Monitoring Growth of Microorganisms using the Methods of Mass-Energy Balance and Utilization of Computer on Line with Fermenter in Real Time Processing (in Russian)

The effective means of microbial culture monitoring is the measurement of low-inertial parameters (respiration rate, rates of supply of alkali for pH maintenance and the limiting substrate) and utilization of computer on line with fermenter for recalculation of these rates into the instant values of mass and energy cell yields, specific rates of cell growth and substrate and oxygen consumption, using the method of mass-energy balance. In this paper, the equations of mass-energy balance are presented both in general form and in the form of numerical algorythms for computer programming. The installation for automation of microbial cultivation experiment is described. Experimental data are presented which indicate the effectiveness of the method of indirect measurement of cell biomass yield and specific rates of physiological processes.     

Growing Phototrophic Cells without Light

Many phototrophic microorganisms contain large quantities of high-value products such as n-3 polyunsaturated fatty acids and carotenoids but phototrophic growth is often slow due to light limitation. Some phototrophic microorganisms can also grow on cheap organic substrate heterotrophically. Heterotrophic cultivation can be well controlled and provides the possibility to achieve fast growth and high yield of valuable products on a large scale. Several strategies have been investigated for cultivation of phototrophic microorganisms without light. These include trophic conversion of obligate photoautotrophic microorganisms by genetic engineering, development of efficient cultivation systems and optimization of culture conditions. This paper reviews recent advances in heterotrophic cultivation of phototrophic cells with an emphasis on microalgae.     

Modelling of agrobacteran production in a batch culture ofAgrobacterium radiobacter

A deterministic model of production of the exopolysaccharide agrobacteran, including the effect of the product on oxygen transfer into the medium, was used for evaluation of a batch cultivation ofAgrobacterium radiobacter. Application of mass-energy balance made it possible to reduce the number of identified parameters. The resulting yield coefficients and rate constants were largely independent of the method of aeration of the culture, with the exception of the maximum specific rate of agrobacteran production which was lower if the culture in an exponential growth phase was subjected to oxygen limitation.     

Partitioning of CO2 Incorporation Among Planktonic Microbial Guilds and Estimation of In Situ Specific Growth Rates

Partitioning of CO₂ incorporation into oxygenic phototrophic, anoxygenic phototrophic, and chemolithoautotrophic guilds was determined in a freshwater lake (Lake Cisó, Banyoles, Spain). CO₂ incorporation into the different types of microorganisms was studied at different depths, during diel cycles, and throughout the year. During winter holomixis, the whole lake became anoxic and both the anoxygenic and chemolithoautotrophic guilds were more active at the surface of the lake, whereas the activity of the oxygenic guild was negligible. During stratification, the latter guild was more active in the upper metalimnion, whereas the anoxygenic guild was more active in the lower metalimnion. Specific growth rates and doubling times were estimated for the most conspicuous phototrophic microorganisms. Doubling times for Cryptomonas phaseolus ranged between 0.5 and 192 days, whereas purple sulfur bacteria (Chromatiaceae-like) ranged between 1.5 and 238 days. These growth rates were similar to those calculated with a different approach in previous papers and indicate slow-growing populations with very large biomass. Overall, the annual total CO₂ incorporation in Lake Cisó was 220 g C m⁻². Most of the CO₂ incorporation, however, was due to the chemolithoautotrophic guild (61% during holomixis and 56% during stratification), followed by the anoxygenic phototrophic guild (35 and 19%, respectively) and the oxygenic phototrophs (4 and 25%, respectively), making dark carbon fixation the key process in the autotrophic metabolism of the lake.     

In situ specific loss and growth rates of purple sulfur bacteria in Lake Cisó

Abstract Growth rates and population dynamics of phototrophic bacteria in Lak Cisó were analysed by measuring bacterial abundances and determining specific rates of growth and loss. Net growth rates were calculated from actual changes in biomass assuming exponential growth. Values ranged between −0.072 and 0.037 per day (d⁻¹) for Chromatium , and between −0.043 and 0.022 d⁻¹ for Amoebobacter . Exponential loss rates through sedimentation, decomposition and washout were determined independently. Values ranged between 0 and 0.025 d⁻¹ in the case of Chromatium and between 0 and 0.015 d⁻¹ in the case of Amoebobacter . Finally, gross growth rates were calculated by adding net growth to losses. Maximal values were 0.063 d⁻¹ for Chromatium and 0.037 d⁻¹ for Amoebobacter . In the case of Chromatium , population growth rates were found to be correlated with the amount of light available per unit of growing biomass. It was concluded that growth of phototrophic bacteria in Lake Cisó was limited by light availability. Altogether, purple sulfur bacteria seemed to maintain a very large biomass with very slow growth, thanks to very slow losses during stratification. During holomixis the situation was more dynamic. Washout of cells and disappearance of algal cells allowed more light to reach the bacteria. Therefore, high growth rates were found towards the end of the winter. A similar pattern repeated itself from year to year. These are the first estimates of in situ growth rates for populations of phototrophic bacteria.     

