Microcalorimetric study of the biodegradation of cellulolytic compounds

Microcalorimetry can be used as a tool for the analysis of microbial growth on soluble or insoluble substrates. Modern apparatus allows studies under aerobic or anaerobic growth culture conditions. The heat quantity evolved during any microbial growth is dependent on catabolic and anabolic reactions according to:△H _met=(1-α)△H _cat+α△H _an In this relationship, δH_met, δH_cat, and δH_an are the enthalpy variations associated with metabolic, catabolic, and anabolic reactions, respectively, and a represents the molar fraction of the energy source that is incorporated into cellular carbon. The value of α is strongly influenced by culture conditions. Under anaerobic conditions, the α coefficient is low and does not exceed 0.2. Under aerobic conditions, α can reach a value as high as 0.6. Some experimental results were presented and discussed. Modern microcalorimeters record the power evolved by microbial growth as a function of time. The curves obtained are called power-time curves. These curves can be used for the determination of growth parameters. However, power-time curves allow visualization of some catabolic phenomena that are not related to growth because they occur during the stationary growth phase. Some examples were shown. More recently, microcalorimetry has been used for studies of degradation of insoluble substrates, such as lignocellulosic compounds, by pure or mixed cultures of microbes. The results obtained with pure cultures of bacteria growing on cellulose were interpreted using the same relationships that have been used for studies on soluble sugars. It was possible to show the influence of the structure of the cellulose on the kinetics of its degradation. The results obtained with mixed cultures were difficult to interprete at the present time. The power-time curves are very complex, showing many heat peaks that cannot be related to any known physiological phenomena. However, the total heat evolved during lignocellulosic fermentation by mixed cultures is proportional to the quantity of sugar fermented; this was demonstrated and discussed. Thus it can be concluded that microcalorimetry can be used for the estimation of the biodegradability of any solid substrate. This technique can also be used for evaluation of the efficiency of the pretreatments of lignocellulosic compounds that are done to increase the biodegradability of these substrates. Recent data will be shown.     

Design and testing of a calorimeter for biological uses

A rotatory microcalorimeter of the conduction type has been designed for the study of microbial metabolic processes under both aerobic and anaerobic conditions. The instrument can be performed in either batch mode or flow mode by changing the calorimeter vessels and the tube connections. The sensitivity and the time constant were determined by electrical calibrations. The heat sensitivity was 0.12 mV/mW with both yeast and other fungi. Because of the sufficient aeration and agitation, the calorimeter is available for studies on the fungi growth experiments of biotechnical interest.     

The effect of solid-liquid effluents from anaerobic digesters on soilmicrobial activity

A calorimetric procedure is developed to study the effect on the soil of the effluents resulting for the anaerobic digestion of slaughtering houses residues. DSC was used to study the pyrolysis properties of the effluent and the soil while isothermal calorimetry is applied to study the microbial activity in the effluent and to assess on its effect on the microbial activity of the soil where the industrial digester will be situated. The calorimetric data were studied together with the chemical and biological properties of that residue. Results showed that effluent is constituted by low levels of carbon and high levels of nitrogen. The power-time curves of the effluent have the typical shape of microbial growth yielding microbial growth rate constants between 0.37 and 0.53 h⁻¹ for about 4 and 11 h. The addition of the effluent to the soil decreases the heat of pyrolysis with time and stimulates the heat flow rate of the microbial metabolism.     

Chromatographic determination of phosphine (PH3) and hydrogen sulfide (H2S) in the headspace of anaerobic bacterial enrichments using flame photometric detection

Summary A gas chromatographic method is presented which distinguishes phosphine from hydrogen sulfide and other possible headspace gases of anaerobic microbial cultures. In anaerobic cultures spiked with phosphine, this gas is recovered in the liquid and gaseous phase down to 10 pg per ml of gas or liquid. No biogenically produced phosphine was found. Phosphine in amounts as small as 30 ng per 1 can be stored for several days in glass bottles covered with rubber septa, filled with nitrogen, in the presence or absence of water or of an anaerobic bacterial culture. Due to the selectivity of the detector and the retention characteristics of the porous layer open tubular polymer column alkanes, alkenes and organosulfur compounds routinely found in anaerobic bacterial headspaces do not interfere with the analytical quantification of phosphine.     

