or uterine anomalies apart from genetic abnormalities reflected by inherited disorders. It has been reported that about 15-20 % of all pregnancies end in spontaneous miscarriages and the contribution of chromosomal abnormalities among such cases is as high as 70 %. The contribution of chromosomal abnormalities and the frequency of chromosome inversions and novel chromosome insertion have reported 11.25 % of chromosomal abnormalities. The other causes associated with RSA and genetic abnormalities can be classified into three categories, namely the maternal age, prevalence of consanguineous marriage, distribution of number of abortions and the gestational age of the abortion. The influence of maternal characteristics especially risk of fetal death with increased maternal age has been reported in many studies. Women of 30 years or older continue to have higher risk of fetal death compared to their younger counterparts. Advanced maternal age continues to be a risk factor for fetal death. Studies conducted 'hitherto' have shown no significant correlation between recurrent miscarriage and consanguinity. Therefore consanguineous marriage is not an important etiologic factor in relation to recurrent miscarriage. It has been reported that the rate of pregnancy losses increases with increased risk of subsequent abortion. Studies have concluded that even a single pregnancy loss doubles the risk of abortion in the next pregnancy. History of one or more first trimester abortion was related to an increased risk of abortion for the following pregnancy. Studies have suggested that women with second trimester loss are significantly more likely to have recurrent second trimester loss in subsequent pregnancy than women with preterm or full term births. Keywords: Recurrent Spontaneous Abortion, Cytogenetics, Chromosomal Abnormalities, and Genetic Factors. Authors:Deepa Parvathi V, Neeli S, Solomon FD Paul. Research Article In vitro Antioxidant Assessment of Saliva from Non Smokers and Smokers Abstract The assay of saliva is an increasing area of research with implications for basic and clinical purposes. Although this biological fluid is easy to manipulate and collect, careful attention must be directed to limit variation in specimen integrity. Recently, the use of saliva has provided a substantial addition to the diagnostic armamentarium as an investigative tool for disease processes and disorders. In this study the antioxidant activity of saliva from smokers and non smokers of both the sex of various age groups were assessed. The FRAP assay revealed that the reducing power of non smokers were high when compared to that of both the acute and chronic smokers. Other antioxidant assays like CUPRAC and Ce (IV) sulphate assay also supported the FRAP assay. A two way ANOVA for all the in vitro antioxidant assays proved to be significant at 5% level (p < 0.05) between the smokers and non smokers. Overall, it was observed that a high reduction in the antioxidant power in the smokers may later on, leads to SCC Keywords: Saliva, antioxidant, Cigarette smoke (CS), Oral squamous cell carcinoma (SCC), oral cancer, FRAP Introduction Atmospheric oxygen is a diatomic molecule with the formula O2, in which the two oxygen atoms are doubly bonded to each other (triplet oxygen). This form of oxygen has two unpaired electrons, making it as a radical. Whereas most radicals are highly reactive molecules, triplet oxygen is fortunately unreactive (Navdeep et al., 2007). Approximately 5% of O2 involved with normal processes like metabolic respiration, strenuous exercise and biotransformation of xenobiotics (Peter Møller and Steffen Loft, 2006) is responsible for the generation of free radicals or reactive oxygen species (ROS). A free radical is a molecule containing one or more unpaired electrons in atomic or molecular orbitals (Halliwell and Gutteridge, 1999) that includes superoxide ions (O2-), hydroxyl radicals (OH-) and H2O2 (Mazumder et al., 2006; Zeynep Alpay et al, 2006). These ROS may induce oxidative damage to various macromolecules like polyunsaturated fatty acids in cell membranes (Halliwell and Gutteridge, 1999), carbohydrates, proteins and DNA (Stadtman, 1992), which results in homeostatic imbalance (Bonnefont et al., 2000). Author:T.Sathishkumar. Research Article Production of Bacillus anthracis Recombinant Protective Antigen in Escherichia coli Abstract Over the century anthrax has been a fundamental model for the studies of infectious diseases. The anthrax toxin consists of three