Biomedical subjects

P F Greenfield

Publications and source records attributed to P F Greenfield.

At least 19 recordsLinked to original sources

PubMed2005

Anaerobic digestion: impact of future greenhouse gases mitigation policies on methane generation and usage.

The debate as to whether carbon dioxide, methane, nitrous oxide and other greenhouse gas emissions will become subject to increasing regulation, increased restrictions, and probably to some form of carbon tax, has moved from a simple "yes" or "no" to "when". Wastewater treatment plants will be significantly impacted by increased energy costs and by specific regulations and/or penalties associated with emissions of methane and nitrous oxide. In this paper, the greenhouse gases emissions of different wastewater process options are estimated. The paper outlines the increasing need for wastewater treatment plants to factor greenhouse gas mitigation issues into their medium- as and long-term strategies, and identifies anaerobic enhouse as processes as being at the core of such strategies. Further, the paper identifies a number of key research challenges to be addressed if such strategies are to play a larger role in attenuating the likely impacts of GHG mitigation requirements on wastewater treatment plant design and operation.

Air Pollutants↗

PubMed2004

Performance evaluation of hybrid and conventional sequencing batch reactor and continuous processes.

Bench-scale systems, using conventional and compact hybrid activated sludge configurations, were set up to evaluate the systems' nitrification-denitrification performance, operating sludge age/MLSS concentration and sludge settleability at a Hong Kong municipal STW. Configurations tested were the continuous clarifier modified Ludzack Ettinger (MLE) and the sequencing batch reactor (SBR) with and without hybrid suspended biofilm carriers. Results demonstrated that the hybrid SBR and MLE systems consistently achieved close to complete nitrification (effluent NH4-N = 2.4 and 6.9 mg/L) and 75% and 67% removal of nitrogen (N) (effluent NO3-N < 10 mg/L) with an overall hydraulic retention time of only 7.5 hours, operating sludge age as short as 5.2 days, and mixed liquor suspended solids concentration of approximately 1,300 mg/L with a sludge volume index of 109 and 229 mL/g, respectively. The most sensitive and slowest growing nitrifiers attached to the hybrid biofilm carriers. This allowed the hybrid processes to be operated at a sludge age shorter than the critical nitrifying sludge age while still retaining near complete nitrification. In contrast, to achieve complete nitrification, the conventional MLE system needed to be operated at 1.5 to 2.5 times the critical sludge age. These results indicate that the hybrid MLE configuration is a suitable process for use in upgrading existing conventional works for N removal and for increasing hydraulic capacity of existing N removal works, without major civil works modifications, in Hong Kong. For new works, consideration might be given to the use of the hybrid SBR, which shows a more stable N removal performance than the MLE process due to its inherent in-basin equalization capacity and better reaction conditions for nitrification in terms of higher initial NH4-N level. It was also observed that the conventional SBR produced better nitrification performance than the hybrid MLE process tested. Design parameters and operating conditions of the hybrid systems should be subjected to further full-scale trial for higher hydraulic capacity and N removal performance.

Ammonia↗

PubMed2002

Effect of mixing on biomethanation of cattle-manure slurry.

The benefits and extent of mixing required during biomethanation of cattle-manure slurry was studied by investigating the effect of: 1) continuous and intermittent mixing, 2) agitator impeller speed and position; 3) not providing assisted mixing; 4) mixing on production of extracellular polymeric substances; and 5) mixing on the ultimate anaerobic biodegradability. Biomethanation was not adversely affected: during intermittent mixing; or when only sufficient mixing was provided to maintain off-bottom suspension of digester contents; or by doubling impeller speed. In fact continuous digestion of cattle-manure slurry without mechanical stirring was superior in terms of gas production. This can be attributed to increased loss of active volatile solids during stirring. Moreover, long-term batch digestion studies showed that the rate of biomethanation in a continuously stirred digester was inferior to that of a non-stirred one. Mixing was found to decrease production of extracellular polymeric substances (EPS). The presence of an increased level of EPS during the quiescent state could indicate increased attachment of cells to each other, resulting in larger agglomerates with better settling properties thus increasing biomass retention time.

