Microbioreactors and Perfusion Bioreactors for Microbial and Mammalian Cell Culture

Screening for novel producer strains and enhanced therapeutic production at reduced cost has been the focus of most of the biopharmaceutical industries. The obligation to carry out prolonged intensive pilot scale experiments gave birth to micro-scale bioreactor systems. Screening large number of microorganisms using shake flasks and benchtop bioreactors is tedious and consumes resources. Microbioreactors that mimic benchtop bioreactors are capable not only of high throughput screening of producer strains, but also aid in optimizing the growth kinetics and expression of proteins. Modern technology has enabled the collection of precise online data for variables such as optical density (OD), pH, temperature, dissolved oxygen (DO), and adjusting in mixing inside microreactors. Microbioreactors have become an irreplaceable tool for biochemical engineers and biotechnologists to perform a large number of experiments simultaneously. Another aspect that is vital to any industry is the product yield and subsequent downstream processing. Perfusion bioreactors are one of the upcoming advances in bioreactor systems that have the potential to revolutionize biologics production. This chapter intends to take a review of different aspects of microbioreactors and perfusion bioreactors including their potential in high throughput pilot studies and microbial and mammalian cell cultivation technologies

Enhanced production of amidase from Rhodococcus erythropolis MTCC 1526 by medium optimisation using a statistical experimental design

In the present work, statistical experimental methodology was used to enhance the production of amidase from Rhodococcus erythropolis MTCC 1526. R. erythropolis MTCC 1526 was selected through screening of seven strains of Rhodococcus species. The Placket-Burman screening experiments suggested that sorbitol as carbon source, yeast extract and meat peptone as nitrogen sources, and acetamide as amidase inducer are the most influential media components. The concentrations of these four media components were optimised using a face-centred design of response surface methodology (RSM). The optimum medium composition for amidase production was found to contain sorbitol (5 g/L), yeast extract (4 g/L), meat peptone (2.5 g/L), and acetamide (12.25 mM). Amidase activities before and after optimisation were 157.85 units/g dry cells and 1,086.57 units/g dry cells, respectively. Thus, use of RSM increased production of amidase from R. erythropolis MTCC 1526 by 6.88-fold

Media optimization for biosurfactant production by Rhodococcus erythropolis MTCC 2794: artificial intelligence versus a statistical approach

This paper entails a comprehensive study on production of a biosurfactant from Rhodococcus erythropolis MTCC 2794. Two optimization techniques-(1) artificial neural network (ANN) coupled with genetic algorithm (GA) and (2) response surface methodology (RSM)-were used for media optimization in order to enhance the biosurfactant yield by Rhodococcus erythropolis MTCC 2794. ANN and RSM models were developed, incorporating the quantity of four medium components (sucrose, yeast extract, meat peptone, and toluene) as independent input variables and biosurfactant yield [calculated in terms of percent emulsification index (% EI24)] as output variable. ANN-GA and RSM were compared for their predictive and generalization ability using a separate data set of 16 experiments, for which the average quadratic errors were ~3 and ~6%, respectively. ANN-GA was found to be more accurate and consistent in predicting optimized conditions and maximum yield than RSM. For the ANN-GA model, the values of correlation coefficient and average quadratic error were ~0.99 and ~3%, respectively. It was also shown that ANN-based models could be used accurately for sensitivity analysis. ANN-GA-optimized media gave about a 3.5-fold enhancement in biosurfactant yield

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Development of ultralight and ultrafine grained Mg-Li-Ca alloy by compositional optimisation and severe plastic deformation