proteins namely, protective antigen (PA), lethal factor, and edema factor. These are produced by the Gram-positive bacterium, Bacillus anthracis. Current vaccines against anthrax consist of protective antigen as the primary component. Production of recombinant PA of Bacillus anthracis was carried out by fermentation process in E. coli at 370C, pH 7.2 for 8 hours. The dissolved oxygen level was maintained at 30-40 % using inlet air as well as pure oxygen whenever required. Expression of the recombinant PA was induced with 1 mM IPTG and further grown for five hours before harvesting. Expression of PA in E. coli yielded an insoluble protein aggregating to form inclusion bodies. The inclusion bodies were solubilized in 8 M Urea and the protein was purified under denaturing conditions using nickel nitrilotriacetic acid (Ni-NTA) affinity chromatography. The recombinant E. coli resulted in cell dry weight of about 8.43 g per litre of culture. This translates into an expression yield of about 36.46 mg of the PA per litre of culture. The purity of the recombinant PA was checked by SDS-PAGE analysis and reactivity of this protein was determined by Western blotting and ELISA. Thus, the recombinant PA may be used for further development as a diagnostic reagent as well as vaccine candidate against anthrax. Keywords : Protective antigen; Bacillus anthracis; Escherichia coli; Fermentation Introduction The bacterium, Bacillus anthracis is the causative agent for the disease called anthrax (Dixon et al., 1999). Recently, B. anthracis has attracted attention as an agent for bioterrorism (Inglesby et al., 1999; Lane et al., 2001). The toxic effects of B. anthracis are primarily due to the “anthrax toxin,” which consists of a complex of three proteins: protective antigen (PA), edema factor (EF), and lethal factor (LF). The three proteins of the toxin are encoded by plasmids pXO1 and the polyglutamate capsule is encoded by pXO2. PA is the most extensively characterised component of anthrax toxin. The molecular weight of PA is 83 kDa. Protective antigen (PA) of Bacillus anthracis is the central moiety of the anthrax toxin complex and it elicits antibody response useful for serodiagnosis of the disease. The primary immunogenic component of the human vaccine is the protective antigen (PA) (Hambleton and Turnbull, 1990). It is also the main immunogen of the cell-free vaccine against anthrax. Immunization with the human vaccine can induce local pain, edema, and erythema, and frequent boosters are required (Brachman et al., 1962). The current licensed vaccine consists of a cell-free filtrate of Bacillus anthracis culture grown to maximize PA content, the major immunogen (Ahuja et al., 2001; Ramirez et al., 2002). There is considerable under reporting of the disease largely because of lack of adequate microbiological facilities and test systems for diagnosis of anthrax (Kumar et al., 2000). Authors:Ambuj Shrivastva, Nagesh K. Tripathi, K. Sathyaseelan, Asha M. Jana, P.V. Lakshmana Rao. Research Article Production of pectin lyase by solid state fermentation of sugarcane bagasse using Aspergillus niger Abstract Pectin lyase is an important enzyme that finds application in food processing industries, particularly in increasing the juice content of the fruits by breaking the glycosidic bonds of the long carbon chains present in the pectin molecule. In this work sugarcane bagasse was used as raw material for the production of pectin lyase by Aspergillus niger through solid state fermentation. Sugarcane bagasse was used in various compositions to optimize the media for the maximum productivity. The physico chemical parameters of the production media such as pH, temperature and fermentation time were also optimized. The result shows that the maximum activity of pectin lyase 229.07 U/ml was achieved from the medium containing 50% of sugarcane bagasse. The effect of pH temperature and time on the production of pectin lyase was found to be pH6, 35oC and 84 hrs respectively. For the optimized conditions the maximum activity of Pectin lyase was found to be 290.88 U/ml. Key words: Pectin Lyase, Aspergillus niger, Sugarcane Bagasse, Pectic Enzymes, Solid State Fermentation Introduction Proteins are the highly complex structures with conjugations formed with metals, carbohydrates or lipids. In a similar way all enzymes are proteins, but all proteins are not enzymes. The enzymes are called biocatalyst since they increase the rate of biochemical reactions without affecting the kinetics of the