Microbial Fermentation Process Research Articles

Overproduction of free fatty acids in E. coli: Implications for biodiesel production

Whereas microbial fermentation processes for producing ethanol and related alcohol biofuels are well established, biodiesel (methyl esters of fatty acids) is exclusively derived from plant oils. Slow cycle times for engineering oilseed metabolism and the excessive accumulation of glycerol as a byproduct are two major drawbacks of deriving biodiesel from plants. Although most bacteria produce fatty acids as cell envelope precursors, the biosynthesis of fatty acids is tightly regulated at multiple levels. By introducing four distinct genetic changes into the E. coli genome, we have engineered an efficient producer of fatty acids. Under fed-batch, defined media fermentation conditions, 2.5 g/L fatty acids were produced by this metabolically engineered E. coli strain, with a specific productivity of 0.024 g/h/g dry cell mass and a peak conversion efficiency of 4.8% of the carbon source into fatty acid products. At least 50% of the fatty acids produced were present in the free acid form.

Development of a Robust, Versatile, and Scalable Inoculum Train for the Production of a DNA Vaccine

For many microbial fermentation processes, the inoculum train can have a substantial impact on process performance in terms of productivity, profitability, and process control. In general, it is understood that a well-characterized and flexible inoculum train is essential for future scale-up and implementation of the process in a pilot plant or manufacturing setting. A fermentation process utilizing E. coli DH5 for the production of plasmid DNA carrying the HIV gag gene for use as a vaccine is currently under development in our laboratory. As part of the development effort, we evaluated inoculum train schemes that incorporate one, two, or three stages. In addition, we investigated the effect of inoculum viable-cell concentrations, either thawed or actively growing, over a wide range (from 2.5 x 10(4) to 1.0 x 10(8) viable cells/mL or approximately 0.001% to 4% of final working volume). The various inoculum trains were evaluated in terms of final plasmid yield, process time, reproducibility, robustness, and feasibility at large scale. The results of these studies show that final plasmid yield remained in the desired range, despite the number of stages or inoculation viable-cell concentrations comprising the inoculum train. On the basis of these observations and because it established a large database, the first part of these investigations supports an exceptional flexibility in the design of scalable inoculum trains for this DNA vaccine process. This work also highlighted that a slightly higher level of process reproducibility, as measured by the time for the culture to reach mid-exponential growth, was observed when using actively growing versus frozen cells. It also demonstrated the existence of a viable-cell concentration threshold for the one-stage process, since we observed that inoculation of the production stage with very low amounts of viable cells from a frozen source could lead to increased process sensitivity to external factors such as variation in the quality of the raw materials used in the medium formulation. However, our analysis indicates that, despite this slight disadvantage, a one-stage inoculum train was a viable option in many situations, especially if the inoculation viable-cell concentration was kept above 4.8 x 10(6) viable cells/mL. Because it leads to a reduction in process steps and eliminates some capital investments (i.e., inoculum fermenter), when feasible a one-stage process configuration will positively impact process economics.

Harnessing plant biomass for biofuels and biomaterials

Harnessing plant biomass for biofuels and biomaterials

Thermal profiling for parallel on-line monitoring of biomass growth in miniature stirred bioreactors

Recently we have described the design and operation of a miniature bioreactor system in which 4-16 fermentations can be performed (Gill et al., Biochem Eng J 39:164-176, 2008). Here we report on the use of thermal profiling techniques for parallel on-line monitoring of cell growth in these bioreactors based on the natural heat generated by microbial culture. Results show that the integrated heat profile during E. coli TOP10 pQR239 fermentations followed the same pattern as off-line optical density (OD) measurements. The maximum specific growth rates calculated from off-line OD and on-line thermal profiling data were in good agreement, at 0.66+/-0.04 and 0.69+/-0.05 h(-1) respectively. The combination of a parallel miniature bioreactor system with a non-invasive on-line technique for estimation of culture kinetic parameters provides a valuable approach for the rapid optimisation of microbial fermentation processes.