Evaluating microbial activity in composts using microcalorimetry

Microcalorimetric isothermal monitoring was carried out for compost and compost-containing growing medium, to provide fingerprints of compost microbial activity at different conditions. Microbial activity is a key property of composts used in agricultural practice. Two aspects are addressed in this study: (1) microcalorimetric evaluation of compost response to glucose as a model stimulating agent; (2) examination of the impact of compost pre-drying and re-wetting on its biological activity.

Addition of glucose solution to the compost involved a strong increase in microbial activity, which was associated with a significant heat evolution without a lag period. In certain cases, this heat evolution was of complicated shape thus manifesting the heterogeneity of microbial populations in composts. Relation between cumulative energy evolved and the amount of added glucose was found to be helpful in distinguishing between aerobic and anaerobic regime of compost microbial activity. As distinct from non-dried compost or growing mixture, glucose addition to samples pre-dried at 65 °C resulted in delayed heat response with initial exponential-like heat evolution. This delay in heat evolution suggests that biological activity was significantly suppressed upon compost drying. Such a temperature-induced inactivation process might also result in dominance of a relatively homogeneous microbial population which survived the heating, thus involving a smooth, exponential-like initial step of the heat evolution. Noteworthy, addition of water (without glucose) to pre-dried compost results in an outburst of activity as compared with non-dried compost. This heat evolution outburst was also characterized by a lag period and an initial exponential-like phase. The heat evolution obtained with pre-dried compost upon water addition was not related to the compost wetting energy but rather reflected the formation of new carbon source due to the changes in compost organic matter upon heating or to the dead biomass of those microbial populations that did not resist the compost heating/drying.     

Design and operation of anaerobic membrane bioreactors: development of a filtration testing strategy

Historically, high performance anaerobic wastewater treatment processes have been developed by maximizing the retention of microorganisms either by fixing them on porous material or by favoring the growth of easy-settling microbial granules. While numerous studies have been directed towards the comprehension of the various processes involved in the fixation of microor-ganisms or the formation of microbial granules, much less work has been devoted to the improvement of solid-liquid separation techniques. the recent development of a new generation of ultra-filtration membranes more productive and less expensive has however prompted the emergence of a new concept in the wastewater treatment technology: the membrane bioreactor.     

Methanogenesis mediated by methylotrophic mixed culture

Enrichment of methanogenic cultures on methanol from the microbial population in the anaerobic digesters operated on agricultural wastes revealed a high rate of biomethanation efficiency. Routine maintenance of this enrichment in a minimal basal medium at room temperature resulted in maximal growth in 40–50 d, and indicated pigment production toward the end of the growth phase. The cultures grown in three different media, with different substrates under light and dark conditions, were analyzed for protein, pigment, and gaseous products, and morphological studies were carried out by light, phase-contrast, fluorescence, and electron microscopy. In different media with methanol as substrate, growth and pigment production were maximal for the light-grown cells, decreasing in the order: phototrophic (PS(m)) > mineral > basal medium. Methanation and phototrophic growth were inversely correlated under lightgrown conditions. In contrast, growth in the dark was predominently methanogenic in the decreasing order: mineral > basal > PS (m). Among other growth conditions tested, utilization of phototrophic substrates under light and dark conditions indicated the following:

1. Basal and mineral media were supportive of methanogenic growth under both light and dark conditions, although methane yields under light-grown conditions were low;
2. Among the different substrates tested, methanol-grown cells gave the highest methane yield in the dark and;
3. Phototrophic growth in PS medium with succinate, malate, and pyruvate was better than that with methanol.

