Journal of Bioprocessing & Biotechniques

Background: Chinese Hamster Ovary cells (CHO) are the most preferred host cells to meet the increasing demand for high quality ‘human-like’ complex biologics production, but is faced with the challenge of achieving high yield at an affordable price. Secreted proteins critically impact cell growth and product quality and quantity and an integral part of secretome is the packaged microvesicles. In spite of numerous efforts to characterize spent-media proteome, none have identified specific contribution of microvesicles, necessitating further differential analysis of these defined fractions of spent-media proteome, specifically packaged microvesicles. Methods: We have investigated proteome of microvesicles isolated from lag, log, stationary and death phase of CHO batch culture using LC-MS/MS based-proteomic approach to identify proteins that may be involved in regulation of cell growth, viability and productivity in culture. Results: A total of 89 unique proteins were identified in the microvesicles isolated from lag, log, stationary and death-phase of culture; of these only 8.9% were categorized as secretory proteins leaving ~91% proteins of intracellular and non-secretory nature. Microvesicles were observed to contain a number of culture phase-specific proteins which included cell-signaling molecules, transcription and translation regulators and molecular chaperons; many of which are known growth regulators, indicating the potency of microvesicles in regulating culture health. Conclusions: This is the first report of CHO microvesicular proteome and this knowledge is critical in developing rationale design of perfusion process, downstream purification process for rendering improved product stability and also novel cell engineering approaches to maximize growth and improved media formulations to maximize yield and minimize product degradation.

The potential of activated sludge microbial lipid technology as a sustainable energy platform has been recognized in the past years, but it has been challenged by the cost of carbon sources. This study hypothesized that Short Chain Fatty Acids (SCFAs) that can be derived from organic wastes can be alternative carbon sources. Therefore, this work evaluated the capability of activated sludge microbial consortia to accumulate microbial lipid by fed-batch feeding of SCFAs acetic acid, propionic acid, and butyric acid that were fed every 12 h period in 5 liter bioreactors. Activated sludge microbial consortia can accumulate microbial lipid by feeding on acetic acid. Acetic acid at 1.5 g/L loading per feeding enhanced the lipid content of activated sludge up to around 20% (w/w) dry biomass. This is comparable to that of oleaginous microorganisms. The feeding of nitrogen source (ammonium) at molar C/N of 70 only at the start resulted in significant lipid accumulation as compared to that from feeding of nitrogen for every feeding of the carbon source (acid-substrates). Fatty Acid Methyl Esters (FAMEs) profiles of the extracted lipids changed during cultivation. A biodiesel volumetric yield increase of 325% (w/w) from initial culture was achieved. This microbial lipid enhancement was confirmed using fluorescence microscopy imaging of neutral lipids, which also showed that the neutral lipid-containing cells are in the size range of yeasts. This work proved the hypothesis that activated sludge microbial consortia can accumulate microbial lipid by feeding on SCFAs.

Silica has a variety of uses, including functioning as a catalytic support and utility in drug delivery systems. It is traditionally obtained from mineral sources through costly extraction processes, which has led to a search for alternative sources. One of the main features of freshwater sponges is that they contain spicules composed mainly of amorphous silica. The species Metania reticulata is found in the Negro river (Amazonas, Brazil) in great abundance, reaching volumes above 3 dm³. Different purification processes employing oxidizing solutions and subsequent heat treatment are proposed in order to extract high purity silica from freshwater sponges. With these processes, it was possible to obtain high purity (99.92%) amorphous silicon at a concentration higher than silica commercially used for column chromatography (99.71%) support material while eliminating the other elements present in the sponges (Fe, K, Ca, Ti and S). This study described low-cost techniques for the production and purification of high purity amorphous silica as alternatives to production methods currently in use.

Background: On January 20th, 2015, President Obama proposed "Precision Medicine Initiative" in State of the Union address, which lead to close attention and rapid follow-up of many counties around the world, including China. Methods: This paper deeply analyzes the background and motivation of the "precision medical plan" of the United States. On the basis of systematic exposition of the connotation, development history and development conditions of Precision Medicine, the advantages and disadvantages of the development of precision medicine in China are analyzed, and the thinking of the development of precision medicine in China is put forward. Results: Five supporting conditions should be satisfied to ensure the sound development of precision medicine: To establish a new inspiring mechanism for innovations; To establish a new disease classification system incorporating molecular genetic data; To optimize the regulatory system of new drug research; Correct interpretation of genetic data; Encouraging patients to actively participate through appropriate regulations and regulatory measures. Conclusions: During developing the precision medicine research initiative of China, the following factors should be taken into account: focusing on prevention and control of China's major diseases; Closely combining basic medical research with clinical needs; To establish a scientific environment based on big data; To launch research on the making of relevant regulatory policies; Strengthening population-based cohort study.

Four different autochthonous calves’ strains: Lactobacillus johnsonii CRL1693, L. murinus CRL1695, L. mucosae CRL1696 and L. salivarius CRL1702 were evaluated by their resistance and survival to dairy farm conditions and were used to prepare probiotic fermented milk for young calves. The strains were previously isolated from calf’s faeces and selected by their beneficial properties. The resistance of the microorganisms to water, colostrum and raw milk was by using those of the environment of a dairy farm. Compatibility assays were performed to know if the strains can be combined in the final fermented product. For the elaboration of fermented milk, different inoculum, incubation times and acidifying capability of the strains were determined and also their survival and the maintenance of beneficial properties during storage at low temperature. Antibiotic resistance profiles were applied to differentiate the strains throughout the experiments. The results indicate that bacteria survive in colostrum (1, 3 or 5 days after calving) and water for 2 or 4 hours. All the strains grow in raw milk, and were compatible between them. The optimum fermentation time was 8 hours at 37°C, reaching 3.78 × 10⁸ CFU/ml from an inoculum of 3 × 10⁹ CFU of each strain in sterile milk. Surface properties as auto-aggregative and hydrophobicity patterns were maintained after the process. Bacteria remained viable during 30 days at refrigeration temperature at a concentration of 3.37 × 10⁷ CFU/ml. The dose suggested is 10 ml of fermented milk prepared at the laboratory and stored at 4°C for younger animals. For older calves, a second fermentation in the farm is proposed to reach 3.94 × 10⁶ CFU/ml. Animal experiments are being performed to determine the efficacy of the fermented milk for diarrhea prevention.

