Parameter oscillation attenuation and mechanism exploration for continuous VHG ethanol fermentation.

A bioreactor system composed of a stirred tank and three tubular bioreactors in series was established, and continuous ethanol fermentation was carried out using a general Saccharomyces cerevisiae strain and a very high gravity medium containing 280 g L(-1) glucose, supplemented with 5 g L(-1) yeast extract and 3 g L(-1) peptone. Sustainable oscillations of glucose, ethanol, and biomass were observed when the tank was operated at the dilution rate of 0.027 h(-1), which significantly affected ethanol fermentation performance of the system. After the tubular bioreactors were packed with 1/2'' Intalox ceramic saddles, the oscillations were attenuated and quasi-steady states were achieved. Residence time distributions were studied for the packed bioreactors by the step input response technique using xylose as a tracer, which was added into the medium at a concentration of 20 g L(-1), indicating that the backmixing alleviation assumed for the packed tubular bioreactors could not be established, and its contribution to the oscillation attenuation could not be verified. Furthermore, the role of the packing's yeast cell immobilization in the oscillation attenuation was investigated by packing the tubular bioreactors with packings with significant difference in yeast cell immobilization effects, and the experimental results revealed that only the Intalox ceramic saddles and wood chips with moderate yeast cell immobilization effects could attenuate the oscillations, and correspondingly, improved the ethanol fermentation performance of the system, while the porous polyurethane particles with good yeast cell immobilization effect could not. And the viability analysis for the immobilized yeast cells illustrated that the extremely lower yeast cell viability within the tubular bioreactors packed with the porous polyurethane particles could be the reason for their inefficiency, while the yeast cells loosely immobilized onto the surfaces of the Intalox ceramic saddles and wood chips could be renewed during the fermentation, guaranteeing their viability and making them more efficient in attenuating the oscillations. The packing Raschig rings without yeast cell immobilization effect did not affect the oscillatory behavior of the tubular bioreactors, further supporting the role of the yeast cell immobilization in the oscillation attenuation.

Ethanol fermentation technologies from sugar and starch feedstocks.

This article critically reviews some ethanol fermentation technologies from sugar and starch feedstocks, particularly those key aspects that have been neglected or misunderstood. Compared with Saccharomyces cerevisiae, the ethanol yield and productivity of Zymomonas mobilis are higher, because less biomass is produced and a higher metabolic rate of glucose is maintained through its special Entner-Doudoroff pathway. However, due to its specific substrate spectrum as well as the undesirability of its biomass to be used as animal feed, this species cannot readily replace S. cerevisiae in ethanol production. The steady state kinetic models developed for continuous ethanol fermentations show some discrepancies, making them unsuitable for predicting and optimizing the industrial processes. The dynamic behavior of the continuous ethanol fermentation under high gravity or very high gravity conditions has been neglected, which needs to be addressed in order to further increase the final ethanol concentration and save the energy consumption. Ethanol is a typical primary metabolite whose production is tightly coupled with the growth of yeast cells, indicating yeast must be produced as a co-product. Technically, the immobilization of yeast cells by supporting materials, particularly by gel entrapments, is not desirable for ethanol production, because not only is the growth of the yeast cells restrained, but also the slowly growing yeast cells are difficult to be removed from the systems. Moreover, the additional cost from the consumption of the supporting materials, the potential contamination of some supporting materials to the quality of the co-product animal feed, and the difficulty in the microbial contamination control all make the immobilized yeast cells economically unacceptable. In contrast, the self-immobilization of yeast cells through their flocculation can effectively overcome these drawbacks.

Application of statistical design for the optimization of amino acid separation by reverse-phase HPLC.

Modified resolution and overall separation factors used to quantify the separation of complex chromatography systems are described. These factors were proven to be applicable to the optimization of amino acid resolution in reverse-phase (RP) HPLC chromatograms. To optimize precolumn derivatization with phenylisothiocyanate, a 2(5-1) fractional factorial design in triplicate was employed. The five independent variables for optimizing the overall separation factor were triethylamine content of the aqueous buffer, pH of the aqueous buffer, separation temperature, methanol/acetonitrile concentration ratio in the organic eluant, and mobile phase flow rate. Of these, triethylamine concentration and methanol/acetonitrile concentration ratio were the most important. The methodology captured the interaction between variables. Temperature appeared in the interaction terms; consequently, it was included in the hierarchic model. The preliminary model based on the factorial experiments was not able to explain the response curvature in the design space; therefore, a central composite design was used to provide a quadratic model. Constrained nonlinear programming was used for optimization purposes. The quadratic model predicted the optimal levels of the variables. In this study, the best levels of the five independent variables that provide the maximum modified resolution for each pair of consecutive amino acids appearing in the chromatograph were determined. These results are of utmost importance for accurate analysis of a subset of amino acids.

