Label-Free Proteomics of a Defined, Binary Co-culture Reveals Diversity of Competitive Responses Between Members of a Model Soil Microbial System.

Interactions among members of microbial consortia drive the complex dynamics in soil, gut, and biotechnology microbiomes. Proteomic analysis of defined co-cultures of well-characterized species provides valuable information about microbial interactions. We used a label-free approach to quantify the responses to co-culture of two model bacterial species relevant to soil and rhizosphere ecology, Bacillus atrophaeus and Pseudomonas putida. Experiments determined the ratio of species in co-culture that would result in the greatest number of high-confidence protein identifications for both species. The 281 and 256 proteins with significant shifts in abundance for B. atrophaeus and P. putida, respectively, indicated responses to co-culture in overall metabolism, cell motility, and response to antagonistic compounds. Proteins associated with a virulent phenotype during surface-associated growth were significantly more abundant for P. putida in co-culture. Co-culture on agar plates triggered a filamentous phenotype in P. putida and avoidance of P. putida by B. atrophaeus colonies, corroborating antagonistic interactions between these species. Additional experiments showing increased relative abundance of P. putida under conditions of iron or zinc limitation and increased relative abundance of B. atrophaeus under magnesium limitation were consistent with patterns of changes in abundance of metal-binding proteins during co-culture. These results provide details on the nature of interactions between two species with antagonistic capabilities. Significant challenges remaining for the development of proteomics as a tool in microbial ecology include accurate quantification of low-abundance peptides, especially from rare species present at low relative abundance in a consortium.

Bacillus spp. from rainforest soil promote plant growth under limited nitrogen conditions.

AIMS: The aim of this study was to evaluate effects of PGPR (plant growth-promoting rhizobacteria) isolated from rainforest soil on different plants under limited nitrogen conditions. METHODS AND RESULTS: Bacterial isolates from a Peruvian rainforest soil were screened for plant growth-promoting effects on Arabidopsis (Col-0). Four selected isolates including one Bacillus subtilis, two B. atrophaeus and one B. pumilus significantly promoted growth of Zea mays L. and Solanum lycopersicum under greenhouse conditions. Moreover, the PGPRs significantly promoted growth of S. lycopersicum in both low and nitrogen-amended soil conditions. These PGPR strains were further studied to obtain insights into possible mechanisms of plant growth promotion. Volatile chemicals from those isolates promoted Arabidopsis growth, and the expression of genes related to IAA production was induced in the Arabidopsis plants treated with PGPRs. Further, selected PGPR strains triggered induced systemic resistance (ISR) against Pseudomonas syringae pv tomato DC3000 in Arabidopsis. CONCLUSIONS: PGPR strains isolated from the rainforest soil promoted the plant growth of Arabidopsis, corn and tomato. SIGNIFICANCE AND IMPACT OF THE STUDY: New PGPR that have wider adaptability to different crops, soils and environmental conditions are needed to decrease our reliance on agricultural amendments derived from fossil-based fuels. The PGPRs isolated from a nonagricultural site constitute new plant growth-promoting strains that could be developed for agricultural uses.

Detection and quantification of functional genes of cellulose- degrading, fermentative, and sulfate-reducing bacteria and methanogenic archaea.