The present study aims at screening and isolates thermostable alkaline protease producing bacteria from soil sample in the northern part of Cameroon, and characterizes their enzyme. Samples of soil were collected from four (04) North Cameroonian towns (Ngaoundere, Garoua, Figuil and Maroua). Six (06) isolates showed important proteolytic activity on skim milk agar. The strain N2.3.5 from Ngaoundere showed the largest hydrolysis halo (24 mm) and was selected for protease production. The partial identification of strain N2.3.5 through morphological and biochemical characterization enabled us to classify it as belonging to the genus Bacillus sp. The growth of the strain N2.3.5 and its enzyme production profile showed that the growth of the bacteria was a diauxic type, and that the protease production was not directly associated with the microbial growth. In a medium composed of soya flour, corn flour, yeast extract and at alkaline pH, following the monitoring of each of these parameters by a complete factorial experimental design, the pH value of 10 was found to be significant for an optimal growth. Meanwhile, the corn flour, the soya flour and the yeast extract did not have a consequent influence on the protease production by the bacteria, under the chosen experimental conditions. The extracellular protease produced by Bacillus sp N235 was partially purified using a precipitation with ammonium sulphate, a dialysis and a gel filtration on sephadex G-75. A specific activity of 1516.7 U/mg and a purification yield of 37.63% were obtained. The partially purified enzyme was able to maintain its activity after been heat-treated at 80°C for 30 min. The maximal activity was exhibited at temperature of 80°C and to a pH 12. The enzyme activity and stability was enhanced in presence of the Ca²⁺.

Cellulolytic bacteria were isolated and screened from municipal solid wastes and rice straw wastes using Carboxy Methyl Cellulose (CMC) agar medium as a selective medium. Production of clear zones by the bacterial isolates on CMC agar medium supplemented with 1% CMC was considered as indicative of extracellular cellulase activity. The size of transparent zone diameter was considered as proportional to the level of cellulase production. These bacterial isolates were identified as Bacillus sp., Pseudomonas sp. and Serratia sp. based on morphological, cultural and biochemical characteristics. A basal medium containing CMC, KH₂PO₄, K₂HPO₄, MgSO₄, (NH₄)₂SO₄, CaCl₂ and FeSO₄ at pH 7.0 was used for cellulase production. The assay of cellulase in term of CMCase was performed by measuring the release of reducing sugar. Different physicochemical parameters were optimized for cellulase production at shake flask fermentation. Time course study revealed that maximum level of cellulase was produced by Bacillus and Serratia isolates after 24 h of cultivation and by Pseudomonas isolates after 42 h of cultivation. Optimum level of cellulase was produced by Pseudomonas and Bacillus isolates at 37°C, and that by Serratia isolate was at 35°C. Optimum pH for cellulase production by these bacterial isolates was 7.0. Optimum temperature and pH for the activity of cellulase from these isolates were 40°C and 7.0, respectively. The cellulase from these isolates was found almost stable up to 55°C and at pH 7.0 for 1 h. The crude cellulase could liberate reducing sugar from filter paper through hydrolysis. Results showed that bacterial isolates produced significant level of cellulase with cellulose degrading capability.

Ethanol was produced from saw dust of Masonia wood by means of simultaneous saccharification and fermentation process. The Ethanol produced was blended with Diesel in different proportions. The fuel properties of the Ethanol-Diesel (ED) blends at different temperatures and load conditions were experimentally investigated. The properties determined were relative density, cloud point, pour point, flash point, viscosity and the calorific value. The Diesel Engine Test Bed (Petter: PJ2W-type, 7227/22.5 BS) was used with ED blends having 5, 10, 15 and 20% ethanol with respectively 95, 90, 85 and 80% diesel on a volume basis to know the performance of the blends. The experimental results of the engine’s performance which include the brake power, brake specific fuel consumption, brake thermal efficiency for the fuel blends were analyzed to know the suitability of using ED blend in Compression Ignition engine. The results show that both the relative density and viscosity of the blends decreased as the ethanol content in the blends was increased. All the blends were found to have the same cloud point of 5°C with that of diesel while their pour points vary and differ from that of diesel. All the blends have flash points 65% lower than that of diesel. The calorific values for ED5, ED10, ED15 and ED20 blends were 2, 3, 4 and 6% respectively less than that of diesel. The engine’s performance analysis indicated that there was an increase in brake thermal efficiency of the engine with increased proportion of ethanol in the fuel blends. The ED20 gave higher brake thermal efficiency than the diesel fuel at all load conditions. It was observed that at all loads conditions; the mass flow rate of ED20 was low so, resulting in decreased in specific fuel consumption. At all loads conditions, carbon dioxide emissions increased while hydrocarbon emissions decreased with increased amount of ethanol in the fuel blends, with ED20 showing the least emissions levels. Also, at all loads conditions, NOX emission of the blends was found to be higher than that of standard diesel due to the oxygen concentration and combustion timing. The results found showed that the ED20 (20% ethanol and 80% diesel) can be used in CI engine without any modification.