Large scale protein separations: engineering aspects of chromatography

The engineering considerations common to large scale chromatographic purification of proteins are reviewed. A discussion of the industrial chromatography fundamentals is followed by aspects which affect the scale of separation. The separation column geometry, the effect of the main operational parameters on separation performance, and the physical characteristics of column packing are treated. Throughout, the emphasis is on ion exchange and size exclusion techniques which together constitute the major portion of commercial chromatographic protein purifications. In all cases, the state of current technology is examined and areas in need of further development are noted.

The physico-chemical advances now underway in chromatographic separation of biopolymers would ensure a substantially enhanced role for these techniques in industrial production of products of new biotechnology.     

Spectrophotometric determination of mycelial biomass

The optical density (450 nm) of samples of homogenized fungal biomass correlated linearly with the dry weight of the biomass in the samples. As shown for broths of the filamentous microfungus Neurospora sitophila, the sensitivity of the technique depended on the extent of fragmentation of fungal hyphae during homogenization: increased fragmentation increased sensitivity. The method applied during all phases of growth, was as accurate as the conventional dry weight technique and permitted rapid and simple measurement of biomass concentration.     

Fermentation of cellulosic materials to mycoprotein foods

A new bioprocess is described in which a cellulolytic, food-grade fungus Neurospora sitophila converts cellulosic materials to protein-rich products for food and fodder. The optimal conditions for the conversion are identified: 35-37 degrees C temperature, pH 5.5, 2.35 ms(-1) agitator tip speed. Scale-up of the production process to 1,300 L is reported. The mycoprotein production data on several types of cellulosic materials (sugarcane bagasse, corn stover, wood cellulose) are presented. The performance of N. sitophila is found to compare favourably with that of Chaetomium cellulolyticum, another cellulolytic organism previously reported on by us.     

Changing Emergence of Shigella Sero-Groups in Bangladesh: Observation from Four Different Diarrheal Disease Hospitals

Background

Shigellosis continues to be a public health challenge for developing countries, including Bangladesh. The aim of the study is to demonstrate recent changes in Shigella sero-groups and their geographical diversity.

Methods

Data were extracted from data archive of four diarrheal disease surveillance systems. A 2% sub sample from urban Dhaka Hospital (2008–2011; n = 10,650), and 10% from urban Mirpur Treatment Centre (2009–2011; n = 3,585), were enrolled systematically; whereas, all patients coming from the Health and Demographic Surveillance System area in rural Matlab (2008–2011; n = 6,399) and rural Mirzapur (2010–2011; n = 2,812) were included irrespective of age, sex, and disease severity. A fresh stool specimen was collected for identification of Shigella spp. Of them, 315 (3%) were positive for Shigella in Dhaka, 490 (8%) from Matlab, 109 (3%) from Mirpur and 369 (13%) from Mirzapur and considered as analyzable sample size.

Results

Among all Shigella isolates regardless of age, significant decreases in percentage of S. flexneri over time was observed in Mirpur (55→29%; p value of χ²-for trend = 0.019) and Mirzapur (59→47%; p = 0.025). A non-significant decrease was also seen in Dhaka (58→48%), while in Matlab there was a non-significant increase (73→81%). Similar patterns were observed among under-5 children at all sites. Emergence of S. sonnei was found in Dhaka (8→25%; p<0.001) and Mirpur (10→33%; p = 0.015), whereas it decreased in Mirzapur (32→23%; p = 0.056). The emergence of S. boydii was seen in all ages in Mirzapur [(3→28%; p<0.001); (3→27%; p<0.001)]. On the other hand, we saw non-significant percent reductions in S. boydii in Dhaka [overall (25→16%); under-5 (16→9%)]. Decreasing rates of Shigella dysenteriae were observed in Matlab, Mirpur and Mirzapur; whereas, in Dhaka it remained unchanged.

Conclusion and Significance

Emergence of S. sonnei and S. boydii as important infectious diarrhea etiologies and variations in geographical diversity underscore the need for monitoring, with possible implications for vaccine development.     

