Equipment for the automatic aseptic inoculation and sampling of microbial cultures

Equipment has been designed and constructed for the automatic aseptic inoculation and sampling of microbial cultures. It enables fermentations to be started and samples to be taken round-the-clock, according to a predetermined program. The equipment is suitable for microbial processes on both a laboratory and a factory scale.     

Identification of critical batch operating parameters in fed-batch recombinant E. coli fermentations using decision tree analysis

To develop a useful fermentation process model, it is first necessary to identify which batch operating parameters are critical in determining the process outcome. To identify critical processing inputs in large databases, we have explored the use of Decision Tree Analysis with the decision metrics of Gain (i.e., Shannon Entropy changes), Gain Ratio, and a multiple hypergeometric distribution. The usefulness of this approach lies in its ability to treat "categorical" variables, which are typical of archived fermentation databases, as well as "continuous" variables. In this work, we demonstrate the use of Decision Tree Analysis for the problem of optimizing recombinant green fluorescent protein production in E. coli. A database of 85 fermentations was generated to examine the effect of 15 process input parameters on final biomass yield, maximum recombinant protein concentration, and productivity. The use of Decision Tree Analysis led to a considerable reduction in the fermentation database through the identification of the significant as well as insignificant inputs. However, different decision metrics selected different inputs and different numbers of inputs to classify the data for each output.     

Optimization of batch fermentation processes. I. Development of mathematical models for batch penicillin fermentations

Two kinds of mathematical models have been developed for batch penicillin fermentations: (1) general models, based on averaged, nondimensionalized cell and penicillin synthesis curves from plant, scale fermentors and (2) particular models developed from specific sets of experimental data from two sources. Parameter-temperature functions used with the general models were assumed to have general shapes which could apply to many fermentations, i.e., they were based on the familiar temperature response of enzyme-catalyzed reactions. Parameter-temperature functions for the particular models were determined from experimental data for batch runs at various temperatures.     

Modeling oxygen dissolution and biological uptake during pulse oxygen additions in oenological fermentations

Discrete oxygen additions during oenological fermentations can have beneficial effects both on yeast performance and on the resulting wine quality. However, the amount and time of the additions must be carefully chosen to avoid detrimental effects. So far, most oxygen additions are carried out empirically, since the oxygen dynamics in the fermenting must are not completely understood. To efficiently manage oxygen dosage, we developed a mass balance model of the kinetics of oxygen dissolution and biological uptake during wine fermentation on a laboratory scale. Model calibration was carried out employing a novel dynamic desorption-absorption cycle based on two optical sensors able to generate enough experimental data for the precise determination of oxygen uptake and volumetric mass transfer coefficients. A useful system for estimating the oxygen solubility in defined medium and musts was also developed and incorporated into the mass balance model. Results indicated that several factors, such as the fermentation phase, wine composition, mixing and carbon dioxide concentration, must be considered when performing oxygen addition during oenological fermentations. The present model will help develop better oxygen addition policies in wine fermentations on an industrial scale.     

Comparison of different options for harvest of a therapeutic protein product from high cell density yeast fermentation broth

Recovery of therapeutic protein from high cell density yeast fermentations at commercial scale is a challenging task. In this study, we investigate and compare three different harvest approaches, namely centrifugation followed by depth filtration, centrifugation followed by filter-aid enhanced depth filtration, and microfiltration. This is achieved by presenting a case study involving recovery of a therapeutic protein from Pichia pastoris fermentation broth. The focus of this study is on performance of the depth filtration and the microfiltration steps. The experimental data has been fitted to the conventional models for cake filtration to evaluate specific cake resistance and cake compressibility. In the case of microfiltration, the experimental data agrees well with flux predicted by shear induced diffusion model. It is shown that, under optimal conditions, all three options can deliver the desired product recovery ( >80%), harvest time ( <15 h including sequential concentration/diafiltration step), and clarification ( <6 NTU). However, the three options differ in terms of process development time required, capital cost, consumable cost, ease of scale-ability and process robustness. It is recommended that these be kept under consideration when making a final decision on a harvesting approach.     