Thermostable enzymes.

In general, enzyme thermostability is an intrinsic property, determined by the primary structure of the protein. However, external environmental factors including cations, substrates, co-enzymes, modulators, polyols and proteins often increase enzyme thermostability. With some exceptions, enzymes present in thermophiles are more stable than their mesophilic counterparts. Some organisms produce enzymes with different thermal stability properties when grown at lower and higher temperatures. There are commercial advantages in carrying out enzymic reactions at higher temperatures. Some industrial enzymes exhibit high thermostability. More stable forms of other industrial enzymes are eagerly being sought.

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.

Biosensors: recent trends.

One of the major bottlenecks in automation and process control of industrial bioprocesses is the lack of suitable sensing devices to accurately measure the concentrations of biomolecules. The measurement of ions (e.g., H(+), NH(4)(+)) and gases (e.g., O(2), CO(2), NH(3)) using standard ion-selective and gas sensing electrodes respectively, is well established. Chemical analysis of biomolecules off-line is generally unreliable, labour intensive and may lead to contamination of the biological systems. Problems of maintaining sterile conditions are especially important when dealing with slow growing mammalian or plant cells in culture. Active research in the development of biosensors for monitoring fermentation processes, food production and pollution control, and for medical and veterinary applications is currently underway. This paper reviews recent approaches toward the development of biosensors which involve a biochemical interaction to measure the concentrations of biomolecules, primarily for the on-line monitoring and control of fermentation processes.

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.

Microbial desulphurization of heavy oils and bitumen.

Most oil producing countries have extensive reserves of heavy oil and bitumen. As easily accessible sources of conventional crudes decline, these reserves will become more important in supplementing the energy requirements. Heavy oil and bitumen are highly viscous and contain 3 to 6% sulphur. These objectionable quantities of sulphur must be removed before being acceptable as refinery feedstock. This paper addresses the potential of biological desulphurization of heavy oil and bitumen. The aerobic and anaerobic processes to remove organic as well as inorganic sulphur have been reviewed. To date, most studies were performed with model substrates, particularly dibenzothiophene (DBT) in a synthetic medium. Early work concerned with the isolation of microorganisms, identification and characterization of intermediate metabolites, and the development of growth media. No commercially viable process has emerged since the engineering details of the process have not been addressed conclusively. Due to high utility and catalyst cost conventional hydrodesulphurization processes are reported to be uneconomic in case of high sulphur oils. Microbial desulphurization, on the other hand, appears to be promising due to the inherent low energy requirement. This process may become more attractive by the application of genetically modified bacteria and improvements in bioreactor design.

Tissue-type plasminogen activator: characteristics, applications and production technology.

Plasminogen activators have immense clinical significance as thrombolytic agents for management of stroke and myocardial infarction. Tissue-type plasminogen activator (tPA) is generally preferred as being effective and safer than either urokinase or streptokinase type activators. Large-scale production of tPA became possible through groundbreaking developments in cell lines and bioprocess technology. Nevertheless, at thousands of dollars per treatment, tPA remains expensive. Enhancing cellular productivity and downstream product recovery through new approaches continue to be major challenges as discussed in this review. Recent clinical experience suggests the need for yet better fibrinolytic agents and attempts are underway to modify the tPA molecule to second generation products. Emerging trends in this field are outlined.

Plasmid stability in recombinant Saccharomyces cerevisiae.

Because of many advantages, the yeast Saccharomyces cerevisiae is increasingly being employed for expression of recombinant proteins. Usually, hybrid plasmids (shuttle vectors) are employed as carriers to introduce the foreign DNA into the yeast host. Unfortunately, the transformed host often suffers from some kind of instability, tending to lose or alter the foreign plasmid. Construction of stable plasmids, and maintenance of stable expression during extended culture, are some of the major challenges facing commercial production of recombinant proteins. This review examines the factors that affect plasmid stability at the gene, cell, and engineering levels. Strategies for overcoming plasmid loss, and the models for predicting plasmid instability, are discussed. The focus is on S. cerevisiae, but where relevant, examples from the better studied Escherichia coli system are discussed. Compared to free suspension culture, immobilization of cells is particularly effective in improving plasmid retention, hence, immobilized systems are examined in some detail. Immobilized cell systems combine high cell concentrations with enhanced productivity of the recombinant product, thereby offering a potentially attractive production method, particularly when nonselective media are used. Understanding of the stabilizing mechanisms is a prerequisite to any substantial commercial exploitation and improvement of immobilized cell systems.