Animals↗

PubMed2002

The impact of sea water flushing on biological nitrification-denitrification activated sludge sewage treatment process.

The process performance of the two largest activated sludge processes in Hong Kong, the Sha Tin and the Tai Po Sewage Treatment Works (STW), deteriorated in the initial period after the introduction of seawater flushing in 1995 and 1996, respectively. High effluent ammonia nitrogen (NH4-N) and total suspended solids (TSS) in excess of the discharge standards resulted from incomplete nitrification and changes in floc characteristics. A desktop study on the inhibitory effects of salinity, particularly on nitrification, was subsequently conducted using the Tai Po STW operating data. To assist the upgrade of the Sha Tin STW a five-month extensive bench-scale investigation on a simple but flexible modified Ludzack-Ettinger configuration with bio-selector was conducted to quantify the inhibitory effects due to the saline concentration. The Sha Tin STW upgrade consists of restoration of its original design capacity (conventional process) of 205,000 m3/day from its currently much reduced capacity as a Bardenpho process. Only the volume of the existing biological process and clarifier is to be utilized. The saline concentration ranges from 3,500 up to 6,500 mg Cl-/L, both daily and seasonally. High and greatly fluctuating saline concentrations have been known to inhibit nitrification. Design consideration should also be given to the peak daily and seasonal TKN loading of up to three times the average. Although the nitrifiers maximum specific growth rate was significantly reduced to a low 0.25 day(-1), the inhibition was considered to be tolerable with effluent NH4-N and NO3-N consistently at < 1 and < 6 mg/L. The bio-selector was demonstrated to be efficient in control of sludge foaming and bulking with SVI consistently < or = 125 mL/g. Results from the IAWO Model No. 1 and the hydraulic model of the secondary clarifiers allowed overall process capacity maximization. With an anoxic mass fraction of 25-30%, operating sludge age of 9-14 days and SVI < or = 125 mL/g, both the design requirements and the effluent discharge standards could be met. Without these investigations, an unnecessarily large reaction basin and secondary clarifier volume, and hence capital investment, would have resulted.

Ammonia↗

PubMed2001

Managing the Brisbane River and Moreton Bay: an integrated research/management program to reduce impacts on an Australian estuary.

The Brisbane River and Moreton Bay Study, an interdisciplinary study of Moreton Bay and its major tributaries, was initiated to address water quality issues which link sewage and diffuse loading with environmental degradation. Runoff and deposition of fine-grained sediments into Moreton Bay, followed by resuspension, have been linked with increased turbidity and significant loss of seagrass habitat. Sewage-derived nutrient enrichment, particularly nitrogen (N), has been linked to algal blooms by sewage plume maps. Blooms of a marine cyanobacterium, Lyngbya majuscula, in Moreton Bay have resulted in significant impacts on human health (e.g., contact dermatitis) and ecological health (e.g., seagrass loss), and the availability of dissolved iron from acid sulfate soil runoff has been hypothesised. The impacts of catchment activities resulting in runoff of sediments, nutrients and dissolved iron on the health of the Moreton Bay waterways are addressed. The Study, established by 6 local councils in association with two state departments in 1994, forms a regional component of a national and state program to achieve ecologically sustainable use of the waterways by protecting and enhancing their health, while maintaining economic and social development. The Study framework illustrates a unique integrated approach to water quality management whereby scientific research, community participation and the strategy development were done in parallel with each other. This collaborative effort resulted in a water quality management strategy which focuses on the integration of socioeconomic and ecological values of the waterways. This work has led to significant cost savings in infrastructure by providing a clear focus on initiatives towards achieving healthy waterways. The Study's Stage 2 initiatives form the basis for this paper.

Australia↗

PubMed1999

Optimized production of recombinant bluetongue core-like particles produced by the baculovirus expression system.