Microbial fermented tea – a potential source of natural food preservatives

Antimicrobial activities of microbial fermented tea are much less known than its health beneficial properties. These antimicrobial activities are generated in natural microbial fermentation process with tea leaves as substrates. The antimicrobial components produced during the fermentation process have shown inhibitory effects against several food-borne and pathogenic bacteria. With the trend of increasing use of natural and biological preservatives in food products, natural antimicrobial agents from microbial fermented tea may offer an innovative and interesting measure for such applications. However, a breakthrough in this field can only be realised after several critical aspects are clarified and further studied. Only then, the application of these potential, novel and natural antimicrobial substances from microbial fermented tea can be industrialized. The present review describes some unique microbial fermentation of tea and the antimicrobial activities formed during the fermentation process. Moreover, future needs in research and development of these antimicrobial compounds from microbial fermentation of tea are discussed for potential industrial applications.

Design and characterisation of a miniature stirred bioreactor system for parallel microbial fermentations

The establishment of a high productivity microbial fermentation process requires the experimental investigation of many interacting variables. In order to speed up this procedure a novel miniature stirred bioreactor system is described which enables parallel operation of 4–16 independently controlled fermentations. Each miniature bioreactor is of standard geometry (100 mL maximum working volume) and is fitted with a magnetically driven six-blade miniature turbine impeller ( d i = 20 mm, d i/ d T = 1/3) operating in the range 100–2000 rpm. Aeration is achieved via a sintered sparger at flow rates in the range of 0–2 vvm. Continuous on-line monitoring of each bioreactor is possible using miniature pH, dissolved oxygen and temperature probes, while PC-based software enables independent bioreactor control and real-time visualisation of parameters monitored on-line. In addition, a new optical density probe is described that enables on-line estimation of biomass growth kinetics without the need for repeated sampling of individual bioreactors. Initial characterisation of the bioreactor involved quantification of the volumetric oxygen mass transfer coefficient as a function of agitation and aeration rates. The maximum k L a value obtained was 0.11 s −1. The reproducibility of E. coli TOP10 pQR239 and B. subtilis ATCC6633 fermentations was shown in four parallel fermentations of each organism. For E. coli (1000 rpm, 1 vvm) the maximum specific growth rate, μ max, was 0.68 ± 0.01 h −1 and the final biomass concentration obtained, X final, was 3.8 ± 0.05 g L −1. Similarly for B. subtilis (1500 rpm, 1 vmm) μ max was 0.45 ± 0.01 h −1 and X final was 9.0 ± 0.06 g L −1. Biomass growth kinetics increased with increases in agitation and aeration rates and the oxygen enrichment for control of DOT levels enabled μ max and X final as high as 0.93 h −1 and 8.1 g L −1 respectively to be achieved. Preliminary, scale-up studies with E. coli in the miniature bioreactor (100 mL working volume) and a laboratory scale 2 L bioreactor (1.5 L working volume) were performed at matched k L a values. Very similar growth kinetics were observed at both scales giving μ max values of 0.94 and 0.97 h −1, and X final values of 5.3 and 5.5 g L −1 respectively. The miniature bioreactor system described here thus provides a useful tool for the parallel evaluation and optimisation of microbial fermentation processes.

The scale-up of microbial batch and fed-batch fermentation processes

Publisher Summary The scale-up of single-celled aerobic microbial fermentation processes is complicated that can lead to unpredictable process performance. However, this is not due to the introduction of fluid dynamic generated stresses (or so-called “shear damage”), whether arising from agitator generated turbulence or bursting bubbles, rather it is because the large-scale fed-batch bioreactor provides a very dynamic environment with large spatial and temporal heterogeneities. Such environmental heterogeneities can induce multiple physiological responses in cells. These responses consume energy and resources such that biomass concentration and product yields can be reduced. These phenomena are not observed in well-mixed homogeneous laboratory-scale reactors where much process development is done and their effects are difficult to model mathematically. Therefore, the ability to obtain data on how a recombinant laboratory process may perform at the large scale, dependent on feeding regime employed or controlling action taken is invaluable for any detailed and informed development program.