Morphology and viability analysis of<Emphasis Type="Italic"> Streptomyces clavuligerus</Emphasis> in industrial cultivation systems

The effects of varying inoculum age and production scale upon the morphology and viability of Streptomyces clavuligerus were studied by analyzing visible and fluorescent light images acquired throughout pilot-plant and pre-industrial scale fermentations. Changes in production scale reveal that in 5 m³ fermentors, the maximum hyphal area obtained is double the value obtained in 0.5 m³ fermentors. It is probably due to the higher shear stresses acting upon hyphae in the 0.5 m³ fermentor caused by higher tip speeds observed in these. The morphological quantification based on elongation and branching rates allowed fermentations to be pattern classified into distinct physiological time zones namely elongation, branching, fragmentation, etc. The general pattern observed for fermentations inoculated with late exponential phase inocula was similar to the pattern of fermentations run with stationary phase inocula except that both the elongation and branching periods started earlier in the former case. Using the available staining technique and image acquisition system, the viability seemed to be generally high and constant throughout the time course of all the studied fermentations.An erratum to this article can be found at     

Genomic characterization and selection of wine yeast to conduct industrial fermentations of a white wine produced in a SW Spain winery

Aims: To analyse the diversity of wild yeast in spontaneous fermentations of a white wine and to select the most suitable autochthonous starter yeasts. The selected yeasts would be used for inoculation of industrial fermentations in several years. Methods and Results: Yeasts were characterized by applying electrophoretic karyotyping. This technique was chosen because it can reveal the large‐scale mutations in the yeast genome induced by gross chromosomal rearrangements. This type of mutation is considered one of the main forces behind the rapid evolution of industrial yeasts. A heterogeneous population of yeast strains was observed in the spontaneous fermentations during two consecutive years. Four of the most abundant strains were isolated and tested for microbiological features of industrial importance. The selected autochthonous strains were used as starter yeasts for the following 7 years. In the majority of these experiences, we obtained homogeneous yeast populations, in which the karyotype of one of the inoculated strains – karyotype V – emerged as clearly dominant. Conclusions: The inoculation of the selected strain with karyotype V and a proper handling of the inoculum scaling‐up process led to the substitution of the spontaneous fermentations by controlled fermentations producing a highly satisfactory final product. Significance and Impact of the Study: We monitored the wine yeast population of an industrial system for a total of 9 years. Our work is one of the first examples made at industrial scale showing how molecular techniques can be successfully applied to improve the efficiency of the winemaking process.     

Estuary ecosystem restoration: implementing and institutionalizing adaptive management

We implemented and institutionalized an adaptive management (AM) process for the Columbia Estuary Ecosystem Restoration Program, which is a large‐scale restoration program focused on improving ecosystem conditions in the 234‐km lower Columbia River and estuary. For our purpose, “institutionalized” means the AM process and restoration programs are embedded in the work flow of the implementing agencies and affected parties. While plans outlining frameworks, processes, or approaches to AM of ecosystem restoration programs are commonplace, their establishment for the long‐term is not. This article presents the basic AM process and explains how AM was implemented and institutionalized. Starting with a common goal, we pursued a well‐understood governance and decision‐making structure, routine coordination and communication activities, data and information sharing, commitment from partners and upper agency management to the AM process, and meaningful cooperation among program managers and partners. The overall approach and steps to implement and institutionalize AM for ecosystem restoration explained here are applicable to situations in which it has been incomplete or, as in our case, the restoration program is just getting started.     

Evaluation of high butanol/acetone ratios in ABE fermentations with cassava by graph theory and NADH regeneration analysis

Higher butanol/acetone ratio is always desirable in ABE fermentation, and this ratio is closely associated with the complicated patterns of metabolic reactions and NADH generation rate. The patterns of acetate/butyrate formation and re-assimilation in multiple closed reaction loops, as well as NADH regeneration in ABE fermentation using different substrates varies. In this study, we evaluated butanol/acetone ratio in ABE fermentations utilizing cassava and corn based media by graph theory and NADH regeneration analysis. The theoretical calculations and experimental data revealed that a lower metabolic strength in butyrate loop and enhanced NADH generation rate were responsible for the achievement of higher butanol/acetone ratio when fermenting cassava based substrate. In traditional fermentations and extractive fermentations with oleyl alcohol/bio-diesel as the extractants when using cassava based substrate, butanol/acetone ratios reached 2.24, 2.84, and 2.19 with the increasing increments of 14.9, 61.4, and 6.8% respectively, while butanol productivities stayed at comparably high levels as compared with those of the fermentations when cultivating on corn based substrate.     

Solid state fermentation—An overview

Since ancient times many solid state fermentations have utilized fungi and bacteria, almost always in mixed culture. The discovery and development of penicillin led to extensive use of liquid fermentations using actinomycetes and fungi and to subsequent neglect of research on solid state fermentations and the use of mixed cultures. This paper reviews the types of solid state fermentations, equipment used, products made, as well as the advantages and disadvantages of solid state fermentations. Products resulting from old and new solid and liquid substrate fermentations are enumerated.     