The baculovirus-expression vector system (BEVS) has been widely used for the experimental production of many human and animal single- and multi-unit vaccines, heterologous proteins, and viral insecticides. In this work, the production of recombinant bluetongue virus core-like particles (CLPs), using Sf9 cells in shaker-suspension culture with the SF900 II medium (GIBCO, NY), has been studied. This system involved the simultaneous production of two proteins, VP7 and VP3, and was shown to achieve high volumetric productivities. The key parameters of the time of infection (TOI), and the multiplicity of infection (MOI) were studied. The results show that the peak-volumetric yields and cell-specific yields achieved using low MOIs at low-cell densities were the same as those obtained following infections with a high MOI at high-cell densities. This work establishes the feasibility of using low MOIs in the baculovirus system to produce complex multiprotein particles.

Animals↗

PubMed1999

Quantification of recombinant core-like particles of bluetongue virus using immunosorbent electron microscopy.

Immunosorbent electron microscopy was used to quantify recombinant baculovirus-generated bluetongue virus (BTV) core-like particles (CLP) in either purified preparations or lysates of recombinant baculovirus-infected cells. The capture antibody was an anti-BTV VP7 monoclonal antibody. The CLP concentration in purified preparations was determined to be 6.6 x 10(15) particles/l. CLP concentration in lysates of recombinant baculovirus-infected cells was determined at various times post-infection and shown to reach a value of 3 x 10(15) particles/l of culture medium at 96 h post-infection. The results indicated that immunosorbent electron microscopy, aided by an improved particle counting method, is a simple, rapid and accurate technique for the quantification of virus and virus-like particles produced in large scale in vitro systems.

Animals↗

PubMed1998

Optimising fed-batch production of recombinant proteins using the baculovirus expression vector system.

Fed-batch culture can offer significant improvement in recombinant protein production compared to batch culture in the baculovirus expression vector system (BEVS), as shown by Nguyen et al. (1993) and Bedard et al. (1994) among others. However, a thorough analysis of fed-batch culture to determine its limits in improving recombinant protein production over batch culture has yet to be performed. In this work, this issue is addressed by the optimisation of single-addition fed-batch culture. This type of fed-batch culture involves the manual addition of a multi-component nutrient feed to batch culture before infection with the baculovirus. The nutrient feed consists of yeastolate ultrafiltrate, lipids, amino acids, vitamins, trace elements, and glucose, which were added to batch cultures of Spodoptera frugiperda (Sf9) cells before infection with a recombinant Autographa californica nuclear polyhedrosis virus (AcNPV) expressing beta-galactosidase (beta-Gal). The fed-batch production of beta-Gal was optimised using response surface methods (RSM). The optimisation was performed in two stages, starting with a screening procedure to determine the most important variables and ending with a central-composite experiment to obtain a response surface model of volumetric beta-Gal production. The predicted optimum volumetric yield of beta-Gal in fed-batch culture was 2.4-fold that of the best yields in batch culture. This result was confirmed by a statistical analysis of the best fed-batch and batch data (with average beta-Gal yields of 1.2 and 0.5 g/L, respectively) obtained from this laboratory. The response surface model generated can be used to design a more economical fed-batch operation, in which nutrient feed volumes are minimised while maintaining acceptable improvements in beta-Gal yield.

Animals↗

PubMed1996

Improved method for counting virus and virus like particles.

An improved method for counting virus and virus like particles by electron microscopy (EM) was developed. The procedure involves the determination of the absolute concentration of pure or semi-pure particles once deposited evenly on EM grids using either centrifugation or antibody capture techniques. The counting of particles was done with a Microfiche unit which enlarged approximately 50 x the image of particles on a developed negative film which had been taken at a relatively low magnification (2500 x) by EM. Initially, latex particles of a known concentration were counted using this approach, to prove the accuracy of the technique. The latex particles were deposited evenly on an EM grid using centrifugation (Modified Beckmen EM-90 Airfuge technique). Subsequently, recombinant Bluetongue virus (BTV) core-like particles (CLPs) captured by a Monoclonal antibody using a novel sample loading method were counted by the Microfiche unit method and by a direct EM method. Comparison of the simplified counting method developed with a conventional method, showed good agreement. The method is simple, accurate, rapid, and reproducible when used with either pure particles or with particles from crude cell culture extracts.