Cellulose degradation, fermentation, sulfate reduction, and methanogenesis are microbial processes that coexist in a variety of natural and engineered anaerobic environments. Compared to the study of 16S rRNA genes, the study of the genes encoding the enzymes responsible for these phylogenetically diverse functions is advantageous because it provides direct functional information. However, no methods are available for the broad quantification of these genes from uncultured microbes characteristic of complex environments. In this study, consensus degenerate hybrid oligonucleotide primers were designed and validated to amplify both sequenced and unsequenced glycoside hydrolase genes of cellulose-degrading bacteria, hydA genes of fermentative bacteria, dsrA genes of sulfate-reducing bacteria, and mcrA genes of methanogenic archaea. Specificity was verified in silico and by cloning and sequencing of PCR products obtained from an environmental sample characterized by the target functions. The primer pairs were further adapted to quantitative PCR (Q-PCR), and the method was demonstrated on samples obtained from two sulfate-reducing bioreactors treating mine drainage, one lignocellulose based and the other ethanol fed. As expected, the Q-PCR analysis revealed that the lignocellulose-based bioreactor contained higher numbers of cellulose degraders, fermenters, and methanogens, while the ethanol-fed bioreactor was enriched in sulfate reducers. The suite of primers developed represents a significant advance over prior work, which, for the most part, has targeted only pure cultures or has suffered from low specificity. Furthermore, ensuring the suitability of the primers for Q-PCR provided broad quantitative access to genes that drive critical anaerobic catalytic processes.

Identification of stress-related proteins in Escherichia coli using the pollutant cis-dichloroethylene.

AIMS: To complement our proteome study, whole-transcriptome analyses were utilized here to identify proteins related to degrading cis-1,2-dichloroethylene (cis-DCE). METHODS AND RESULTS: Metabolically engineered Escherichia coli strains were utilized expressing an evolved toluene ortho-monooxygenase along with either (i) glutathione S-transferase and altered gamma-glutamylcysteine synthetase or (ii) a rationally engineered epoxide hydrolase. cis-DCE degradation induced 30 known stress genes and 32 uncharacterized genes. Because of the reactive cis-DCE epoxides formed, we hypothesized that some of these uncharacterized genes may be related to a variety of stresses. Using isogenic mutants, IbpB, YchH, YdeI, YeaR, YgiW, YoaG and YodD were related to hydrogen peroxide, cadmium and acid stress. Additional whole-transcriptome studies with hydrogen peroxide stress using the most hydrogen peroxide-sensitive mutants, ygiW and ychH, identified that FliS, GalS, HcaR, MglA, SufE, SufS, Tap, TnaB, YhcN and YjaA are also involved in the stress response of E. coli to hydrogen peroxide, cadmium and acid, as well as are involved in biofilm formation. CONCLUSION: Seventeen proteins are involved in the stress network for this organism, and YhcN and YchH were shown to be important for the degradation of cis-DCE. SIGNIFICANCE AND IMPACT OF THE STUDY: Six previously uncharacterized proteins (YchH, YdeI, YgiW, YhcN, YjaA and YodD) were shown to be stress proteins.

Bioremediation of nitroexplosive wastewater by an yeast isolate Pichia sydowiorum MCM Y-3 in fixed film bioreactor.

Nitroexplosives are essential for security and defense of the nation and hence their production continues. Their residues and transformed products, released in the environment are toxic to both terrestrial and aquatic life. This necessitates remediation of wastewaters containing such hazardous chemicals to reduce threat to human health and environment. Bioremediation technologies using microorganisms become the present day choice. High Melting Explosive (HMX) is one of the nitroexplosives produced by nitration of hexamine using ammonium nitrate and acetic anhydride and hence the wastewater bears high concentration of nitrate and acetate. The present investigation describes potential of a soil isolate of yeast Pichia sydowiorum MCM Y-3, for remediation of HMX wastewater in fixed film bioreactor (FFBR). The flask culture studies showed appreciable growth of the organism in HMX wastewater under shake culture condition within 5-6 days of incubation at ambient temperature (28 +/- 2 degrees C). The FFBR process operated in both batch and continuous mode, with Hydraulic Retention Time (HRT) of 1 week resulted in 50-55% removal in nitrate, 70-88% in acetate, 50-66% in COD, and 28-50% in HMX content. Continuous operation of the reactor showed better removal of nitrate as compared to that in the batch operation, while removal of acetate and COD was comparable in both the modes of operation of the reactor. Insertion of baffles in the reactor increased efficiency of the reactor. Thus, FFBR developed with baffles and operated in continuous mode will be beneficial for bioremediation of high nitrate and acetate containing wastewater using the culture of P. sydowiorum.

