The auxiliary substrate concept — an approach for overcoming limits of microbial performances

In biotechnological processes, fundamental performances of microorganisms are used. The economy of these processes is essentially determined by the efficiency, velocity (productivity) and quality of the products. Therefore it is a permanent task and challenge for basic and biotechnological research to seek out measures for improving the actually attained parameters. The auxiliary substrate concept supplics an approach. It is based on the fact that chemo-organo-heterotrophic substrates differ in the carbon: energy ratio, thus, growth yield is limited in energy and/or reducing power. It says that, by simultaneous utilization of physiologically similar substrates (mixed substrates), the growth yield increases. The substrates are to combine in such a way that with their simultaneous utilization a minimum of carbon is dissimilated merely for the purpose of the generation of biologically useful energy and/or reducing power. Since all chemo-organo-heterotrophic substrates are more or less energy-deficient, an increase in growth efficiency can be expected if the individual substrates of the mixture are assimilated more efficiently than the respective substrates alone. This may result, for instance, from an immediate assimilation of a substrate (according to the “manner of finished part construction”). An increased growth rate is rather the rule than the exception in mixed substrate utilization. In product syntheses the substrates are, depending on the concrete product and metabolic pathway, either energy-excess or energy-excess or energy-deficient. or, in other words, the processes are energy-generating or energy-consuming, respectively. If this is responsible for discrepancies between the possible yields determined by the carbon metabolism and the experimentally obtained yields, the discrepancies should be able to be decreased and the yields increased by mixing substrates. The substrates are to choose and combine so that, due to simultaneous utilization, the product formation process becomes energy neutral. As a rule, the enhanced efficiency is accompanied by an increased velocity. This does not only apply to syntheses, but also to degradation (and detoxification) reactions. Even supposedly inert compounds or persistent substances can be activated by simultaneous (co-)metabolization of another (an auxiliary substrate, victim substrate or co-substrate) and converted at a considerable rate. It is of interest for syntheses of products but in particular for degradation and decontamination of harmful and waste products in the environment that the residual concentrations of the substrates are smaller than those achieved if the compounds of a mixture are metabolized separately. The auxiliary substrate concept has proven to be fruitful, both for theoretical and practical questions. It was practically already being used before it was formulated (mixed substrate utilization, cometabolism). However, an abundance of regulatory and energetic aspects are waiting to be investigated in more detail.     

The mixed substrate concept, applied for microbial syntheses of metabolites

Microbial overproduction of metabolites is a response to suboptimal conditions for growth and multiplication. It is an energy-wasting process in terms of life insofar as a part of energy of the carbon source remains in the metabolite. From an energetic point of view microbial overproduction can be divided into two categories: i) energy-consuming, ii) energy-yielding. The amount of energy required or made available is considered to be responsible for discrepancies between carbon metabolism-determined possible and experimentally obtained yields. Since the expenditure of energy must be provided by oxidation of carbon source more substrate is consumed than required according to the metabolic pathway. In the case of energy-yielding synthesis energy must be discharged. Various possibilities exist. Since metabolic sequences not involved in the synthesis of the proper product are not switched off completely other synthetic processes and even growth can occur. The energy is thus discharged at the expense of substrate. To increase the experimental yield the energy produced or consumed has to be maintained low. This can be achieved by means of substrate mixtures. The synthesis of by-products and growth are difficult to prevent completely. However, growth can be quite desirable since the catalyst is renewed thus making the product synthesis possible.     

Microbial export of lactic and 3-hydroxypropanoic acid: implications for industrial fermentation processes

Lactic acid and 3-hydroxypropanoic acid are industrially relevant microbial products. This paper reviews the current knowledge on export of these compounds from microbial cells and presents a theoretical analysis of the bioenergetics of different export mechanisms. It is concluded that export can be a key constraint in industrial production, especially under the conditions of high product concentration and low extracellular pH that are optimal for recovery of the undissociated acids. Under these conditions, the metabolic energy requirement for product export may equal or exceed the metabolic energy yield from product formation. Consequently, prolonged product formation at low pH and at high product concentrations requires the involvement of alternative, ATP-yielding pathways to sustain growth and maintenance processes, thereby reducing the product yield on substrate. Research on export mechanisms and energetics should therefore be an integral part of the development of microbial production processes for these and other weak acids.     