Paper pulpmill sludge utilization: techno-economic potential for fuel ethanol, methane and scp production.

Various processes have been developed or proposed for converting cellulosic residues from pulp and paper mills into products which can be used for fuel or food. Among the promising practical possibilities are processes for ethanol, methane and microbial protein production by fermentation technology. Given the current Canadian financial climate and product demand, the results of techno-economic sensitivity analyses of these three process options indicate that microbial protein production for animal food applications is the most attractive followed by methane then ethanol, the last being quite uneconomical at present. Ironically, research emphasis seems to be placed in the reverse order. It is evident that the relevant costs of upstream and downstream processing in the various process proposals have not been adequately addressed. Case studies of several scenarios illustrate the problems.

Media for hybridoma growth and monoclonal antibody production.

For the economical production of monoclonal antibodies (MAbs), the cell-culture medium must be optimized for three different phases: growth of the hybridomas, MAb productivity of the hybridomas, and MAb purification or downstream processing. Medium improvements are necessary to meet these requirements for large-scale MAb production. Information bearing on this issue is being addressed in two research areas, cell biology and biochemical engineering, and is reviewed in this article.

Bioprocessing with genetically modified and other organisms: case studies in processing constraints.

Whereas the gene stability related considerations are important in bioprocessing with recombinant cultures, bioreactor design and scale-up require attention to the often reduced shear tolerance of the genetically altered biocatalysts relative to the corresponding wild strains. In addition, the peculiarities of expression of the rDNA product impact the downstream recovery methods. As a consequence, a bioprocessing scheme and the process machinery designed for a naturally occurring organism may need significant modifications for use with a genetically modified variety of the same organism. The case studies described highlight some of the processing constraints and consideration of general relevance.

Plasmid instability kinetics in continuous culture of a recombinant Saccharomyces cerevisiae in airlift bioreactor.

Plasmid instability of a recombinant Saccharomyces cerevisiae C468/pGAC9 (ATCC 20690) was examined during continuous culture in a nonselective medium in an airlift bioreactor. The recombinant strain contained a 2-micron based shuttle vector pGAC9 and expresses Aspergillus awatnori glucoamylase gene under the control of the yeast enolase I (ENO1) promoter. The changes in the fraction of plasmid-bearing cells and glucoamylase activity followed first-order kinetics. Expressed as a function of time, the decay rates of both the plasmid-bearing cell fraction and glucoamylase expression increased with increasing dilution rates. If expressed as a function of cell generations, the decay rates were nearly constant over the dilution rates tested. The results indicated that the growth rate difference between plasmid-bearing and plasmid-free cells was negligible. This was probably due to the low copy number of the 2-micron based yeast shuttle vector. Thus the contribution of preferential growth to apparent plasmid instability was negligible. A novel numerical method is proposed to evaluate the parameters related to plasmid stability. The estimated values of probability of plasmid loss (P = 0.0499) were nearly constant at different dilution rates. No significant effect of growth rates on plasmid instability was observed. The proposed kinetics agreed well with experimental observations.

Effects of the hydrodynamic environment and shear protectants on survival of erythrocytes in suspension.

Survival of media-suspended porcine erythrocytes exposed to various hydrodynamic environments was investigated with and without such shear protectant additives as bovine serum albumin, dextran and the non-ionic surfactant Pluronic F68. Erythrocytes provided a model cell population with cells of a uniform size, metabolic state and shear tolerance. Because the cells were non-growing, any shear adaptation effects were avoided. Cell lysis was followed by microscopic counts or release of haemoglobin. The cells were susceptible to agitation damage in unaerated shake flasks agitated at 100 rpm or greater. Relative to additives-free operation, the presence of 0.1% (w/v) dextran or albumin prolonged cell survival, but Pluronic F68 actually enhanced cell lysis in flasks agitated at 100 rpm. The protective effect of the additives depended on the hydrodynamic conditions. The protective effect of albumin was demonstrated also in aerated conditions in a split-cylinder airlift bioreactor (aspect ratio of 8.8; riser-to-downcomer cross-sectional area ratio of 1.0; specific power input of 0.34 W m-3). Comparison of the cell lysis characteristics in the airlift device and the best case performance of the shake flask showed longer survival in the flask (100 rpm); however, the length of survival in the reactor (approx. 70 h) was sufficient for practical purposes. In all cases, the cell lysis pattern conformed initially to zero-order dependence in cell concentration, becoming first-order after varying degrees of exposure to hydrodynamic forces. Fatigue failure of cells was inferred.

Continuous ethanol production and evaluation of yeast cell lysis and viability loss under very high gravity medium conditions.