Comparison of microbial community composition and activity in sulfate-reducing batch systems remediating mine drainage.

Five microbial inocula were evaluated in batch tests for the ability to remediate mine drainage (MD). Dairy manure (DM), anaerobic digester sludge, substrate from the Luttrell (LUTR) and Peerless Jenny King (PJK) sulfate-reducing permeable reactive zones (SR-PRZs) and material from an MD-treatment column that had been inoculated with material from a previous MD-treatment column were compared in terms of sulfate and metal removal and pH neutralization. The microbial communities were characterized at 0, 2, 4, 9, and 14 weeks using denaturing gradient gel electrophoresis and quantitative polymerase chain reaction to quantify all bacteria and the sulfate-reducing bacteria of the genus Desulfovibrio. The cultures inoculated with the LUTR, PJK, and DM materials demonstrated significantly higher rates of sulfate and metal removal, and contained all the microorganisms associated with the desired functions of SR-PRZs (i.e., polysaccharide degradation, fermentation, and sulfate reduction) as well as a relatively high proportion of Desulfovibrio spp. These results demonstrate that inoculum influences performance and also provide insights into key aspects of inoculum composition that impact performance. This is the first systematic biomolecular examination of the relationship between microbial community composition and MD remediation capabilities.

Proteomic analysis of diaminochlorotriazine adducts in wistar rat pituitary glands and LbetaT2 rat pituitary cells.

Atrazine (ATRA) is the most commonly applied herbicide in the United States and is frequently detected in drinking water at significant levels. After oral exposure, ATRA metabolism yields diaminochlorotriazine (DACT), an electrophilic molecule that has been shown to form covalent protein adducts. This research was designed to identify ATRA-induced protein adducts formed in the pituitary gland of ATRA-exposed rats and in DACT-exposed LbetaT2 rat pituitary cells. Immunohistochemistry showed diffuse cytoplasmic and nuclear staining in both pituitary sections and LbetaT2 cells indicating the formation of DACT protein adducts. Protein targets from both rat pituitaries and LbetaT2 cell culture were identified following two-dimensional electrophoresis (2DE), immunodetection, and matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. Western blots from both exposed rats and LbetaT2 cells revealed over 30 DACT-modified spots that were not present in control animals. Protein spots were matched to concurrently run 2DE gels stained with Sypro Ruby, excised, and in-gel-digested with trypsin. Mass spectrometry analysis of digest peptides resulted in the identification of 19 spots and 8 unique proteins in the rats and 21 spots and 19 unique proteins in LbetaT2 cells. The identified proteins present in both sample types included proteasome activator complex subunit 1, ubiquitin carboxyl-terminal hydrolase isozyme L1, tropomyosin, ERp57, and RNA-binding proteins. Each of these proteins contains active-site or solvent-exposed cysteine residues, making them viable targets for covalent modification by DACT.

The effect of inoculum on the performance of sulfate-reducing columns treating heavy metal contaminated water.

Sulfate-reducing permeable reactive zones (SR-PRZs) are a passive means of immobilizing metals and neutralizing the pH of mine drainage through microbially mediated reactions. In this bench-scale study, the influence of inoculum on the performance of columns simulating SR-PRZs was investigated using chemical and biomolecular analyses. Columns inoculated from two sources (bovine dairy manure (DM) and a previous sulfate-reducing column (SRC)) and uninoculated columns (U) were fed a simulated mine drainage and compared on the basis of pH neutralization and removal of cadmium, zinc, iron, and sulfate. Cadmium, zinc, and sulfate removal was significantly higher in SRC columns than in the DM and U columns, while there was no significant difference between the DM and U columns. Denaturing gradient gel electrophoresis (DGGE) analysis revealed differences in the microbial community composition among columns with different inocula, and indicated that the microbial community in the SRC columns was the first to reach a pseudo-steady state. In the SRC columns, a higher proportion of the DGGE band DNA sequences were related to microorganisms that carry out cellulose degradation, the rate-limiting step in SR-PRZ energy flow, than was the case in the other columns. The proportion of sulfate-reducing bacteria of the genus Desulfobacterium was monitored using real-time quantitative PCR and was observed to be consistently higher in the SRC columns. The results of this study suggest that the inoculum plays an important role in SR-PRZ performance. This is the first report providing a detailed analysis of the effect of different microbial inocula on the remediation of acid mine drainage.