Microbial production of bulk chemicals: development of anaerobic processes

Innovative fermentation processes are necessary for the cost-effective production of bulk chemicals from renewable resources. Current microbial processes are either anaerobic processes, with high yield and productivity, or less-efficient aerobic processes. Oxygen utilization plays an important role in energy generation and redox metabolism that is necessary for product formation. The aerobic productivity, however, is relatively low because of rate-limiting volumetric oxygen transfer; whereas the product yield in the presence of oxygen is generally low because part of the substrate is completely oxidized to CO?. Hence, new microbial conversion processes for the production of bulk chemicals should be anaerobic. In this opinion article, we describe different scenarios for the development of highly efficient microbial conversion processes for the anaerobic production of bulk chemicals.     

The efficiency of recombinant Escherichia coli as biocatalyst for stereospecific epoxidation

Styrene is efficiently converted into (S)-styrene oxide by growing Escherichia coli expressing the styrene monooxygenase genes styAB of Pseudomonas sp. strain VLB120 in an organic/aqueous emulsion. Now, we investigated factors influencing the epoxidation activity of recombinant E. coli with the aim to improve the process in terms of product concentration and volumetric productivity. The catalytic activity of recombinant E. coli was not stable and decreased with reaction time. Kinetic analyses and the independence of the whole-cell activity on substrate and biocatalyst concentrations indicated that the maximal specific biocatalyst activity was not exploited under process conditions and that substrate mass transfer and enzyme inhibition did not limit bioconversion performance. Elevated styrene oxide concentrations, however, were shown to promote acetic acid formation, membrane permeabilization, and cell lysis, and to reduce growth rate and colony-forming activity. During biotransformations, when cell viability was additionally reduced by styAB overexpression, such effects coincided with decreasing specific epoxidation rates and metabolic activity. This clearly indicated that biocatalyst performance was reduced as a result of product toxicity. The results point to a product toxicity-induced biological energy shortage reducing the biocatalyst activity under process conditions. By reducing exposure time of the biocatalyst to the product and increasing biocatalyst concentrations, volumetric productivities were increased up to 1,800 micromol/min/liter aqueous phase (with an average of 8.4 g/L(aq) x h). This represents the highest productivity reported for oxygenase-based whole-cell biocatalysis involving toxic products.     

Kinetics of the xanthan fermentation

Xanthan gum, a heteropolysaccharide with unusual and useful properties, is now produced commercially by fermentation with Xanthomonas compestris NRRL B–1459 in a medium containing glucose, minerals, and a complex nitrogen source—distillers' dried solubles (DDS). Understanding the kinetics of the fermentation should contribute to process improvements and increase the market potential for the gum. Earlier studies showed that although DDS determined initial growth rate, growth was stopped by some mechanism other than substrate exhaustion, probably an effect related to product formation. Product formation did not require active growth, but its rate increased with cell concentration. Specific product formation rate declined at high viscosities. Varying glucose concentration from 0.5 to 5.0% and dissolved O₂ tension between 20 and 90% air saturated had no effect on the rates, but pH had to be maintained near 7 and temperature near 28°C to permit continued product formation. Xanthan yield could be explained by the energy required for growth and polymerization, that energy coming from dissimilation of the part of the carbohydrate substrate not converted to polymer.     

Bioproduction of butanol from biomass: from genes to bioreactors

Butanol is produced chemically using either the oxo process starting from propylene (with H2 and CO over a rhodium catalyst) or the aldol process starting from acetaldehyde. The key problems associated with the bioproduction of butanol are the cost of substrate and butanol toxicity/inhibition of the fermenting microorganisms, resulting in a low butanol titer in the fermentation broth. Recent interest in the production of biobutanol from biomass has led to the re-examination of acetone-butanol-ethanol (ABE) fermentation, including strategies for reducing or eliminating butanol toxicity to the culture and for manipulating the culture to achieve better product specificity and yield. Advances in integrated fermentation and in situ product removal processes have resulted in a dramatic reduction of process streams, reduced butanol toxicity to the fermenting microorganisms, improved substrate utilization, and overall improved bioreactor performance.     

Lactate metabolism in the muscle of the crab Chasmagnathus granulatus during hypoxia and post-hypoxia recovery

The present study showed that the lactate/glucose ratio in the hemolymph of Chasmagnathus granulatus maintained in normoxia (controls) was 4.9, suggesting that lactate is an important substrate for this crab. Periods of hypoxia are part of the biological cycle of this crab, and lactate is the main end product of anaerobiosis in this crab. Our hypothesis was that this lactate would be, therefore, used by gluconeogenic pathway or can be oxidized or excreted to the aquatic medium during hypoxia and post-hypoxia periods in C. granulatus. The concentrations of hemolymphatic lactate in animals in normoxia are high, and are used as an energy substrate. In hypoxia, muscle gluconeogenesis and excretion of lactate to the aquatic medium would contribute significantly in regulating the concentration of circulating lactate. Utilization of these pathways would serve the objective of maintaining the acid-base equilibrium of the organism. Muscle gluconeogenesis participates, during the recovery process, in metabolizing the lactate produced during the period of hypoxia. Lactate excretion to the external medium, was one of the strategies used to decrease the higher hemolymphatic lactate levels. However, oxidation of lactate in the muscle is not a main strategy used by this crab to metabolize lactate in the recovery periods.     