Absorption spectra of light-grown cells indicated the presence of bacteriochlorophyll a (Bchl a), as a doublet in the 800–0 nm region, which was absent in the dark-grown cells. Spectra of extracted pigments confirmed the presence of Bchl a with a 770-nm peak and carotenoid absorption bands in the 400–500 nm region indicative of the presence of a pigment of the spirilloxanthin type. Collective evidence for the predominant growth of a phototrophic organism under lightgrown conditions and microscopic examination under all conditions indicated the possible presence in the mixed culture of purple nonsulfur bacteria of theRhodopseudomonas type. In addition, the enrichment culture was found to contain other morphological forms, such as short and long rods, both individually and in clusters and coccoid cells. The presence of such different forms of microbial population in a methylotrophic enrichment along with phototrophic bacteria is interesting and is of ecological significance. Considering the uphill task of methanol oxidation under anaerobic conditions, the studies on the present enrichment signify metabolic partnerships in the methylotrophic biochemical mechanisms operative toward energy recovery.     

On-line calorimetry as a technique for process monitoring and control in biotechnology

This contribution reviews laboratory-scale investigations carried out on the usefulness of biological heat release measurements, as a means for monitoring and controlling the metabolic state of microbial cultures. Such studies are carried out in high-quality bench-scale calorimeters, but measuring heat generation rates by establishing energy balances ought to be applicable to large-scale bioreactors without resorting to sophisticated instrumentation. The signal received can either be interpreted by more qualitative correlation with the evolution of the culture, or it may be quantitatively exploited - together with other on-line measurements - in order to assess the rates at which various types of metabolisms proceed in the culture. The work described shows how this can be used to keep a culture in a desired metabolic state during fed-batch and transient continuous cultures of the yeast, S. cerevisae, and how a bacterial fed-batch culture can be controlled in order to optimize biosynthesis of an antibiotic.     

A mathematical model of ethanol fermentation from cheese whey

The cybernetic approach to modeling of microbial kinetics in a mixedsubstrate environment has been used in this work for the fermentative production of ethanol from cheese whey. In this system, the cells grow on multiple substrates and generate metabolic energy during product formation. This article deals with the development of a mathematical model in which the concept of cell maintenance was modified in light of the specific nature of product formation. Continuous culture data for anaerobic production of ethanol byKluyveromyces marxianus CBS 397 on glucose and lactose were used to estimate the kinetic parameters for subsequent use in predicting the behavior of microbial growth and product formation in new situations.     

The use of cationic surfactants and ionic liquids in the detection of microbial contamination by capillary electrophoresis

A rapid test of whether a laboratory sample contains any microorganisms is important and necessary for many areas of science and technology. Currently, most of the standard procedures for the detection of aerobic bacteria, anaerobic bacteria and fungi, require the preparation of microbial cultures in respective growth media, which are dramatically slow. Different approaches providing fast analysis such as CE are becoming more desired. To compensate for the natural electrophoretic heterogeneity of microbes, various buffer additives were examined to stack all bacteria and fungi in a sample plug into a single peak. This peak was removed from the molecular contaminants in the sample to prevent false positives. Both cationic surfactants and ionic liquids (IL) were investigated as run buffer additives and they are both widely applicable to different species of bacteria and fungi. Given that high concentrations of surfactants can potentially lyse cells, dicationic IL offer attractive auxiliary buffer additives for use in CE-based sterility tests. The analysis can be completed in 10 min, thus providing a great advantage over traditional direct inoculation methods that require several weeks to complete.     

Modeling and computational simulation of dilution and biochemical materials balance equations for partially emptied batch reactors

Sequencing batch reactors (SBRs) including aerobic SBRs and anaerobic SBRs (ASBRs) are partially emptied batch reactors that are widely used as bioprocesses in pollution control. We present dilution and biochemical materials balance modeling equations and simulation results for the partially emptied batch reactors, especially for ASBR treatment of low-strength wastewater. The simulated substrate and microbial concentrations for both dilution and materials balance equations follow the same pattern during both feeding and reaction times. However, the results of the materials balance equations show microbial activities during feeding as well as during reaction times and were found to be more appropriate for the biologic system in which substrate removal is associated with microbial growth. Further-more, the simulation results point to the need to foster high microbial accumulation in the system during startup to optimize the process performance and the need to operate the system at a short reaction time, especially for low substrate concentrations. The results were found to be in agreement with the results of prior laboratory studies.     