Flux Balance Analysis of Cyanobacterial Metabolism: The Metabolic Network of Synechocystis sp. PCC 6803

Cyanobacteria are versatile unicellular phototrophic microorganisms that are highly abundant in many environments. Owing to their capability to utilize solar energy and atmospheric carbon dioxide for growth, cyanobacteria are increasingly recognized as a prolific resource for the synthesis of valuable chemicals and various biofuels. To fully harness the metabolic capabilities of cyanobacteria necessitates an in-depth understanding of the metabolic interconversions taking place during phototrophic growth, as provided by genome-scale reconstructions of microbial organisms. Here we present an extended reconstruction and analysis of the metabolic network of the unicellular cyanobacterium Synechocystis sp. PCC 6803. Building upon several recent reconstructions of cyanobacterial metabolism, unclear reaction steps are experimentally validated and the functional consequences of unknown or dissenting pathway topologies are discussed. The updated model integrates novel results with respect to the cyanobacterial TCA cycle, an alleged glyoxylate shunt, and the role of photorespiration in cellular growth. Going beyond conventional flux-balance analysis, we extend the computational analysis to diurnal light/dark cycles of cyanobacterial metabolism.     

Complete assimilation of cysteine by a newly isolated non-sulfur purple bacterium resembling Rhodovulum sulfidophilum (Rhodobacter sulfidophilus)

A rod-shaped, motile, phototrophic bacterium, strain SiCys, was enriched and isolated from a marine microbial mat, with cysteine as sole substrate. During phototrophic anaerobic growth with cysteine, sulfide was produced as an intermediate, which was subsequently oxidized to sulfate. The molar growth yield with cysteine was 103 g mol^–1, in accordance with complete assimilation of electrons from the carbon and the sulfur moiety into cell material. Growth yields with alanine and serine were proportionally lower. Thiosulfate, sulfide, hydrogen, and several organic compounds were used as electron donors in the light, whereas cystine, sulfite, or elemental sulfur did not support phototrophic anaerobic growth. Aerobic growth in the dark was possible with fructose as substrate. Cultures of strain SiCys were yellowish-brown in color and contained bacteriochlorophyll a, spheroidene, spheroidenone, and OH-spheroidene as major photosynthetic pigments. Taking the morphology, photosynthetic pigments, aerobic growth in the dark, and utilization of sulfide for phototrophic growth into account, strain SiCys was assigned to the genus Rhodovulum (formerly Rhodobacter) and tentatively classified as a strain of R. sulfidophilum. In cell-free extracts in the presence of pyridoxal phosphate, cysteine was converted to pyruvate and sulfide, which is characteristic for cysteine desulfhydrase activity (l-cystathionine γ-lyase, EC 4.4.1.1). Received: 15 December 1995 / Accepted: 1 April 1996     

Mass-energy balance for microbial product synthesis-biochemical and cultural aspects

Interrelations between the rates of the product synthesis, cell biomass growth, respiration, and organic substrate consumption have been studied by the mass-energy balance method. This method is based on the utilization of a special unit of substance reducity, namely redoxon. Biochemical parameters have been found which are involved in these interrelations and which describe the processes of high-energy bond gain and energy expenditure during metabolism. In order to find these, the separation of the whole metabolism into several partial metabolisms has been applied. Equations have been obtained describing the dependences of the product yield and process specific productivity on the biochemical parameters and two macroscopic rates (e.g., rates of dilution and substrate consumption). Both aerobic and anaerobic product syntheses have been considered. The estimate of the upper limit of process productivity has been obtained. Mechanisms of the influence of the producer's intracellular characteristics on the rates of physiological processes and the culture productivity are discussed.     