A biologically based model of growth and senescence of Syrian hamster embryo (SHE) cells after exposure to arsenic.

We modified the two-stage Moolgavkar-Venzon-Knudson (MVK) model for use with Syrian hamster embryo (SHE) cell neoplastic progression. Five phenotypic stages are proposed in this model: Normal cells can either become senescent or mutate into immortal cells followed by anchorage-independent growth and tumorigenic stages. The growth of normal SHE cells was controlled by their division, death, and senescence rates, and all senescent cells were converted from normal cells. In this report, we tested the modeling of cell kinetics of the first two phenotypic stages against experimental data evaluating the effects of arsenic on SHE cells. We assessed cell division and death rates using flow cytometry and correlated cell division rates to the degree of confluence of cell cultures. The mean cell death rate was approximately equal to 1% of the average division rate. Arsenic did not induce immortalization or further mutations of SHE cells at concentrations of 2 microM and below, and chromium (3.6 microM) and lead (100 microM) had similar negative results. However, the growth of SHE cells was inhibited by 5.4 microM arsenic after a 2-day exposure, with cells becoming senescent after only 16 population doublings. In contrast, normal cells and cells exposed to lower arsenic concentrations grew normally for at least 30 population doublings. The biologically based model successfully predicted the growth of normal and arsenic-treated cells, as well as the senescence rates. Mechanisms responsible for inducing cellular senescence in SHE cells exposed to arsenic may help explain the apparent inability of arsenic to induce neoplasia in experimental animals.

Flow cytometry in biotechnology.

Flow cytometry is a general method for rapidly analyzing large numbers of cells individually using light-scattering, fluorescence, and absorbence measurements. The power of this method lies both in the wide range of cellular parameters that can be determined and in the ability to obtain information on how these parameters are distributed in the cell population. Flow cytometric assays have been developed to determine both cellular characteristics such as size, membrane potential, and intracellular pH, and the levels of cellular components such as DNA, protein, surface receptors, and calcium. Measurements that reveal the distribution of these parameters in cell populations are important for biotechnology, because they better describe the population than the average values obtained from traditional techniques. This Mini-Review provides an overview of the principles of flow cytometry, with descriptions of methods used to measure various cellular parameters and examples of the application of flow cytometry in biotechnology. Finally, a discussion of the challenges and limitations of the method is presented along with a future outlook.

Modeling substrate interactions during the biodegradation of mixtures of toluene and phenol by Burkholderia species JS150.

The biodegradation kinetics of toluene, phenol, and a mixture of toluene and phenol by Burkholderia species JS150 was measured and modeled. Both of these compounds can serve as the sole source of carbon and energy for this microorganism. The single-substrate biodegradation kinetics was described well using the Monod model, with model constants of mu(max,T) = 0.39 h(-1) and K(S,T) = 0.011 mM for growth on toluene and mu(max,P) = 0.309 h(-1) and K(S,P) = 0.0054 mM for growth on phenol. Degradation of the mixture of toluene and phenol followed simultaneous utilization kinetics with toluene being the preferred substrate. Toluene was found to inhibit the rate of utilization of phenol while the presence of phenol had little effect on the rate of degradation of toluene. Of the kinetic models that were tested, one developed for microbial degradation of multiple substrates was able to describe substrate interactions and to model the mixture utilization by strain JS150. Simple competitive, noncompetitive, or uncompetitive substrate kinetics were not sufficient to describe the observed inhibitory interactions.

Use of 16S-rRNA to investigate microbial population dynamics during biodegradation of toluene and phenol by a binary culture.