Immobilized Biocatalysts for the Production of Nucleosides and Nucleoside Analogues by Enzymatic Transglycosylation Reactions

The recombinant enzymes uridine phosphorylase (UP) and purine nucleoside phosphorylase (PNP) were over-expressed in high-biomass bacterial fermentations and co-immobilized, without previous purification, on epoxy-activated solid supports by covalent linkages. These preparations are efficient biocatalysts of transglycosylation reactions and have been developed for producting natural and modified nucleosides of pharmaceutical interest in the field of antiviral and antitumoral agents. The new biocatalysts described in this work are suitable for both laboratory and industrial scale applications due to the maintainance of high catalytic efficiency, thermal and solvent stability, reusability and ease of operation in batch as well as in continuous reactions.     

Equations and calculations of product yields and preferred pathways for butanediol and mixed-acid fermentations

Using the available information of fermentation biochemistry, fermentation (stoichiometric) equations are derived for anaerobic saccharolytic fermentations of butanediol and mixed acids. The equations describe the interrelations among the fermentation products, biomass, and consumed substrate (glucose). The validity of the equations is tested using a variety of batch data from the literature. The validity of the equations is expected to extend to steady-state and transient fermentations, as well. Uses, improvements, and extensions of the equations are also discussed in detail. Among others, it is shown that the equations are useful for checking the consistency of experimental data, for calculating maximal yields and selectivities for the fermentation products, and calculating the extent of utilization of the Embden-Meyerhof-Parnas pathway versus the Hexose Monophosphate pathway of glucose utilization.     

Measurement,control, and modeling of submerged acetic acid fermentation

For control and optimization of large scale bioprocesses, mathematical models are needed to describe transient growth and/or product formation. Such models can only be developed from reliable experimental data. A computerized experimental system was applied to submerged acetic acid fermentation with industrial Acetobacter strains in order to obtain quantitatively reproducible long-term data. Automated repeated-batch fermentations were carried out over a period of one year. It was found that consideration of substrate, product, and biomass concentrations alone was not sufficient to describe transient culture conditions. At least one more internal parameter must be taken into account. A delay-time model was developed which takes into consideration the variable concentration of an internal component of the cells, the ribonucleic acid. This model was used to simulate the acetic acid fermentation. The simulation results agreed well with the experimental data. Thus, the validity of the model assumptions could be confirmed. The model was capable of simulating the lag-phase of growth as well as lysis of microorganisms due to product inhibition.     

数据驱动的社会-经济-自然复合生态系统研究:尺度、过程及其决策关联

社会-经济-自然系统是人类赖以生存和发展的复合系统,而数据驱动下的系统研究为加强生态系统的认知提供了新的增值导向。在新的数据语境下,社会-经济-自然复合系统呈现出一些新的特征,研究对象逐渐从单一要素向多要素耦合的方向转变,使支撑量化研究的数据体系更多样化、数据来源更广泛化、数据表达更具可视化,并呈现出研究尺度逐渐扩大化、研究对象更精细化的特征。在对数据的识别、表达和可视化的过程中,既要加强对时间、空间、结构、数量和秩序的耦合,也要注重与决策制定和地方服务的结合。新时期复合生态系统的未来研究方向应该从关键科学问题及支撑技术、尺度作用和多要素耦合以及地方和全球治理的科技支撑等方面展开,在数据的不断革新下,加强对多源数据、长期监测和时间序列的认知仍是需要深入研究的课题。开展复合生态系统的数据驱动分析,不仅能为生态系统的服务及可持续发展提供技术支撑,增强数据的长效共享机制,同时可为实现决策制定和信息传播等方面提供更多的价值支持。     

The influence of impeller type in pilot scale xanthan fermentations

The rheological complexity of Xanthan fermentations presents an interesting problem from a mixing viewpoint, because the phenomena of poor bulk blending and low oxygen mass transfer rates inherent in highly viscous fermentations (and their consequences) can be systematically investigated, even at the pilot plant scale. This study in a 150 L fermentor compares the physical and biological performance of four pairs of impellers: a standard Rushton turbine, a large diameter Rushton turbine, a Prochem Maxflo T, and a Scaba 6SRGT. Accurate in-fermentor power measurements, essential for the comparison of impellers in relation to operating costs are also reported. It is demonstrated that the agitator performance in Xanthan fermentations is very specific and the choice of which impeller to use in bioreactors to obtain enhanced performance is dependant on the applied criterion. None of the criterion favored the use of the standard Rushton turbine, therefore suggesting that there are strong grounds for retrofitting these impellers with either large diameter impellers of similar design or with novel agitators. In addition, fluid dynamic modeling of cavern formation has clearly highlighted the importance of a well mixed and oxygenated region for providing the capacity for high microbial oxygen uptake rates which govern Xanthan productivity and quality. Copyright 1998 John Wiley & Sons, Inc.     