Antibodies, Monoclonal↗

PubMed1994

Hybridoma cells in a protein-free medium within a composite gel perfusion bioreactor.

A composite gel system has been developed combining the chemical and physical properties of calcium alginate and agarose gels. The results of growing composite gel immobilized hybridoma SPO1 cells in a protein-free medium within a fluidized-bed perfusion bioreactor are presented in this paper. During the continuous operation of this system, the total cell density reached 3.9 x 10(7) cells per ml of beads (viability 79.6%). The specific productivity of monoclonal antibody of the immobilized hybridoma cells reached more than 1.5 micrograms per 10(6) viable cells per hour, compared with 0.5 for non-immobilized viable cells grown in a one liter agitated bioreactor with the same medium. Significant increases in cell metabolic activities, including substrate utilization and byproduct formation, were also observed. Leaching of materials from the beads was evident and the major fraction of released materials was alginate.

Alginates↗

PubMed1994

Relationship between oxygen uptake rate and time of infection of Sf9 insect cells infected with a recombinant baculovirus.

Oxygen uptake rates (OUR) of Sf9 insect cells propagated in a serum-free medium (SF900II, Gibco) and of cells infected with a recombinant AcNPV were investigated before and after infection in a laboratory-scale bioreactor. The volumetric OURs of uninfected and exponentially growing cells were found to be proportional to the cell density. For infected cultures, the specific OUR of cells increased immediately after addition of virus and a maximum of 1.3 times the value of uninfected cells was noted for all the cultures between 8 to 30 hours post infection, which coincides with the period at which most viral replication and the majority of DNA synthesis takes place. It was observed that the rate of rise in the specific OUR decreased as the cell density at the time of infection increased, which meant that the later the infection, the later the maximum sOUR was observed. We therefore suggest that OUR measurement can be used to reflect the efficiency of a batch infection. Carbohydrate and amino acid consumption rates from an infected run were analysed in an effort to identify substrate(s) that may be used at increased rates to fuel the rise in oxygen demand observed early in the infection cycle. No observable rise in the consumption rates of glucose or glutamine, which are the major energy sources for animal cells, were seen after infection but an increase in the consumption rates of some amino acids suggests that infected Sf9 cells may utilise amino acids at an enhanced rate for energy post infection.

Amino Acids↗

PubMed1993

Recombinant protein production via fed-batch culture of the yeast Saccharomyces cerevisiae.

Protein production with the recombinant yeast Saccharomyces cerevisiae in fed-batch culture is investigated in this work using beta-galactosidase as a model protein. Segregational instability was negligible during the observed culture periods. The final volumetric productivity, as determined by both cell concentration and gene expression, was strongly affected by the time course of the glucose levels in the bioreactor. It was found that an average glucose feed rate of 1.31 g glucose h-1 resulted in both the maximum beta-galactosidase production rate of 831-950 units ml-1 h-1 and the maximum cell production rate of 0.520-0.524 mg ml-1 h-1.

Cell Division↗

PubMed1993

Operational parameters for packed beds in solid-state cultivation.

Packed bed cultivation systems have potential for widespread application in solid-state cultivation (SSC), but they are poorly characterized. The effects of particle size and substrate loading on the growth of Rhizopus oligosporus on sago-beads in packed bed bioreactors were investigated. Pressure drop and protein were monitored as indicators of fungal growth in cultivations performed in a large column (4.9 cm internal diameter and 60 cm height) and a system of small columns (4.2 cm internal diameter and 5.2 cm height). The differential pressure drop increased to a maximum between 34 and 44 h and then decreased again. The maximum differential pressure drop attained was greatest for the smallest particle size and for the lower substrate loadings. However, since the protein content continued to increase throughout the cultivation, pressure drop could not be used to monitor growth directly.