Interspecies interactions and changes in the rate and extent of biodegradation in mixed culture-mixed substrate studies were investigated. A binary mixed culture of Pseudomonas putida F1 and Burkholderia sp. JS150 degraded toluene, phenol, and their mixture. Both toluene and phenol can serve as sole sources of carbon and energy for both P. putida F1 and strain JS150. To investigate the population dynamics of this system, a fluorescent in-situ hybridization method was chosen because of its ability to produce quantitative data, its low standard error, and the ease of use of this method. When the binary mixed culture was grown on toluene or phenol alone, significant interactions between the species were observed. These interactions could not be explained by a pure-and-simple competition model and were substrate dependent. Strain JS150 growth was slightly inhibited when grown with P. putida F1 on phenol, and P. putida F1 grew more rapidly than expected. Conversely, when the two species were grown together on toluene alone, P. putida F1 was inhibited while strain JS150 was unaffected. During growth of the mixed culture on a combination of toluene and phenol, the interactions were similar to that observed during growth on phenol alone; P. putida F1 growth was enhanced while strain JS150 was unaffected. Because of the observed interspecies interactions, monoculture kinetic parameters were not sufficient to describe the mixed culture kinetics in any experiment. This is one of the first reports of microbial population dynamics in which molecular microbial ecology and mathematical modeling have been combined. The use of the 16S-rRNA-based method allowed for observation and understanding of interspecies interactions that were not observable with standard culture-based methods. These results suggest the need for more investigations that account for both substrate and microbial interactions when predicting the fate of organic pollutants in real systems.

Biodegradation kinetics of benzene, toluene, and phenol as single and mixed substrates for Pseudomonas putida F1.

Although microbial growth on substrate mixtures is commonly encountered in bioremediation, wastewater treatment, and fermentation, mathematical modeling of mixed substrate kinetics has been limited. We report the kinetics of Pseudomonas putida F1 growing on benzene, toluene, phenol, and their mixtures, and compare mathematical models to describe these results. The three aromatics are each able to act as carbon and energy sources for this strain. Biodegradation rates were measured in batch cultivations following a protocol that eliminated mass transfer limitations for the volatile substrates and considered the culture history of the inoculum and the initial substrate to inoculum mass ratio. Toluene and benzene were better growth substrates than phenol, resulting in faster growth and higher yield coefficients. In the concentration ranges tested, toluene and benzene biodegradation kinetics were well described by the Monod model. The Monod model was also used to characterize phenol biodegradation by P. putida F1, although a small degree of substrate inhibition was noted. In mixture experiments, the rate of consumption of one substrate was found to be affected by the presence of the others, although the degree of influence varied widely. The substrates are catabolized by the same enzymatic pathway, but purely competitive enzyme kinetics did not capture the substrate interactions well. Toluene significantly inhibited the biodegradation rate of both of the other substrates, and benzene slowed the consumption of phenol (but not of toluene). Phenol had little effect on the biodegradation of either toluene or benzene. Of the models tested, a sum kinetics with interaction parameters (SKIP) model provided the best description of the paired substrate results. This model, with parameters determined from one- and two-substrate experiments, provided an excellent prediction of the biodegradation kinetics for the three-component mixture.

Species-specific oligonucleotides for enumeration of Pseudomonas putida F1, Burkholderia sp. strain JS150, and Bacillus subtilis ATCC 7003 in biodegradation experiments.

Species-specific sequences were identified within the V4 variable region of 16S rRNA of two bacterial species capable of aromatic hydrocarbon metabolism, Pseudomonas putida F1 and Burkholderia sp. strain JS150, and a third, Bacillus subtilis ATCC 7003, that can function as a secondary degrader. Fluorescent in situ hybridization (FISH) with species-specific oligonucleotides was used for direct counting of these species throughout a phenol biodegradation experiment in batch culture. Traditional differential plate counting methods could not be used due to the similar metabolism and interactions of the primary degraders and difficulties in selecting secondary degraders in mixed culture. In contrast, the FISH method provided reliable quantitative results without interference from those factors.