Aerobic and anaerobic microbial degradation of poly-beta-hydroxybutyrate produced by Azotobacter chroococcum

Food industry wastewater served as a carbon source for the synthesis of poly-β-hydroxybutyrate (PHB) by Azotobacter chroococcum. The content of polymer in bacterial cells grown on the raw materials reached 75%. PHB films were degraded under aerobic, microaerobic, and anaerobic conditions in the presence and absence of nitrate by microbial populations of soil, sludges from anaerobic and nitrifying/denitrifying reactors, and sediment from a sludge deposit site. Changes in molecular mass, crystallinity, and mechanical properties of PHB were studied. Anaerobic degradation was accompanied by acetate formation, which was the main intermediate utilized by denitrifying bacteria or methanogenic archaea. On a decrease in temperature from 20 to 5° C in the presence of nitrate, the rate of PHB degradation was 7.3 times lower. Under anaerobic conditions and in the absence of nitrate, no PHB degradation was observed, even at 11°C. The enrichment cultures of denitrifying bacteria obtained from soil and anaerobic sludge degraded PHB films for a short time (3–7 d). The dominant species in the enrichment culture from soil were Pseudomonas fluorescens and Pseudomonas stutzeri. The rate of PHB degradation by the enrichment cultures depended on the polymer molecular weight, which reduced with time during biodegradation.     

The application of heat conduction microcalorimetry to study the metabolism and pharmaceutical modulation of cultured mammalian cells

The heat produced by animal cells in culture can be used as the primary indicator of the kinetics of their metabolism because the scalar flux of it is a function of the metabolic flux. The validity of the relationship between heat and metabolism was demonstrated theoretically through the concept of thermal advancement and in experiments by the use of continuous cultures. This validation permitted the application of heat flux as a probe of the metabolic state of cells in culture. It consisted of an on-line heat conduction microcalorimeter that measures the instantaneous heat flow and dividing the smoothed signal with one obtained simultaneously using a dielectric spectrometer that records the change in capacitance as an estimate of the amount of viable biomass. In this mini-review, it is shown with Chinese hamster ovary cells (CHO320) genetically engineered to produce interferon-γ (IFN-γ) that heat flux is an early signal of deteriorating metabolism in cultures that produce considerable amounts of toxic lactate under fully aerobic conditions. The early detection favours the use of heat flux as the control variable in fed-batch cultures. This is a particularly useful finding in the context of the pharmaceutical industry because it will help to ensure the high fidelity of the cytokines, antibodies and vaccines produced in large-scale cultures. The monotonic relationship between the fluxes for heat and metabolism means that the enthalpy balance method can be employed to test the validity of the growth reaction for cells in culture. This showed that the crucial ratio between the substrates, glucose and glutamine, in the culture medium was incorrect at 5.5:1 instead of about 3:1, depending on the phase of the culture. Together with other changes to the medium composition, an improved formulation was made that ensured faster cell growth and greater specific rate (flux) of IFN-γ constitutive secretion while decreasing glucose utilisation and, most importantly, halving the excretion of lactate, that is toxic to the cells and harmful to the fidelity of their secondary products. Indirect calorimetry (oxygen uptake rate, OUR) is often favoured over the direct technique, but the former only measures aerobic metabolism. The environmental conditions in cultures favours lactate production even under fully aerobic conditions. Developments in measuring OUR mean that the stationary liquid phase balance can be used successfully to make the calorimetric:respirometric (CR) ratio a valuable tool in optimising cell culture to grow cells that synthesise the maximum amounts of the high fidelity secondary products.

Besides the value of heat flux in improving the cultures of animal cells producing heterologous products, three different techniques are examined that should be valuable in the testing the many compounds that are produced on a speculative basis as potential drugs. They are: (i) a thin-film thermopile transducer as an immunosensor; (ii) infra-red imaging of cells cultured in multi-well microtitre plates and (iii) integrated circuit (IC) calorimetry for small samples and low detection limit. One or more of these methods could well find favour with industry in the near future.     