Journal Article↗

PubMed1992

Avoiding rapid growth at high cell densities: a potentially important optimisation criterion for hybridoma cultures.

Inhibition caused by rapid changes in the environment has earlier been observed in hybridoma cultures following deliberate step-changes in the culture environment. This paper presents evidence of similar effects occurring during the normal span of continuous cultures fed enriched medium at low dilution rates (0.002-0.005 1/h). The effect of this observation on optimisation is discussed. In continuous culture at a dilution rate of 0.013 1/h, a viable cell density of 4 x 10(9) cells/l was achieved by gradually increasing the nutrient concentration in the feed medium. The MAb titre was 200 mg/l representing a 6-fold increase compared to batch culture and a 2-fold increase compared to continuous culture using standard medium.

Antibodies, Monoclonal↗

PubMed1992

Modelling the growth and protein production by insect cells following infection by a recombinant baculovirus in suspension culture.

A mathematical model has been developed to describe the growth and infection of insect cells by recombinant baculoviruses. The model parameters were determined from a series of independent experiments involving batch suspension culture. The profiles generated by the model for cell growth, virus production and protein production agree with those observed in experiments. Presently, the model simulates only systems where cells are not growth-limited. The model is useful in aiding the design and optimization of large-scale systems for production of biological insecticides as well as recombinant proteins and in delineating those areas which are limiting the process and require further, more fundamental, investigation.

Animals↗

PubMed1992

Accuracy of the endpoint assay for virus titration.

The statistics of estimators used with the endpoint assay for virus titration were investigated. For a standard assay with 10 wells/dilution, the graphical estimator traditionally used was found to produce estimates with significant positive bias and a relatively low accuracy. Furthermore, the graphical estimator was found to be inconsistent. A superior estimator based on the maximum likelihood principle was developed. The results are discussed in relation to the choice between the endpoint titration assay and the plaque assay, and an alternative two-stage assay is presented.

Evaluation Studies as Topic↗

PubMed1991

Weaning of three hybridoma cell lines to serum free low protein medium.

A general weaning procedure is described which allowed a range of hybridomas to be weaned readily off serum without loss of antibody production. Initial work was carried out with one cell line only (SPO1 cells) and one serum substitute containing a final protein concentration of 40 mg l-1. The SPO1 cells were first adapted to a range of readily available basal media and then weaned off serum by a range of protocols. From this work an optimal weaning protocol and basal medium for weaning were determined. These were then used to wean the SPO1 cells and two other cell lines off serum with a second, protein free, serum substitute with varying concentrations of defined proteins added. All three cell lines investigated were readily weaned off serum by this protocol at protein concentrations as low as 1 mg l-1. No loss of antibody production was observed with any of the cell lines. The weaning procedure outlined in both simple and rapid and has been successfully adopted in our laboratory by relatively inexperienced cell culture technicians.

Animals↗

PubMed1990

Hybridoma growth limitations: the roles of energy metabolism and ammonia production.

Energy metabolism and the production of ammonia in hybridoma cell culture and its inhibitory effects on cell growth are reviewed. The interactive roles of glucose and glutamine metabolism affect the rate of production of ammonia, and these interactions are described. It is shown that growth inhibition usually occurs between 2-4 mM ammonia although some cell lines have been shown to adapt to much higher concentrations, particularly in continuous culture. In batch cultures cell growth appears to be particularly susceptible to increased ammonia concentrations during the early stages of growth; ammonia increased the rate of cell death in the late stage of batch growth. The specific productivity of monoclonal antibodies is much less sensitive to the released ammonia than is growth; lower volumetric productivities relate to the lower viable cell concentrations which are achieved at the high ammonia levels. Techniques to prevent ammonia accumulation or remove ammonia selectively have been relatively unsuccessful to date.

Ammonia↗