Optimizing the Production of Bacterial Cellulose in Surface Culture: Evaluation of Substrate Mass Transfer Influences on the Bioreaction (Part 1)

The interest in cellulose produced by bacteria from surface cultures has increased steadily in recent years because of its potential for use in medicine and cosmetics. Unfortunately, the low yield of the production process has limited the commercial usefulness of bacterial cellulose. This series of three papers dealing with the production of bacterial cellulose using (batch) surface culture, firstly present a complete and complex analysis of the overall system, which allows a fundamental optimization of the production process to be performed. This material has many applications but the low yield of the process limits its commercial usefulness. In part 1, the effect of the rate of mass transfer of substrate on the microbial process, which is characterized by the growth of the bacteria, product formation, and the utilization of the substrate by the bacteria, is studied. A fundamental model for the diffusion of glucose through the growing cellulose layer is proposed and solved. The model confirmed that the increase in diffusional resistance is indeed significant but other factors will also need to be taken into account.     

Substrate inactivation of enzymes in vitro and in vivo

Some enzymes are inactivated by their natural substrates during catalytic turnover, limiting the ultimate extent of reaction. These enzymes can be separated into three broad classes, depending on the mechanism of the inactivation process. The first type is enzymes which use molecular oxygen as a substrate. The second type is inactivated by hydrogen peroxide, which is present either as a substrate or a product, and are stabilized by high catalase activity. The oxidation of both types of enzymes shares common features with oxidation of other enzymes and proteins. The third type of enzyme is inactivated by non-oxidative processes, mainly reversible loss of cofactors or attached groups. Sub classes are defined within each broad classification based on kinetics and stoichiometry. Reaction-inactivation is in part a regulatory mechanism in vivo, because specific proteolytic systems give rapid turnover of such labelled enzymes. The methods for enhancing the stability of these enzymes under reaction conditions depends on the enzyme type. The kinetics of these inactivation reactions can be used to optimize bioreactor design and operation.     

Fermentation of cashew apple juice to produce high added value products

The use of agriculture substrates in industrial biotechnological processes has been increasing because of its low cost. Cashew apples are considered an agriculture low cost product in the Brazilian Northeast because the cashew cultivation is done mainly to produce cashew nuts. About 90% of the cashew apples production is lost in the field after removing the nut. In this work, the use of clarified cashew apple juice as substrate for microbial cultivation was investigated. The results showed that cashew apple juice is a good source of reducing sugars and can be used to grow Leuconostoc mesenteroides to produce high added value products such as dextran, lactic acid, mannitol and oligosaccharides.     

Comparison of the kinetics of lipopeptide production by Bacillus amyloliquefaciens XZ-173 in solid-state fermentation under isothermal and non-isothermal conditions

This study aimed to compare the kinetics of lipopeptide production in solid-state fermentation (SSF) under isothermal and non-isothermal conditions. Models based on the logistic, modified Gompertz and Luedeking–Piret-like equations were developed to describe the time course of fermentation under different conditions. The experiments were conducted in 250 mL flasks and a 50 L fermenter. The results showed that the non-isothermal process had higher levels of product formation rate and substrate utilization rate compared to the isothermal process. The part of substrate carbon to meet microbial maintenance—energy, biomass and lipopeptides formation requirements got increased using the non-isothermal technique. In addition, fermenter conditions positively influenced the lipopeptides formation rate with significantly higher levels of substrate for the microbial growth and product formation, though the product productivity and biomass both decreased as compared to flask. This is the first report that investigates the effects of temperature changing on the kinetics of lipopeptide production by Bacillus amyloliquefaciens strain under SSF condition using soybean flour and rice straw as major substrates in flask and in fermenter.     