A Microstructure Heat Exchanger for Quenching the Metabolism of Mammalian Cells

AbstractMetabolomics, the quantification of intracellular metabolites, is a powerful tool that has proven useful in microbial strain and fermentation process development. The crucial step is the rapid quenching of the cellular metabolism to preserve the intracellular situation as it is in the bioreactor until (MS‐based) analysis. For microbial systems and plants this can be achieved by using ice‐cold methanol (–40 °C) or liquid nitrogen, but for mammalian cells in suspension no reliable technique is yet available. Here, a metallic microstructure heat exchanger is presented which allows cooling of a CHO cell suspension from cultivation temperature (37 °C) to 0 °C in less than one second without any detectable cell damage or cell disruption even at very high cell densities up to 8 · 106 cells/mL.

Development of a microbial fermentation process for detoxification of gossypol in cottonseed meal

The objective was to study effects of selected fungi, carbohydrate sources, urea, heat treatment and minerals on the reduction of free gossypol (FG) levels during solid substrate fermentation of cottonseed meal (CSM), and to determine the crude protein (CP) and amino acid (AA) content as well as the in vitro digestible CP and AA of CSM substrate fermented under optimal conditions. Six groups of autoclaved CSM substrate were incubated for 48 h after inoculation with either of the fungi Candida capsuligena ZD-1, Candida tropicalis ZD-3, Saccharomyces cerevisae ZD-5, Aspergillus terricola ZD-6, Aspergillus oryzae ZD-7, or Aspergillus niger ZD-8. One non-inoculated group was the control. Levels of initial and final FG and CP were assayed. Results indicated that microbial fermentation could greatly decrease FG levels in CSM, but it differed among species of microorganisms with C. tropicalis ZD-3 the most effective. The CSM substrate was supplemented with carbohydrate sources, urea and minerals, and one group of non-autoclaved CSM substrate was used to investigate effects of heat treatment. The treated CSM substrates were inoculated with C. tropicalis ZD-3 and incubated for 24 or 48 h. Results showed that CSM substrate supplemented with starch and sucrose enhanced detoxification of gossypol, but heat treatment and minerals were also effective in reducing FG levels during solid substrate fermentation of CSM. The detoxification effect with minerals was the best among treatments.

Some methodological and analytical considerations regarding application of the gas production technique

The in vitro gas production technique is used widely in animal nutrition for feed evaluation and to study the kinetics of microbial fermentation processes in the digestive tract. This technique is based on the assumption that gas produced in batch cultures inoculated with mixed microorganisms from ruminal or hindgut contents or faeces is directly related to the amount of substrate fermented. The technique relies on mathematical modelling to estimate the rate and extent of feed digestion from cumulative gas production profiles. Section 1 of this article deals with the application of gas laws to calculate the volume of gas produced from pressure measurements when batch cultures are incubated in gas-tight bottles. Calculation of the amount of a specific gas (methane) produced at a given incubation time from total gas production and composition is presented in Section 2. Then, the definition of specific rate of substrate degradation and that of gas production are considered. Next, a piecewise linear model based on the assumption that microbial growth and fermentation follows zero-order kinetics is derived and evaluated by comparison with other non-linear models with regard to goodness-of-fit of experimental data and estimation of rate and extent of degradation in the rumen of some feedstuffs. Finally, the possibility of using procedures to account for the inevitable cyclic trends in gas production profiles by data smoothing are discussed. All these considerations provide an insight into some of the mathematical and methodological aspects of the in vitro gas production technique and should contribute to facilitating the description and interpretation of experimental data.