Clinical Characteristics,Etiology and Antimicrobial Susceptibility among Overweight and Obese Individuals with Diarrhea: Observed at a Large Diarrheal Disease Hospital,Bangladesh

Background

The present study aimed to determine the clinical characteristics and etiology of overweight and obese (OO) individuals with diarrhea attending an urban Dhaka Hospital, International Centre for Diarrheal Disease Research (icddr,b), Bangladesh.

Methods

Total of 508 under-5 children, 96 individuals of 5–19 years and 1331 of >19 years were identified as OO from the Diarrheal Disease Surveillance System (DDSS) between 1993–2011. Two comparison groups such as well-nourished and malnourished individuals from respective age stratums were selected.

Results

Isolation rate of rotavirus was higher among OO under-5 children compared to malnourished group (46% vs. 28%). Rotavirus infection among OO individuals aged 5–19 years (9% vs. 3%) (9% vs. 3%) and >19 years (6% vs. 4%) (6% vs. 3%) was higher compared to well-nourished and malnourished children. Conversely, Vibrio cholerae was lower among all OO age groups compared to well-nourished and malnourished ones. Shigella (4% vs. 6%) (4% vs. 8%), and Campylobacter (3% vs. 5%) (3% vs. 5%) were lower only among OO in >19 years individuals compared to their counterparts of the same age stratum. Salmonella was similarly isolated in all age strata and nutritional groups. In multinomial logistic regression among under-5 children, significant association was observed only with use of antimicrobials at home [OR-1.97] and duration of hospital stay [OR-0.68]. For individuals aged 5–19 years, use of antimicrobials at home (OR-1.83), some or severe dehydration (OR-3.12), having received intravenous saline (OR-0.46) and rotavirus diarrhea (OR-2.96) were found to be associated with OO respectively. Moreover, significant associations were also found for duration of diarrhea before coming to hospital (>24 hours) (OR-1.24), Shigella (OR-0.46), and Campylobacter (OR-0.58) among >19 years OO individuals along with other associated co-variates in 5–19 years group (all p<0.05).

Conclusion and significance

Higher proportion of OO were infected with rotavirus and a greater proportion of them used antimicrobials before coming to the hospital.     

Botryococcus braunii: a renewable source of hydrocarbons and other chemicals

Botryococcus braunii, a green colonial microalga, is an unusually rich renewable source of hydrocarbons and other chemicals. Hydrocarbons can constitute up to 75% of the dry mass of B. braunii. This review details the various facets of biotechnology of B. braunii, including its microbiology and physiology; production of hydrocarbons and other compounds by the alga; methods of culture; downstream recovery and processing of algal hydrocarbons; and cloning of the algal genes into other microorganisms. B. braunii converts simple inorganic compounds and sunlight to potential hydrocarbon fuels and feedstocks for the chemical industry. Microorganisms such as B. braunii can, in the long run, reduce our dependence on fossil fuels and because of this B. braunii continues to attract much attention.     

A process for high yield and scaleable recovery of high purity eicosapentaenoic acid esters from microalgae and fish oil

A low expense process is developed for recovering esterified eicosapentaenoic acid (EPA) from microalgae and fish oil. Over 70% of the EPA content in the esterified crude extract of microalgae were recovered at purities exceeding 90%. The recovery scheme utilizes either wet or freeze-dried algal biomass. The process consists of only three main steps: 1) simultaneous extraction and transesterification of the algal biomass; 2) argentated silica gel column chromatography of the crude extract; and 3) removal of pigments by a second column chromatographic step. Argentated silica gel chromatography recovered about 70% of the EPA ester present in the crude fatty ester mixture of fish oil, but at a reduced purity ( approximately 83% pure) compared to the microalgal derived EPA. The optimal loading of the fatty ester mixture on the chromatographic support was about 3% (w/w) but loadings up to 4% did not affect the resolution significantly. The process was scaled up by a factor of nearly 320 by increasing the diameter of the chromatography columns. The elution velocity remained constant. Compared to the green alga Monodus subterraneus, the diatom Phaeodactylum tricornutum had important advantages as a potential commercial producer of EPA. For a microalgal EPA process to be competitive with fish oil derived EPA, P. tricornutum biomass (2.5% w/w EPA) needs to be obtained at less than $4/kg. If the EPA content in the alga are increased to 3.5%, the biomass may command a somewhat higher price. The quality of microalgal EPA compares favorably with that of the fish oil product. Compared to free fatty acid, EPA ester is more stable in storage. Shelf-life is extended by storing in hexane. The silver contamination in the final purified EPA was negligibly small (<210 ppb).     