A kinetic model for product formation of microbial and mammalian cells

Growth of microbial and mammalian cells can be classified into substrate-limited and substrate-sufficient growth according to the relative availability of the substrate (carbon and energy source) and other nutrients. It has been observed for a number of microbial and mammalian cells that the consumption rate of substrate and energy (ATP) is generally higher under substratesufficient conditions than under substrate limitation. Accordingly, the product formation under substrate excess often exhibits different patterns from those under substrate limitation. The extent of increase or decrease in product formation may depend not only on the nature of limitation and cell growth rate but also on the residual substrate concentration in a relatively wide range. The product formation kinetic models existing in literature cannot describe these effects. In this study, the Luedeking-Piret kinetic is extended to include a term describing the effect of residual substrate concentration. The extended model has a similar structure to the kinetic model for substrate and energy consumption rate recently proposed by Zeng and Deckwer. The applicability of the extended model is demonstrated with three microbial cultures for the production of primary metabolites and three hybridoma cell cultures for the production of ammonia and lactic acid over a wide range of substrate concentration. The model describes the product formation in all these cultures satisfactorily. Using this model, the range of residual substrate concentration, in which the product formation is affected, can be quantitatively assessed. (c) 1995 John Wiley & Sons, Inc.     

Estimating cell and product yields

The balance equations for carbon, reduction potential, and energy during cell growth and product formation are rederived in a general form. Cells are treated simply as a very complex product, and the Y(ATP) concept is extended to products. Limitations on the theoretical yield are discussed for different product types. Simple aerobic products cannot be energy limited unless the maintenance requirement is large, while complex products cannot be reduction limited. A maximum yield is defined for products much more oxidized than their substrate (carbon limited) because the theoretical yield conditions may violate the energy balance. For reduced complex products the yield on available electrons is related to Y(ATP), the P/O ratio, and the product composition. Narrow bounds are established on the actual yields in simple anaerobic fermentations, and the significance of the yields in the linear growth equation is discussed.     

Microbiological fermentation of lignocellulosic biomass: current state and prospects of mathematical modeling

The anaerobic fermentation process has achieved growing importance in practice in recent years. Anaerobic fermentation is especially valuable because its end product is methane, a renewable energy source. While the use of renewable energy sources has accelerated substantially in recent years, their potential has not yet been sufficiently exploited. This is especially true for biogas technology. Biogas is created in a multistage process in which different microorganisms use the energy stored in carbohydrates, fats, and proteins for their metabolism. In order to produce biogas, any organic substrate that is microbiologically accessible can be used. The microbiological process in itself is extremely complex and still requires substantial research in order to be fully understood. Technical facilities for the production of biogas are thus generally scaled in a purely empirical manner. The efficiency of the process, therefore, corresponds to the optimum only in the rarest cases. An optimal production of biogas, as well as a stable plant operation requires detailed knowledge of the biochemical processes in the fermenter. The use of mathematical models can help to achieve the necessary deeper understanding of the process. This paper reviews both the history of model development and current state of the art in modeling anaerobic digestion processes.     

Strategy tool trial for office furniture

Purpose

A strategic product development tool combining REACH and environmental and financial factors was previously developed for a coatings company. This paper presents results from refining this tool for an office furniture company, using life cycle assessment (LCA)-based environmental information, addressing the research questions: ? Is it possible to combine information from REACH with the LCA approach to provide useful information for a furniture producer in their environmental product development process? ? Does the approach developed for substances in mixtures need to be adapted for articles? ? Is there a correlation between energy consumption and the environmental impacts analysed? ? Will product designers get the same information independent of the environmental impact category used? ?C Will the strategy tool indicate the same ranking of products for all environmental impacts? ?C Does REACH information indicate the same set of priorities as those arising from LCA environmental data alone? (Do they agree, or is there a conflict?) ? Will strategic decisions differ if different environmental indicators are in focus? The strategy tool??s purpose is to analyse company product portfolios, identifying products that need redevelopment or redesign because of issues concerning hazardous substances, or environmental performance.

Methods

The LCA data used is cradle-to-gate data from type III environmental declarations for 11 seating solutions. REACH Complexity, health hazard and environmental class indicators (based on risk phrases) are combined with financial data and LCA-based indicators. Correlations between energy consumption and environmental impact factors for these specific furniture products are investigated. Establishing any such correlations serves to simplify subsequent analysis in the product development process, by effectively reducing the number of indicators that need to be taken into consideration.

Results

Correlations between energy consumption and the environmental impacts global warming, acidification, eutrophication and heavy metals are presented. Strategy tool figures are shown for energy consumption, ozone depletion potential and photochemical oxidation potential. The results for office chairs and conference/visitor chairs are presented separately, as the two types of chairs fulfil different functions.