Characterization and Application of a Miniature 10 mL Stirred-Tank Bioreactor, Showing Scale-Down Equivalence with a Conventional 7 L Reactor

The aim of this study was to characterize the engineering environment of an instrumented 10 mL miniature stirred-tank bioreactor and evaluate its potential as a scale-down device for microbial fermentation processes. Miniature bioreactors such as the one detailed in this work have been developed by several research groups and companies and seek to address the current bottleneck at the screening stage of bioprocess development. The miniature bioreactor was characterized in terms of overall volumetric oxygen transfer coefficient and mixing time over a wide range of impeller speeds. Power input to the miniature bioreactor was directly measured, and from this the power number of each impeller was calculated and specific power input estimated, allowing the performance of the miniature bioreactor to be directly compared with that of a conventional 7 L bioreactor. The capability of the miniature bioreactor to carry out microbial fermentations was also investigated. Replicate batch fermentations of Escherichia coli DH5alpha producing plasmid DNA were performed at equal specific power input, under fully aerobic and oxygen-limiting conditions. The results showed a high degree of equivalence between the two scales with regard to growth and product kinetics. This was underlined by the equal maximum specific growth rate and equal specific DNA product yield on biomass obtained at the two scales of operation, demonstrating the feasibility of scaling down to 10 mL on the basis of equivalent specific power input.

Microbial community analysis of a biogas-producing completely stirred tank reactor fed continuously with fodder beet silage as mono-substrate

The bioconversion of renewable raw material to biogas by anaerobic microbial fermentation processes in completely stirred tank reactors (CSTR) is a valuable alternative resource of energy especially for rural areas. However, knowledge about the microorganisms involved in the degradation of plant biomass is still poor. In this study, a first analysis of the biogas-forming process within a CSTR fed continuously with fodder beet silage as mono-substrate is presented in the context of molecular data on the microbial community composition. As indicated by the conventional process parameters like pH value, content of volatile fatty acids, N:P ratio and the biogas yield, the biogas-forming process within the CSTR occurred with a stable and efficient performance. The average biogas yield based on volatile solids was 0.87 m 3 kg −1 at an organic loading rate of 1.2–2.3 kg m −3 d −1. This amounts to 94% of the theoretical maximum. In order to identify microorganisms within the CSTR, a 16S rDNA clone library was constructed by PCR amplification applying a prokaryote-specific primer set. One hundred and forty seven clones were obtained and subsequently characterized by amplified rDNA restriction analysis (ARDRA). The sequences of 60 unique ARDRA patterns were estimated in a length of approximately 800–900 bp each. Four of them were assigned to the domain Archaea and 56 to the domain Bacteria. Within the domain Archaea, all clones showed a close relationship to methanogenic species. Major bacterial groups represented in the clone library were the class Clostridia of the phylum Firmicutes (22% of all 16S rDNA clones), the class Deltaproteobacteria of the phylum Proteobacteria (24%), the class Bacilli of the phylum Firmicutes (22%) and members of the phylum Bacteroidetes (21%). Within these major groups, the highest biodiversity was found within the class Clostridia (35% of all operational taxonomic units). Members of the phyla Actinobacteria and Spirochaetes were represented only by 5 and 2 clonal sequences, respectively.

Modeling of the pyruvate production with Escherichia coli: comparison of mechanistic and neural networks-based models