A combined bioreactor system, composed of a stirred tank and a three-stage tubular bioreactor in series and with a total working volume of 3260 ml, was established. Continuous ethanol production was carried out using Saccharomyces cerevisiae and a very high gravity (VHG) medium containing 280 g l(-1) glucose. An average ethanol concentration of 124.6 g l(-1) or 15.8% (v) was produced when the bioreactor system was operated at a dilution rate of 0.012 h(-1). The yield of ethanol to glucose consumed was calculated to be 0.484 or 94.7% of its theoretical value of 0.511 when ethanol entrapped in the exhaust gas was incorporated. Meanwhile, quasi-steady states and non-steady oscillations were observed for residual glucose, ethanol and biomass concentrations for all of these bioreactors during their operations. Models that can be used to predict yeast cell lysis and viability loss were developed.

Increased heterologous protein production in Aspergillus niger fermentation through extracellular proteases inhibition by pelleted growth.

The dependence of filamentous fungal protease secretion on morphology was investigated by employing the recombinant Aspergillus niger strain AB4.1[pgpdAGLAGFP] which contains a gene for the glucoamylase-GFP (green fluorescence protein) fusion protein. Different inoculum levels were used to obtain different sizes of pellet or free mycelia. The extracellular protease activity of the cultures varied with the pellet size and decreased dramatically when the morphology was changed from free mycelia to pellets. The culture with an optimal pellet size of 1.6 mm was obtained from an inoculum of 4 x 10(6) spores/mL. It resulted in a specific protease activity of 158 units/L, only one-third of that in free mycelial growth, and a maximum specific GFP yield of 0.98 mg/g (cell mass) compared to 0. 29 mg/g for free mycelial growth with an inoculum of 10(7) spores/mL. The results indicate that this bioprocessing strategy can be effectively used to inhibit protease activity in filamentous fungal fermentation and thereby to enhance heterologous protein production.

Antimicrobial activity of bark extracts of Bridelia ferruginea (Euphorbiaceae).

Water and ethanol extracts of Bridelia ferruginea were examined for phytochemical and antimicrobial properties. The extracts, which were tested at a final concentration of 5 mg/ml, produced in vitro antimicrobial activities in assays against hospital strains of Staphylococcus aureus, Candida albicans, Staphylococcus epidermidis, Escherichia coli, Streptococcus lactis, Proteus vulgaris, Proteus mirabilis, Streptococcus pyogenes and Klebsiella sp. The zones of inhibition produced by the extracts in agar diffusion assays against the test micro-organisms ranged from 4 to 20 mm, while the chloramphenicol antibiotic control produced zones that measured 15-36 mm. Preliminary phytochemical analysis of the plant extracts showed the presence of phenols and tannins. Sesquiterpenes, anthroquinones, and saponins were not detected in the extracts. The Gram-negative bacteria appeared to be more susceptible (4-20 mm) to the antimicrobial effect of the extracts than the Gram-positive organisms (4-18 mm).

Single versus multiple bioreactor scale-up: economy for high-value products.

The economy of scaling-up a bioreactor by increasing the number of units was investigated with respect to an integrated flowsheet. For the production of t-PA from animal cells, a base case flowsheet using a single large bioreactor was compared to a multiple bioreactor case. Simulation of the complete flowsheets for the two cases showed that a multiple bioreactor approach to scale-up increases the return of investment (ROI) of the base process by 122%. This enormous increase in ROI results from the smaller size of the downstream units compared to the base case, since downstream processing accounts for about 80% of the total cost for high value products like t-PA. Proper scheduling of the downstream units allowed sharing of the equipment by the bioreactors. A breakdown of the equipment purchase cost showed that cost related to cell culture equipment increased from 14% for the base case to about 37% for the multiple bioreactor case. The contribution from chromatography columns to the total equipment purchase cost, on the other hand, decreased from 52 to 33%.

Aeration and mixing in vortex fermenters.

The overall apparent volumetric gas-liquid mass transfer coefficient (kLa) and the mixing time (t95) were determined in a 240 dm3 vortex aerated fermenter over stirrer speed and air flow ranges of 300-800 rpm and 10-45 normal dm3 min-1, respectively. The mass transfer data obtained in an aqueous salt solution (2.5 kg m-3 NaCl in water) compared well with the measurements in a fermentation medium used in culture of certain microaerophilic bacteria. Over the ranges examined, the gas-liquid mass transfer coefficient depended only on air flow rate; the dependence was linear with flow. Mixing time declined with increasing agitation according to a power-law relationship. The mixing and mass transfer characteristics of the vortex aerated system were compared with that of a 'standard' stirred tank fermenter (27 dm3). The mixing time variations with respect to agitation rate were remarkably similar for the two types of fermenters examined.