This PhD thesis aimed at developing a new superlight Mg-Li-Ca alloy with superior properties. Binary Mg-30Ca and Mg-14Li (wt %) master alloys were melted successively in the induction furnace to obtain a Mg-Li-Ca ternary alloy containing 3.99 % Li and 1 % Ca. The alloy is designated Mg-4Li-1Ca (LX41). The as-cast material was homogenised at 350° C for 2 hr followed by furnace cooling. The homogenised alloy was then independently processed via normal hot rolling (60 % reduction in thickness), newly designed two step rolling (TSR, 60 % reduction in thickness) and multi temperature equal channel angular pressing (ECAP, 4 passes). Samples of the alloy in each processing condition were further annealed at 350 °C for 15-90 min followed by air cooling. The microstructures of as-cast, homogenised, conventionally rolled (AR), two step rolled (TSR), Equal channel angular pressed (ECAP) and subsequently annealed conditions were examined by optical, scanning and transmission electron microscopy. It clearly showed the presence of α-Mg and eutectic (α-Mg + Mg2Ca) phases in as-cast and homogenised samples. Fine recrystallised grains along with twins were seen in AR structure which subsequently transformed to equiaxed grains structure upon annealing. TSR and ECAP microstructures showed extensive grain refinement leading to average grain size of 200 and 300 nm, respectively, along with substantial fragmentation of the Mg2Ca phase during deformation. LX41 alloy also showed significant change in the quality of the texture upon different processing. AR alloy showed significant pole splitting towards TD whereas TSR alloy showed substantial spread in the texture along the transverse direction (TD) of rolling. Annealing subsequent to rolling and TSR resulted in substantial weakening of basal texture (texture intensity of 3.7 multiples of random distribution (MRD)) and especially after 30 min of annealing subsequent to TSR (TA30 material), no pronounced texture persisted and a fine equiaxed grain structure with a variety of orientations away from basal pole occurred. ECAP led to development of an even weaker texture, the cross-section and extrusion direction having texture intensities of 3 and 4.5 MRD, respectively. Annealing subsequent to ECAP was found to stabilise the texture. In addition, an extra texture component developed in the pole figure suggesting activation of non-basal slip components to accommodate deformation in the polycrystal. Tensile samples corresponding to all processing histories were deformed to failure at room temperature at a strain rate of 10-4 s-1. ECAP processed alloy showed the greatest strength of 270 MPa, whereas TSR led to a strength of 250 MPa, while that after normal rolling was 208 MPa. Further improvement of the properties was achieved by annealing. Thus, TA30 material showed a remarkable strain hardening ability leading to a true UTS of 250 MPa (engineering UTS of 210 MPa), which is on par with true UTS after TSR (270 MPa). The main gain was a significant improvement in the ductility of the alloy upon 30 min annealing after TSR. The strain to failure achieved was as high as 18.1 % - larger than that after ECAP (11 %). Thus, this microstructural engineering in terms of a well designed TSR and annealing step opened up a new way of obtaining a favourable combination of strength and ductility for LX41 alloy. While investigating the bio-response of the material it was found that pH variation during bio-degradation was significantly changing with starting microstructure for a given time of immersion thereby showing the highest pH value for as-cast alloy and the lowest one for TSR alloy in the early stage of degradation (just after 3 days of immersion). Hence, the important role of microstructure in determining the biodegradation (corrosion) mechanism (uniform or localised) was demonstrated. Also, the release of H2 gas and Li+ and Mg2+ ions after 7 days of immersion was found to be controlled by varying the microstructure of the LX41 alloy. The measured levels of hydrogen and Li+ and Mg2+ ions after immersion in a simulated body fluid (EBSS) were found to be below tolerable levels in the human body. SEM-EDAX and XRD analysis of the surfaces of immersed specimens confirmed formation of hydroxyapatite (HA) containing Mg2+ and Li+ ions and it was also noted that the driving force for the formation of HA was governed by OH- ions released during degradation. Once HA with entrapped Mg2+ and Li1+ ions was formed, these ions, as well as Ca2+, acted as mediators between the cells and the alloy. Hence, as understood from the above discussion, the formation of a HA layer with entrapped Mg2+ and Li1+ ions plays a key role in enhancing cell response and thus, indirectly, promoting cytocompatibility. As the formation of HA is a function of the amount of OH- ions released, which is controlled by microstructure, there exist a strong microstructure-biodegradation-cytocompatibility relationship for LX41 alloy. Finally, development of the new ultralight Mg-4Li-1Ca alloy having an excellent combination of density, strength, ductility and cytocompatibility has resulted as a very promising candidate for use in biodegradable medical implants as well as for lightweight applications. The alloy is considered as a strong competitor to conventional structural Mg alloys of the AZ and ZK series processed in a similar way. Thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy of the Indian Institute of Technology Bombay, India and Monash University, Australia