Three different models: the unstructured mechanistic black-box model, the input-output neural network-based model and the externally recurrent neural network model were used to describe the pyruvate production process from glucose and acetate using the genetically modified Escherichia coli YYC202 ldhA::Kan strain. The experimental data were used from the recently described batch and fed-batch experiments [ Zelić B, Study of the process development for Escherichia coli-based pyruvate production. PhD Thesis, University of Zagreb, Faculty of Chemical Engineering and Technology, Zagreb, Croatia, July 2003. (In English); Zelić et al. Bioproc Biosyst Eng 26:249-258 (2004); Zelić et al. Eng Life Sci 3:299-305 (2003); Zelić et al Biotechnol Bioeng 85:638-646 (2004)]. The neural networks were built out of the experimental data obtained in the fed-batch pyruvate production experiments with the constant glucose feed rate. The model validation was performed using the experimental results obtained from the batch and fed-batch pyruvate production experiments with the constant acetate feed rate. Dynamics of the substrate and product concentration changes was estimated using two neural network-based models for biomass and pyruvate. It was shown that neural networks could be used for the modeling of complex microbial fermentation processes, even in conditions in which mechanistic unstructured models cannot be applied.

Fusion of Mass Spectrometry-Based Metabolomics Data

A general method is presented for combining mass spectrometry-based metabolomics data. Such data are becoming more and more abundant, and proper tools for fusing these types of data sets are needed. Fusion of metabolomics data leads to a comprehensive view on the metabolome of an organism or biological system. The ideas presented draw upon established techniques in data analysis. Hence, they are also widely applicable to other types of X-omics data provided there is a proper pretreatment of the data. These issues are discussed using a real-life metabolomics data set from a microbial fermentation process.

Optimization of submerged culture requirements for the production of mycelial growth and exopolysaccharide by Cordyceps jiangxiensis JXPJ 0109.

The objective of the present study was to investigate the optimal culture requirements for mycelial growth and exopolysaccharide production by Cordyceps jiangxiensis JXPJ 0109 in submerged culture. The effects of medium ingredients (i.e. carbon and nitrogen sources, and growth factor) and other culture requirements (i.e. initial pH, temperature, etc.) on the production of mycelia and exopolysaccharide were observed using a one-factor-at-a-time method. More suitable culture requirements for mycelial growth and exopolysaccharide production were proved to be maltose, glycerol, tryptone, soya bean steep powder, yeast extract, medium capacity 200 ml in a 500-ml flask, agitation rate 180 rev min(-1), seed age 4-8 days, inoculum size 2.5-7.5% (v/v), etc. The optimal temperatures and initial pHs for mycelial growth and exopolysaccharide production were at 26 degrees C and pH 5 and at 28 degrees C and pH 7, respectively, and corresponding optimal culture age were observed to be 8 and 10 days respectively. According to the primary results of the one-factor-at-a-time experiments, the optimal medium for the mycelial growth and exopolysaccharide production were obtained using an orthogonal layout method to optimize further. Herein the effects of medium ingredients on the mycelial growth of C. jiangxiensis JXPJ 0109 were in the order of yeast extract > tryptone > maltose > CaCl2 > glycerol > MgSO4 > KH2PO4 and the optimal concentration of each composition was 15 g maltose (food-grade), 10 g glycerol, 10 g tryptone, 10 g yeast extract, 1 g KH2PO4, 0.2 g MgSO4, and 0.5 g CaCl2 in 1 l of distilled water, while the order of effects of those components on exopolysaccharide production was yeast extract > maltose > tryptone > glycerol > KH2PO4 > CaCl2 > MgSO4, corresponding to the optimal concentration of medium was as follows: 20 g maltose (food-grade), 8 g glycerol, 5 g tryptone, 10 g yeast extract, 1 g KH2PO4, and 0.5 g CaCl2 in 1 l of distilled water. Under the optimal culture requirements, the maximum exopolysaccharide production reached 3.5 g l(-1) after 10 days of fermentation, while the maximum production of mycelial growth achieved 14.5 g l(-1) after 8 days of fermentation. This is the first report on the submerged culture requirements for mycelial growth and exopolysaccharide in C. jiangxiensis, and this two-step optimization strategy in this study can be widely applied to other microbial fermentation processes.

Overproduction of thymidine by recombinant brevibacterium helvolum amplified with thymidine monophosphate phosphohydrolase gene from bacteriophage PBS2.