Fermentation of cellodextrins to ethanol using mixed-culture fermentations

The potential for enhancing ethanol production from cellodextrins by employing mixed-culture (Candida wickerhamii-Saccharomyces cerevisiae) fermentations was investigated. Initially, ethanol production was monitored in fermentation medium containing 50 g/L glucose plus 45 g/L cellobiose. Inoculum levels and times of inoculum addition were varied. Of the conditions tested, the most rapid rates of ethanol formation occurred in fermentations in which either C. wickerhamii and S. cerevisiae were coinoculated at a ratio of 57 : 1 cell/mL or in fermentations in which a 10-fold-greater S. cerevisiae inoculum was added to a pure culture C. wickerhamii fermentation after 1 day incubation. These conditions were used to attempt to enhance fermentations in which cellodextrins produced by trifluoroacetic acid hydrolysis of cellulose served as the sole carbon source. Cellodextrins that were not further purified after cellulose hydrolysis contained compounds that were slightly inhibitory to C. wickerhamii. In this case the mixed-culture fermentations produced 12-45% more ethanol than a pure culture C. wickerhamii fermentation. However, if the substrate was treated with Darco G-60 charcoal, the toxic materials were apparently removed and the pure culture C. wickerhamii fermentations performed as well as the mixed-culture fermentations.     

Evidencing the role of lactose permease in IPTG uptake by Escherichia coli in fed-batch high cell density cultures

The lac-operon and its components have been studied for decades and it is widely used as one of the common systems for recombinant protein production in Escherichia coli. However, the role of the lactose permease, encoded by the lacY gene, when using the gratuitous inducer IPTG for the overexpression of heterologous proteins, is still a matter of discussion. A lactose permease deficient strain was successfully constructed. Growing profiles and acetate production were compared with its parent strain at shake flask scale. Our results show that the lac-permease deficient strain grows slower than the parent in defined medium at shake flask scale, probably due to a downregulation of the phosphotransferase system (PTS). The distributions of IPTG in the medium and inside the cells, as well as recombinant protein production were measured by HPLC-MS and compared in substrate limiting fed-batch fermentations at different inducer concentrations. For the mutant strain, IPTG concentration in the medium depletes slower, reaching at the end of the culture higher concentration values compared with the parent strain. Final intracellular and medium concentrations of IPTG were similar for the mutant strain, while higher intracellular concentrations than in medium were found for the parent strain. Comparison of the distribution profiles of IPTG of both strains in fed-batch fermentations showed that lac-permease is crucially involved in IPTG uptake. In the absence of the transporter, apparently IPTG only diffuses, while in the presence of lac-permease, the inducer accumulates in the cytoplasm at higher rates emphasizing the significant contribution of the permease-mediated transport.     

The influence of acetic acid on penicillin production

Summary Fed-batch fermentations with Penicillum chrysogenum, strain S 3723, were fed with glucose as carbon source or with a mixture of glucose and acetic acid. When 20% of the carbon source was acetic acid, yields of penicillin-V were 25% higher than in fermentations where glucose was the only carbon source in the feed. The increased yield was due to higher specific productivity and/or cell mass. The effect was seen in fermentations where the carbon source was fed at a constant rate and the pH kept automatically at 6.5 by addition of inorganic acid or base, as well as in fermentations where pH controlled the addition of feed.     

Relevance of microbial coculture fermentations in biotechnology

The purpose of this article is to review coculture fermentations in industrial biotechnology. Examples for the advantageous utilization of cocultures instead of single cultivations include the production of bulk chemicals, enzymes, food additives, antimicrobial substances and microbial fuel cells. Coculture fermentations may result in increased yield, improved control of product qualities and the possibility of utilizing cheaper substrates. Cocultivation of different micro‐organisms may also help to identify and develop new biotechnological substances. The relevance of coculture fermentations and the potential of improving existing processes as well as the production of new chemical compounds in industrial biotechnology are pointed out here by means of more than 35 examples.