Methylation of arsenic by anaerobic microbial consortia isolated from lake sediment

Anaerobic enrichment cultures, isolated from arsenic–contaminated lake sediment in the Canadian sub–arctic and grown in five selective media, methylated arsenate/arsenite to produce mono?, di? and tri–methyl arsenicals. The extent of methylation and methylarsenic species produced varied with the type of enrichment. Iron–reducing, manganese–reducing, sulfate–reducing and broad–spectrum anaerobic heterotrophic mixed cultures all produced methylarsenicals. Sulfate–reducing cultures produced higher concentrations of methylarsenicals (especially trimethyl species) than iron- or maganese–reducers. There is evidence that several of the methylarsenicals, which were hydride–reactive at pH 6, were methylarsenic(III) thiols. The organoarsenicals produced by enrichment cultures were the same as those detected in the porewater of the lake sediments used to initiate the enrichment cultures. Overall, this study demonstrates that microbes from anaerobic lake sediments can methylate (and demethylate) arsenic, a capability shared by manganese?, iron?, and sulfate–reducing microbial consortia.     

Potentiometric measurement of lipoic acid reduction in microbial cultures: Part I. Influence of bacterial growth on the potential-time signals

In this paper, we investigate the theoretical relations between microbial growth and the time evolution of the zero-current potential of a gold electrode in microbial cultures provided with exogenic lipoic acid. A linear relationship was elicited by computer calculation between simple potential versus time data and inverses of apparent growth rates of organisms. To verify this relation experimentally, we carried out potential-time measurements in cultures supplied with increasing concentrations of different sugars, and followed the kinetics of bacterial growth by classical photometric measurements. Judging from the theory, the potentiometric and growth photometric data were in good agreement. These results show that the potentiometric data may be used as reliable indexes for bacterial growth.     

Heat flux and the calorimetric-respirometric ratio as measures of catabolic flux in mammalian cells

It is advocated that cellular heat flow rate (Ø = dQ/dt, where Q is heat) be expressed as an intensive quantity specific to cell size (X) and termed heat flux (J_Ø/X). It has been the practice to cite such data on a ‘per cell’ basis, but it would be preferable to use biomass (cellular volume or mass). This quantity is shown to be a measure of metabolic activity and, more accurately, catabolic rate coupled to the demand for ATP in anabolic processes and work in the cell. Recent developments in flow microcalorimetry and dielectric spectroscopy reveal that heat flux can be measured on-line, with the potential of industrial use as a control variable in the growth of hybridoma and genetically engineered cells. This is because the enthalpy change of growth can be regarded as a unique kind of stoichiometric coefficient directly related to the mass coefficients in the growth reaction. This can be verified by an enthalpy balance comparing data for material fluxes of catabolites with the value for heat flux. Information revealed by the stoichiometric growth equation can be used to improve medium design.

The ratio of heat flux to oxygen consumption (flux) is known as the calorimetric-respirometric (CR) ratio. It detects anaerobic processes when the value is more negative than −450 (±5%) kJ mol⁻¹ O₂. These processes are found in cells growing under fully aerobic conditions, because glycolysis provides biosynthetic precursors with lactate as the byproduct. It is suggested that the CR ratio would be a powerful on-line control variable for the growth of animal cells in bioreactors.     

Biosynthesis of Poly-(3-hydroxybutyrate) under the Control of an Anaerobically Induced Promoter by Recombinant Escherichia coli from Sucrose

Poly-(3-hydroxybutyrate) (PHB) is a polyester with biodegradable and biocompatible characteristics and has many potential applications. To reduce the raw material costs and microbial energy consumption during PHB production, cheaper carbon sources such as sucrose were evaluated for the synthesis of PHB under anaerobic conditions. In this study, metabolic network analysis was conducted to construct an optimized pathway for PHB production using sucrose as the sole carbon source and to guide the gene knockout to reduce the generation of mixed acid byproducts. The plasmid pMCS-sacC was constructed to utilize sucrose as a sole carbon source, and the cascaded promoter P_3nirB was used to enhance PHB synthesis under anaerobic conditions. The mixed acid fermentation pathway was knocked out in Escherichia coli S17-1 to reduce the synthesis of byproducts. As a result, PHB yield was improved to 80% in 6.21 g/L cell dry weight by the resulted recombinant Escherichia coli in a 5 L bed fermentation, using sucrose as the sole carbon source under anaerobic conditions. As a result, the production costs of PHB will be significantly reduced.     