biochemical reactions. Enzymes in various forms and ingredients have been used to prepare food since before the recorded history, but the production and usage of purified enzymes has gained importance in the recent past. Pectic enzymes (pectinases or pectinolytic enzymes) are those that catalyses the hydrolysis of pectic substances. Pectic enzymes have two classes namely Pectinesterases and Pectin depolymerases. Pectin esterase has the ability to de-esterify pectin by the removal of methoxy residues. Pectin depolymerases readily split the main chain and it was further classified as- Polygalacturonase (PG) and pectin lyases (PL). Thus on the whole pectinases are hydrolytic enzymes, which hydrolyze the pectin molecules and are readily soluble in water. Pectinases finds its application in food industries, tea industries and textile industries. Other industries like paper and pulp industries, waste management industries, animal feed manufacturing industries, and acid fiber manufacturers also make use of pectic enzymes. Author:Praveen Kumar Ramanujam. Short Communication Bioremediation of Automobile oil effluent by Pseudomonas sp. Abstract Automobile workshops are an important component of the service sector industry. The most significant environmental impact associated with the existing workshops is the seepage of used engine oil and washed water into the soil. Contamination of the soil by oil causes it to lose its useful properties such as fertility, water-holding capacity, permeability and binding capacity. In this study was to degrade the oils from the automobile effluent by Pseudomonas sp. with the best carrier based formulation. The oil degrading Pseudomonas sp. was isolated by enrichment technique from the automobile oil effluent polluted soil, which had been contaminated over a period of time. In this study, two liter capacity of batch type mini fermentor was used for the production of Pseudomonas sp. Two different carrier formulations like Diatomaceous and Diatomaceous earth with lignite (8:2 ratios) were used as a carrier for the preparation of carrier based biodegrader. The automobile effluent was analyzed by various methods like pH, acidity, Total Dissolved and suspended solids, hardness, BOD, COD. Addition of Inorganic salts found to be maximum degradation of 93.18 % at the 6th day. In the combination of Glycerol and Inorganic salts was showed 100 % oil degradation and which was found to be a most effective treatment. 96.97 % of oil degradation was recorded in the treatment of glycerol with inorganic salts. Key words: auto mobile effluent, carrier, Diatomaceous earth, oil, Pseudomonas sp. Author :P. Sathiya moorthi. Tools & Techniques AFM Principles and Application in Biosciences Abstract In recent year, Atomic Force Microscope (AFM) has provided a range of now opportunities for viewing, Manipulating and analyzing biomolecules in the environments. And will hopefully allow application to be developed for AFM in Medicine and biotechnology. Keywords:Atomic Force Microscope (AFM), Biomolecules, Interaction tip, Piconewton (10-12N). Introduction The invention of ATOMIC FORCE MICROSCOPY (AFM) in the mid-1980s6, followed by continuous progress in instrumentation, (AFM). A relatively new form of microscopy in which a sharp tip is scanned over the surface of a sample, while sensing the interaction force between the tip and the sample. Because AFM does not rely on an incident beam, as in electron or light microscopy, the specimen can be directly observed at high Resolution in aqueous solution. The newly developed atomic force microscope is a valuable tool for studying physical and biological structures provides a unique window to the microworld of cells, subcellular structures, and biomolecules. The AFM can image the three-dimensional structure of biological specimens in a physiological environment. This enables real-time biochemical and physiological processes to be monitored at a resolution similar to that obtained for the electron microscope. Atomic force microscopy since the initial reports of considerable progress has been made, but applications in the field of biology are exploring biological structures under conditions in which living organisms exist. AFM is a kind of scanning probe microscope where imaging of the sample is realized by interaction of the probe with the sample surface and no imaging beam (light or electron) is involved in the process. The tip of the probe is mounted on the end of a flexible cantilever (1). Author :R. Rajasekaran.