Principles of the light-limited chemostat: theory and ecological applications

Light is the energy source that drives nearly all ecosystems on planet Earth. Yet, light limitation is still poorly understood. In this paper, we present an overview of the principles of the light-limited chemostat. The theory for light-limited chemostats differs considerably from the standard theory for substrate-limited chemostats. In particular, photons cannot be mixed by vigorous stirring, so that phototrophic organisms experience the light-limited chemostat as a heterogeneous environment. Similar to substrate-limited chemostats, however, light-limited chemostats do reach a steady state. This allows the study of phototrophic microorganisms under well-controlled light conditions, at a constant specific growth rate, for a prolonged time. The theory of the light-limited chemostat is illustrated with several examples from laboratory experiments, and a variety of ecological applications are discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Adaptation and Acclimation of Photosynthetic Microorganisms to Permanently Cold Environments

Persistently cold environments constitute one of our world's largest ecosystems, and microorganisms dominate the biomass and metabolic activity in these extreme environments. The stress of low temperatures on life is exacerbated in organisms that rely on photoautrophic production of organic carbon and energy sources. Phototrophic organisms must coordinate temperature-independent reactions of light absorption and photochemistry with temperature-dependent processes of electron transport and utilization of energy sources through growth and metabolism. Despite this conundrum, phototrophic microorganisms thrive in all cold ecosystems described and (together with chemoautrophs) provide the base of autotrophic production in low-temperature food webs. Psychrophilic (organisms with a requirement for low growth temperatures) and psychrotolerant (organisms tolerant of low growth temperatures) photoautotrophs rely on low-temperature acclimative and adaptive strategies that have been described for other low-temperature-adapted heterotrophic organisms, such as cold-active proteins and maintenance of membrane fluidity. In addition, photoautrophic organisms possess other strategies to balance the absorption of light and the transduction of light energy to stored chemical energy products (NADPH and ATP) with downstream consumption of photosynthetically derived energy products at low temperatures. Lastly, differential adaptive and acclimative mechanisms exist in phototrophic microorganisms residing in low-temperature environments that are exposed to constant low-light environments versus high-light- and high-UV-exposed phototrophic assemblages.     

Adaptation and acclimation of photosynthetic microorganisms to permanently cold environments.

Persistently cold environments constitute one of our world's largest ecosystems, and microorganisms dominate the biomass and metabolic activity in these extreme environments. The stress of low temperatures on life is exacerbated in organisms that rely on photoautrophic production of organic carbon and energy sources. Phototrophic organisms must coordinate temperature-independent reactions of light absorption and photochemistry with temperature-dependent processes of electron transport and utilization of energy sources through growth and metabolism. Despite this conundrum, phototrophic microorganisms thrive in all cold ecosystems described and (together with chemoautrophs) provide the base of autotrophic production in low-temperature food webs. Psychrophilic (organisms with a requirement for low growth temperatures) and psychrotolerant (organisms tolerant of low growth temperatures) photoautotrophs rely on low-temperature acclimative and adaptive strategies that have been described for other low-temperature-adapted heterotrophic organisms, such as cold-active proteins and maintenance of membrane fluidity. In addition, photoautrophic organisms possess other strategies to balance the absorption of light and the transduction of light energy to stored chemical energy products (NADPH and ATP) with downstream consumption of photosynthetically derived energy products at low temperatures. Lastly, differential adaptive and acclimative mechanisms exist in phototrophic microorganisms residing in low-temperature environments that are exposed to constant low-light environments versus high-light- and high-UV-exposed phototrophic assemblages.     

Phototrophic Fe(II) oxidation promotes organic carbon acquisition by Rhodobacter capsulatus SB1003

Anoxygenic phototrophic Fe(II) oxidation is usually considered to be a lithoautotrophic metabolism that contributes to primary production in Fe-based ecosystems. In this study, we employed Rhodobacter capsulatus SB1003 as a model organism to test the hypothesis that phototrophic Fe(II) oxidation can be coupled to organic carbon acquisition. R. capsulatus SB1003 oxidized Fe(II) under anoxic conditions in a light-dependent manner, but it failed to grow lithoautotrophically on soluble Fe(II). When the strain was provided with Fe(II)-citrate, however, growth was observed that was dependent upon microbially catalyzed Fe(II) oxidation, resulting in the formation of Fe(III)-citrate. Subsequent photochemical breakdown of Fe(III)-citrate yielded acetoacetic acid that supported growth in the light but not the dark. The deletion of genes (RRC00247 and RRC00248) that encode homologs of atoA and atoD, required for acetoacetic acid utilization, severely impaired the ability of R. capsulatus SB1003 to grow on Fe(II)-citrate. The growth yield achieved by R. capsulatus SB1003 in the presence of citrate cannot be explained by lithoautotrophic growth on Fe(II) enabled by indirect effects of the ligand [such as altering the thermodynamics of Fe(II) oxidation or preventing cell encrustation]. Together, these results demonstrate that R. capsulatus SB1003 grows photoheterotrophically on Fe(II)-citrate. Nitrilotriacetic acid also supported light-dependent growth on Fe(II), suggesting that Fe(II) oxidation may be a general mechanism whereby some Fe(II)-oxidizing bacteria mine otherwise inaccessible organic carbon sources.     