Photoautotrophic Production of Lipids by Some <Emphasis Type="Italic">Chlorella</Emphasis> Strains

The microalgae Chlorella protothecoides UTEX 25, Chlorella sp. TISTR 8991, and Chlorella sp. TISTR 8990 were compared for use in the production of biomass and lipids under photoautotrophic conditions. Chlorella sp. TISTR 8990 was shown to be potentially suitable for lipid production at 30°C in a culture medium that contained only inorganic salts. For Chlorella sp. TISTR 8990 in optimal conditions in a stirred tank photobioreactor, the lipid productivity was 2.3 mg L⁻¹ h⁻¹ and after 14 days the biomass contained more than 30% lipids by dry weight. To attain this, the nitrogen was provided as KNO₃ at an initial concentration of 2.05 g L⁻¹ and chelated ferric iron was added at a concentration of 1.2 × 10⁻⁵ mol L⁻¹ on the ninth day. Under the same conditions in culture tubes (36 mm outer diameter), the biomass productivity was 2.8-fold greater than in the photobioreactor (0.125 m in diameter), but the lipid productivity was only 1.2-fold higher. Thus, the average low-light level in the photobioreactor actually increased the biomass specific lipid production compared to the culture tubes. A light-limited growth model closely agreed with the experimental profiles of biomass production, nitrogen consumption, and lipid production in the photobioreactor.     

Biotechnology-a sustainable alternative for chemical industry

This review outlines the current and emerging applications of biotechnology, particularly in the production and processing of chemicals, for sustainable development. Biotechnology is “the application of scientific and engineering principles to the processing of materials by biological agents”. Some of the defining technologies of modern biotechnology include genetic engineering; culture of recombinant microorganisms, cells of animals and plants; metabolic engineering; hybridoma technology; bioelectronics; nanobiotechnology; protein engineering; transgenic animals and plants; tissue and organ engineering; immunological assays; genomics and proteomics; bioseparations and bioreactor technologies. Environmental and economic benefits that biotechnology can offer in manufacturing, monitoring and waste management are highlighted. These benefits include the following: greatly reduced dependence on nonrenewable fuels and other resources; reduced potential for pollution of industrial processes and products; ability to safely destroy accumulated pollutants for remediation of the environment; improved economics of production; and sustainable production of existing and novel products.     

Effects of the sporulation conditions on the lovastatin production by Aspergillus terreus

The production of biomass and lovastatin by spore-initiated submerged fermentations of Aspergillus terreus ATCC 20542 was shown to depend on the age of the spores used for inoculation. Cultures started from older spores produced significantly higher titers of lovastatin. For example, the lovastatin titer increased by 52% when the spore age at inoculation rose from 9 to 16 days. The lovastatin titer for a spore age of 16 days was 186.5±20.1 mg L⁻¹. The time to sporulation on surface cultures was sensitive to the light exposure history of the fungus and the spore inoculation concentration levels. A light exposure level of 140 μE m⁻² s⁻¹ and a spore concentration of 1,320 spore cm⁻² produced the greatest extent of sporulation within about 50 h of inoculation. Sporulation was slowed in the dark and with diluted inoculants. A rigorous analysis of the data of statistically designed experiments showed the above observations to be highly reproducible.