Studies on Commercial Aspects of Xylanase from <i>Chainia </i>Species

878-882Production of xylanase from Chainia species in a fermenter was standardized to a one litre scale. The substrate, wheat bran, was varied  from 3 per cent to 5 per cent and the pH profile monitored. When 3 per cent and 4 per cent wheat bran was used the major portion of the xylan was utilized for biomass production. With 5 per cent substrate a maximum activity of 29.78 IU/ml, in 72h was achieved .The conditions were easily scaled up to 101. Many commercial organic nitrogen sources were studied, at shake flask level, 2 per cent soybean meal supplemented with 0.2 per cent yeast extract, gave activities comparable to when 2 per cent yeast extract was used in the media. Separation of enzyme from fermentation broth was tried by two methods, ultrafiltration and solvent precipitation. Ultra filtration was carried out using cellulose acetate membrane cut off 5000. However, a three-old concentration of fermentation broth gave only a little over 50 per cent of the original xylanase activity. The solvents used for precipitation of xylanase from fermentation broth were ethanol and acetone. Three volumes of ethanol gave 99 per cent xylanase recovery with a 20-fold increase in specific activity. Whereas two volumes of acetone gave 93.5 per cent recovery with 10-fold increase in specific activity. Xylanase activity in a clarified fermentation broth, stored at 10oC, retained 90 per cent of the original activity, it reduced to half its value only after three months

Integrated process for extraction and purification of alcohol dehydrogenase from Baker's yeast involving precipitation and aqueous two phase extraction

The potential of aqueous two phase extraction for the purification of alcohol dehydrogenase (ADH) from Baker's yeast (Saccharomyces cerevisiae) is demonstrated. An integrated process involving precipitation followed by aqueous two phase extraction (ATPE) is used for effective recovery of the enzyme. This is carried out in two schemes. In the first scheme, ammonium sulphate precipitation is carried out followed by ATPE of the precipitate after suspending it in buffer. While the second scheme involves polyethylene glycol precipitation followed by ATPE of the supernatant. The effect of various process parameters such as molecular weight of polymer, tie line length, phase volume ratio and neutral salt is investigated for enhancing the activity recovery of ADH. First scheme resulted in about 85% enzyme activity recovery of ADH with purification factor of 4.2 while the second scheme about 90% enzyme activity recovery with 6.6-fold purification

Membrane distillation for the concentration of raw cane-sugar syrup and membrane clarified sugarcane juice

Membrane Distillation (MD) is a process being investigated the world over as a low cost energy saving alternative to conventional separation processes such as distillation and reverse osmosis. MD has a wide range of benefits such as a 100% (theoretical) rejection of ionic species, macromolecules, colloids, cells and other non-volatiles, lower operating pressures, reduced chemical interaction between membrane and process solutions, less demanding membrane mechanical properties and reduced vapour spaces. This process is ideally suited for the concentration of aqueous streams such as fruit juice and sugar solutions. Clarified cane sugar solution (20°Brix) obtained from the sugar mill, immediately after the Dorr filtration was subjected to membrane distillation, whereby the sensible heat from this stream was used to remove water as vapour through a hydrophobic polypropylene membrane. The influence of cross-flow rate and continuous water addition on flux was evaluated

Targeting capsular polysaccharides of <em>Mycoplasma mycoides</em> using synthetic biology tools

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