Biological Hydrogen Production Using Chloroform-treated Methanogenic Granules

In fermentative hydrogen production, the low-hydrogen-producing bacteria retention rate limits the suspended growth reactor productivity because of the long hydraulic retention time (HRT) required to maintain adequate bacteria population. Traditional bacteria immobilization methods such as calcium alginate entrapment have many application limitations in hydrogen fermentation, including limited duration time, bacteria leakage, cost, and so on. The use of chloroform-treated anaerobic granular sludge as immobilized hydrogen-producing bacteria in an immobilized hydrogen culture may be able to overcome the limitations of traditional immobilization methods. This paper reports the findings on the performance of fed-batch cultures and continuous cultures inoculated with chloroform-treated granules. The chloroform-treated granules were able to be reused over four fed-batch cultures, with pH adjustment. The upflow reactor packed with chloroform-treated granules was studied, and the HRT of the upflow reactor was found to be as low as 4 h without any decrease in hydrogen production yield. Initial pH and glucose concentration of the culture medium significantly influenced the performance of the reactor. The optimum initial pH of the culture medium was neutral, and the optimum glucose concentration of the culture medium was below 20 g chemical oxygen demand/L at HRT 4 h. This study also investigated the possibility of integrating immobilized hydrogen fermentation using chloroform-treated granules with immobilized methane production using untreated granular sludge. The results showed that the integrated batch cultures produced 1.01 mol hydrogen and 2 mol methane per mol glucose. Treating the methanogenic granules with chloroform and then using the treated granules as immobilized hydrogen-producing sludge demonstrated advantages over other immobilization methods because the treated granules provide hydrogen-producing bacteria with a protective niche, a long duration of an active culture, and excellent settling velocity. This integrated two-stage design for immobilized hydrogen fermentation and methane production offers a promising approach for modifying current anaerobic wastewater treatment processes to harvest hydrogen from the existing systems.     

Quantitative methods for the gas chromatographic characterization of acidic fermentation by-products of anaerobic bacteria

The present report describes improved chromatographic procedures which are capable of separating and quantitating complex mixtures of acidic fermentation by-products produced by anaerobic bacteria grown in two glucose-containing media. These methods are reliable and are sensitive, being able to detect as low as 0.5 mumoles of each by-product. Sample preparation has been simplified, and the methylation conditions have been optimized. It is also indicated in this investigation that each culture produced different patterns of by-products in each medium, indicating that the types and quantities of by-products produced in one medium cannot be used as a basis for characterization of these same cultures when grown in a different medium. Finally, it is shown that cultures can be characterized by the distinctive molar proportions of by-products they produce within each medium.     

Analytical determination of the microbial utilization and transformation of humic acids extracted from municipal refuse

Humic substances are usually the refractory part of natural organic matter, and in a landfill they can retain inorganic and organic micropollutants. This study has investigated analytically whether humic acids (HA) extracted by use of alkali from either fresh municipal refuse or from refuse disposed of in a landfill for up to 12 months can resist microbial degradation under aerobic conditions. When added as a supplementary nutrient source, up to 63.6% of HA was utilized and this percentage was enhanced to a mean value of 88.5% when different HA preparations were used as the sole source of carbon. In cultures of a soil microbial community containing the same preparations as sole sources of nitrogen, HA was usually completely utilized. The remaining HA re-isolated from some microbial cultures were highly depleted in carbon and, simultaneously, the nitrogen content was enhanced. The FTIR spectra were indicative of strong participation of aliphatic structural units in the refuse-related HA preparations. Because of the microbial activity, different carbonaceous substances were primarily removed from the HA structure, and an increase in nitrogenous molecular groups became apparent. The structural transformations brought about by soil microorganisms "in vitro" corresponded to those occurring naturally in HA obtained from refuse aged for 12 months in a landfill.