A microbial fermentation process could be used to produce thymidine biologically but many of the enzymes related to nucleotide biosynthesis are highly regulated. To overcome the complex regulation steps, an analogue mutant of Brevibacterium helvolum resistant to fluorouracil, hydroxyurea, and trimethoprim was constructed. This mutant accumulated 380 mg thymidine 1(-1) in 16 h in shake-flask culture. However, the accumulation of thymidine monophosphate (TMP) inside the cells suggested a low activity of nucleotidase which degrades TMP to thymidine. This limitation was overcome by cloning the TMP phosphohydrolase (TMPase) gene of the unusual bacteriophage, PBS2. As a result, TMP in recombinant cells decreased from 230 micromol g(-1) cell to 20 micromol g(-1) cell with accumulation of 500 mg thymidine l(-1) in the medium.

Vitamin B3

This chapter is a short review of the production of vitamins with the main focus on the microbial fermentation processes that are currently being employed in the production of vitamins. The state of the art of the industry of vitamins is extensively analysed with a description of both chemical and biotechnological systems that have been developed for the production of each vitamin. Finally, future trends in the microbial production of vitamins are analysed and the recent advances in strain design for whole-cell production of vitamins are discussed.

Population genetics of nine STR loci in the Turkish population

This chapter is a short review of the production of vitamins with the main focus on the microbial fermentation processes that are currently being employed in the production of vitamins. The state of the art of the industry of vitamins is extensively analysed with a description of both chemical and biotechnological systems that have been developed for the production of each vitamin. Finally, future trends in the microbial production of vitamins are analysed and the recent advances in strain design for whole-cell production of vitamins are discussed.

Hematopoietic growth factor mimetics.

Hematopoietic growth factors are glycoproteins of 15-70 kDa. Although much clinical success has been obtained using recombinant proteins produced in mammalian cell lines and in microbial fermentation processes, the full-length polypeptides necessarily are expensive to produce, require parenteral administration, and in some cases have provoked detrimental immune responses. With the availability of high throughput biological function and receptor binding assays it has become possible to screen millions, if not billions, of randomly produced organic compounds and relatively short peptides to identify lead compounds for the development of small molecular mimetics of hematopoietic growth factors. Herein the strategies used to screen libraries of small molecules and peptides and the successes in finding mimetics and antagonists for/to erythropoietin, granulocyte colony-stimulating factor, and thrombopoietin are reviewed. Finally, the structural study of mimetic-receptor complexes has provided us with many molecular details of growth factor-induced receptor activation and is likely to yield new insights into the molecular basis of hematopoietic signal transduction.

Capillary electrophoresis in determination of inorganic anions in microbial fermentation processes; comparison with high-performance liquid chromatography

Capillary electrophoresis (CE) and isocratic high-performance liquid chromatographic (HPLC) methods were optimized and compared for the analysis of several inorganic anions using ultraviolet (UV) and refractive index (RI) detectors, respectively. The chromatographic methods were then applied to the analysis of samples from fermentation processes aimed at microbial purification of flue gases, in particular, anions originating from sulfur and nitrogen oxides. In the CE method, the concentration of electrolyte had a notable effect on the resolution, migration order, and migration times of the anions. The best resolution was achieved in 10 mM chromate with 0.5 mM tetradecyltrimethyl-ammonium bromide (TTAB) as S2O32–, Cl–, NO2–, NO3–, SO42–, SO32–, PO43–, and CO32– were separated within 10 min. With the HPLC method (ION-120 anion analysis column), the combination of 3 mM salicylic acid and 5 mM Tris showed the best resolution for nitrate and nitrite. Sulfite and sulfate did not separate from each other under any of the conditions tested while the total time of chromatography was 25 min. The UV detection of CE was about 10 times more sensitive than the RI detection used in HPLC. The reproducibility of quantitative analysis data with HPLC was generally slightly better than with CE. A distinct advantage of CE is that pretreatment of samples is unnecessary.