Light‐responsive current generation by phototrophically enriched anode biofilms dominated by green sulfur bacteria

The objective of this study was to employ microbial electrochemical cells (MXCs) to selectively enrich and examine anoxygenic photosynthetic bacteria for potential anaerobic respiration capabilities using electrodes. In the process, we designed a novel enrichment strategy that manipulated the poised anode potential, light, nitrogen availability, and media supply to promote growth of phototrophic bacteria while minimizing co‐enrichment of non‐phototrophic anode‐respiring bacteria (ARB). This approach resulted in light‐responsive electricity generation from fresh‐ and saltwater inocula. Under anoxic conditions, current showed a negative light response, suggesting that the enriched phototrophic consortia shifted between phototrophic and anaerobic respiratory metabolism. Molecular, physical, and electrochemical analyses elucidated that anode biofilms were dominated by green sulfur bacteria, and biofilms exhibited anode respiration kinetics indicative of non‐mediated electron transfer, but kinetic parameters differed from values previously reported for non‐phototrophic ARB. These results invite the utilization of MXCs as microbiological tools for exploring anaerobic respiratory capabilities among anoxygenic photosynthetic bacteria. Biotechnol. Bioeng. 2013; 110: 1020–1027. © 2012 Wiley Periodicals, Inc.     

Ratio of heat production to oxygen consumption during the cell cycle of candida maltosa EH 15 grown on ethanol

The experimental technique for measurement of microbial culture heat evolution directly in fermenter has been described and its correctness analysed. Heat-to-oxygen ratio, Q₀, of synchronized yeast culture in the absence of fermentative metabolism has been found to be practically independent of a cell cycle phase and close to the theoretical constant predicted by the mass-energy balance theory. The collection of literature data on the heat-to-oxygen ratio is given. Energetic properties of cell biomass are discussed on the basis of the obtained and the surveyed values of Q₀.     

Influence of material properties and photocatalysis on phototrophic growth in multi-year roof weathering

Phototrophic growth on roofs leads to weathering and impacts their appearance. Roof tiles with various properties are available (natural clay, engobed, varnished or coated with photocatalytic TiO₂). The aim of this study was to examine the influence of materials on the development of phototrophic biofilms. Roof tiles were weathered in six climatic regions in Germany for several years. Phototrophic biomass was periodically determined by PAM-fluorometry, image analysis, and visual evaluation. Roof tiles of natural clay were the most heavily infested, while black varnished roof tiles were hardly covered with any phototrophs. This colonisation pattern was compared to water availability on roof tiles surfaces. In contrast to rough natural clay, varnished black tiles accumulated less water, dried quickly, and were rather resistant to phototrophs. The photocatalytic coating was not effective against phototrophic growth. Materials with appropriate properties may prevent phototrophic growth without biocides through reduced water absorption capacities and by avoiding radiation protected structures.     

Cultivation of Saccharomyces cerevisiae in continuous culture

Summary Growth of Saccharomyces cerevisiae was investigated under aerobic conditions in a glucose limited chemostat. The steady state concentrations of cells, glucose and ethanol were measured in dependence of the dilution rate. The growth rate showed a biphasic dependence from the glucose concentration. A shift from respiratory to fermentative metabolism (Crabtree-effect) altering heavily the cell yield and the ethanol yield took place in the range of dilution rates between 0.3 h^-1 and 0.5 h^-1. Therefore the classical theory of continuous cultures is not applicable on aerobic growth of Saccharomyces cerevisiae under glucose limitation without introducing further premises. On the other hand the steady state cell concentration as a function of the dilution rate fits well the theoretically calculated curves, if cells are cultivated under conditions where only fermentation or respiration is possible.     

Isolation and characterization of transposon Tn5 mutants of Rhodobacter sphaeroides deficient in both nitrate and chlorate reduction.

Abstract The wild-type strain Rhodobacter sphaeroides DSM 158 is a nitrate-reducing bacterium with a periplasmic nitrate reductase. Addition of chlorate to the culture medium causes a stimulation of the phototrophic growth, indicating that this strain is able to use chlorate as an ancillary oxidant. Several mutant strains of R. sphaeroides deficient in nitrate reductase activity were obtained by transposon Tn5 mutagenesis. Mutant strain NR45 exhibited high constitutive nitrate and chlorate reductase activities and phototrophic growth was also increased by the presence of chlorate. In contrast, the stimulation of growth by chlorate was not observed in mutant strains NR8 and NR13, in which transposon Tn5 insertion causes the simultaneous loss of both nitrate and chlorate reductase activities. Tn5 insertion probably does not affect molybdenum metabolism since NR8 and NR13 mutants exhibit both xanthine dehydrogenase and nitrogenase activities. These results that a single enzyme could reduce both nitrate and chlorate in R. sphaeroides DSM 158.