Integrated approaches for the analysis of toxicologic interactions of chemical mixtures.

Although an overwhelmingly large portion of the resources in toxicologic research is devoted to single chemical studies, the toxicology of chemical mixtures, not single chemicals, is the real issue regarding health effects of environmental and/or occupational exposure to chemicals. The relative lack of activities in the area of toxicology of chemical mixtures does not suggest ignorance of the importance of the issue by the toxicology community. Instead, it is a reflection of the difficulty, complexity, and controversy surrounding this area of research. Until recently, much of the literature on the toxicology of chemical mixtures has been either very focused on certain specific interaction studies or slanted toward broad-based, relatively vague theoretical deliberation. The typical interaction study involved binary mixtures at relatively high dose levels with acute toxicities as endpoints. Although the theoretical papers have been valuable contributions, little is available on actual, practical experimental approaches toward a systematic solution of this immensely complex area of research. We present here a broad discussion on the important issues of the toxicology of chemical mixtures. First, we provide some background information with respect to the problem and significance of toxicology of chemical mixtures in relation to some of the real life issues. Second, we review and compare the existing experimental approaches relevant to toxicologic interactions of chemical mixtures. Third, we propose three integrated approaches that involve the combination of physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) modeling with: (1) Monte Carlo simulation, (2) median effect principle (MEP), and (3) response surface methodology (RSM). These modeling approaches, coupled with very focused mechanistically based toxicology studies, could be the basis for solving the problems of toxicology and risk assessment of chemical mixtures.

A proposed approach to study the toxicology of complex mixtures of petroleum products: the integrated use of QSAR, lumping analysis and PBPK/PD modeling.

Mixture toxicity is a topic that has become a matter of concern during the last two decades. One of the major problems with assessing the toxicity of mixtures and the associated human and environmental risk is the large number of possible mixtures, as well as the fact that the actual mixture effect for a given set of constituents might strongly depend on the actual composition of the mixture, i.e., the ratios of the constituent, as well as their nature. This paper presents a possible approach to describe and thereby better understand the pharmacokinetics and dynamics of complex mixtures by combining quantitative structure-activity relationships to predict needed parameters, lumping to reduce the complexity of the problem, and physiologically based pharmacokinetic/pharmacodynamic modeling to integrate all this information into a complete toxicological description of the mixture. It is our hope that by presenting this conceptual approach we might be able to stimulate some criticisms and discussions in the toxicology community regarding this complex and yet very important area of research.

A gas-liquid system for enzyme kinetic studies of volatile organic chemicals. Determination of enzyme kinetic constants and partition coefficients of trichloroethylene.

A gas-liquid system was developed for enzyme kinetic study with volatile organic chemicals (VOCs) by modification of the gas uptake method for the in vivo physiologically based pharmacokinetic experiment. This gas-liquid system, designed in our laboratory, is composed of: 1) a diffusion chamber for adjusting initial vapor concentration by mixing ambient air and the VOCs; 2) a condenser for maintaining the liquid level in the incubation chamber; 3) a stainless-steel metal bellows pump for recirculating vapor in this system; 4) a gas chromatograph equipped with an autosampler and a flame ionization detector; and 5) a computer for controlling automation and data processing. Trichloroethylene (TCE) was used as a model chemical, and enzyme kinetics were studied by measuring the depletion of TCE in the gas phase of the system. TCE-at initial concentrations of 56, 620, and 1240 ppm-was incubated with rat liver microsomes and a NADPH regenerating system in a 100-ml round-bottom flask. Based on parallel enzyme assays using p-nitrophenol as a substrate, cytochrome P450IIE1, activity remained stable up to 3 hr under the incubation conditions (37 degrees C and pH 7.4) whereas addition of glutathione into the incubation mixture did not affect TCE metabolism. Kinetic constants were analyzed using a two-compartment pharmacokinetic model and the computer software SimuSolv. Statistical optimization using the maximum-likelihood method produced apparent in vitro Vmax and KM values of 0.55 nmol/mg protein/min and 0.9 microM, respectively. In addition, this newly developed methodology has a number of advantages over those reported in the literature, including the potential utility of determining tissue partition coefficients of VOCs for physiologically based pharmacokinetic modeling. We conclude that this gas-liquid system is suitable for determination of kinetic constants near realistic environmental concentrations of VOCs including TCE.