Conclusions

The correlation between energy consumption and certain environmental impact indicators affords a simplification of the product development process, since energy consumption can be used as a reasonable proxy for these indicators in this specific case. The results support acknowledged principles of Ecodesign. Energy and materials minimization improves environmental performance??higher recycled material content and proportion of renewable energy resources are also beneficial. Designers have to consider multiple aspects in parallel and the strategy tool is useful for this purpose; the furniture producer has gained useful product development insight. The tool is applicable for strategic choice of products for development or redesign that can be useful across many business sectors.     

Auxiliary phase guidelines for microbial biotransformations of toxic substrate into toxic product

When an industrial process is developed using the microbial transformation of a precursor into a desired chemical compound, high concentrations of substrate and product will be involved. These compounds may become toxic to the cells. In situ product removal (ISPR) may be carried out, using auxiliary phases such as extractants or adsorbents. Simultaneously, in situ substrate addition (ISSA) may be performed. It is shown that for uncharged substrates and products, the aqueous solubilities of substrate and product can be used to predict if ISPR might be required. When a particular auxiliary phase is selected and the distribution coefficients of substrate and product are known, it is possible to estimate a priori if this auxiliary phase might be good enough and how much of it might be needed for an efficient (fed-)batch biotransformation process. For biotransformation products of intermediate polarity (aqueous solubility of about 1-10 g/L) there seems to be a lack of extractants and adsorbents with the capacity to raise the product concentrations to commercially more interesting levels.     

Essential motions in a fungal lipase with bound substrate,covalently attached inhibitor and product

As an aid to understanding the influence of dynamic fluctuations during esterolytic catalysis, we follow protein flexibility at three different steps along the catalytic pathway from substrate binding to product clearance via a covalently attached inhibitor, which represents a transition-state mimic. We have applied a classical approach, using molecular dynamics simulations to monitor protein dynamics in the nanosecond regime. We filter out small amplitude fluctuations and focus on the anharmonic contributions to the overall dynamics. This 'essential dynamics' analysis reveals different modes of response along the pathway suggesting that binding, catalysis and product clearance occur along different energy surfaces. Motions in the enzyme with a covalently attached ligand are more complex and occur along several eigenvectors. The magnitudes of the fluctuations in these individual subspaces are significantly smaller than those observed for the substrate and product molecules, indicating that the energy surface is shallow and that a relatively large number of conformational substates are accessible. On the other hand, substrate binding and product release occur at distinct modes of the protein flexibility suggesting that these processes occur along rough energy surfaces with only a few minima. Detailed energetic analyses along the trajectories indicated that in all cases binding is dominated by van der Waals interactions. The carboxylate form of the product is stabilized by a tight hydrogen bond network involving in particular Ser82, which may be a potential cause of product inhibition. Considerations such as these should aid the understanding of mechanisms of substrate, inhibitor or product recognition and could become of importance in the design of new substrates or inhibitors for enzymes.     

A kinetic model for energy spilling-associated product formation in substrate-sufficient continuous culture

It has been demonstrated that excess substrate can cause uncoupling between anabolism and catabolism, which leads to energy spilling. However, the Luedeking-Piret equation for product formation does not account for the energy spilling-associated product formation due to substrate excess. Based on the growth yield and energy uncoupling models proposed earlier, a kinetic model describing energy spilling-associated product formation in relation to residual substrate concentration was developed for substrate-sufficient continuous culture and was further verified with literature data. The parameters in the proposed model are well defined and have their own physical meanings. From this model, the specific productivity of unit energy spilling-associated substrate consumption, and the maximum product yield coefficient, can be determined. Results show that the majority of energy spilling-associated substrate consumption was converted to carbon dioxide and less than 6% was fluxed into the metabolites, while it was found that the maximum product yield coefficients varied markedly under different nutrient limitations. The results from this research can be used to develop the optimized bioprocess for maximizing valuable product formation.     

Economical factors in the assessment of various cellulosic substances as chemical and energy resources.

Economic factors in the assessment of various cellulosic substances as chemical and energy resources are many and complex. No substrate nor conversion process can be singled out as significantly advantageous. Agricultural wastes appear to have the best volume and availability characteristics. If glucose is to be the end product, then it will probably have to compete with corn syrup. If SCP is to be the end product, then productivities of 2-4 g/liter-hr must be achieved and the protein demand be such that the product can sell for at least $225/ton. If alcohol is to be the end product, then an intermediate product stream of glucose and other sugars must be obtained for 1-3cent/lb of fermentable sugars.