A bioreactor system for the nitrogen loop in a Controlled Ecological Life Support System.

As space missions become longer in duration, the need to recycle waste into useful compounds rises dramatically. This problem can be addressed by the development of Controlled Ecological Life Support Systems (CELSS) (i.e., Engineered Closed/Controlled Eco-Systems (ECCES)), consisting of human and plant modules. One of the waste streams leaving the human module is urine. In addition to the reclamation of water from urine, recovery of the nitrogen is important because it is an essential nutrient for the plant module. A 3-step biological process for the recycling of nitrogenous waste (urea) is proposed. A packed-bed bioreactor system for this purpose was modeled, and the issues of reaction step segregation, reactor type and volume, support particle size, and pressure drop were addressed. Based on minimization of volume, a bioreactor system consisting of a plug flow immobilized urease reactor, a completely mixed flow immobilized cell reactor to convert ammonia to nitrite, and a plug flow immobilized cell reactor to produce nitrate from nitrite is recommended. It is apparent that this 3-step bioprocess meets the requirements for space applications.

Parameters influencing the productivity of recombinant E. coli cultivations.

In the past 10 to 15 years, many of the promises of microbial genetic engineering have been realized: the use of recombinant Escherichia coli has moved from the laboratory to the production facility, and the manufacture of therapeutic recombinant proteins such as human growth hormone and interleukins is a rapidly growing industry. Along with this progress, however, have come new problems to solve: bioreactor operators have discovered that large-scale cultivations of plasmid-containing bacteria do not behave in exactly the same way as those of plasmid-free cells, plasmid stability has been recognized as a major hurdle, and the protein product might not be present in a soluble form but rather as intracellular granules that resist solubilization. These and other difficulties represent a new generation of challenges for genetic engineering. However, genetic engineering can do more than solve these problems. Molecular biological techniques also have the ability to create new opportunities: to produce new compounds, to use cheaper substrates, to facilitate downstream processing, and to optimize production in new ways. The productivity of a cultivation can generally be expressed as the product of the cell density and the specific biological activity. Both of these parameters are influenced by a variety of factors. For recombinant cultivations, though, the level of biological activity, a reflection of the plasmid copy number and expression efficiency, is the more interesting and important consideration and will therefore be given more attention in our review. In this contribution, our general goal is to discuss the factors that influence the productivity of recombinant E. coli cultivations, covering parameters relating to DNA; parameters relating to protein synthesis; parameters relating to proteins; and parameters relating to downstream processing. The object is not to tell the reader how to choose the perfect plasmid, host, and cultivation conditions, but to make known the many variables involved in designing a recombinant process and to point out recent and potential advances made possible by genetic engineering. The discussion focuses on the production of a protein, but many of the same concepts apply to other cultivations of recombinant E. coli, including cases in which the desired product is not a protein or the cells have been designed for a special metabolic capability such as pollutant biodegradation.

Immuno- and flow cytometric analytical methods for biotechnological research and process monitoring.

In this article, the applications of immunoanalysis and flow cytometry for research and process monitoring in biotechnology are discussed. Brief reviews of the two analytical methods are followed by descriptions of actual applications in various areas of biotechnology. In the case of immunoanalysis, emphasis is placed on systems for on-line bioprocess monitoring, and examples are given for a thermostable pullulanase, a mouse IgG, and antithrombin III. Although flow cytometry is not currently an on-line analytical technique, its value as an off-line method is illustrated by examples of the measurement of shear stress effects, lipid content, and sterol content.