Modeling substrate inhibition of microbial growth

This article presents a general equation for substrate inhibition of microbial growth using a statistical thermodynamic approach. Existing empirical models adapted from enzyme kinetics, for example, the Haldane-Andrews equation, often criticized for not being physically based for microbial growth, are shown to derive from the general equation in this article, and their empirical parameters are shown to be well defined physically. Three sets of experimental data from the literature are used to test the modeling abilities of the general equation to represent experimental data. The results are compared with those obtained by fitting the same data set to a widely used empirical model existing in the literature. The general equation is found to represent all three experimental data sets better than the alternative model tested. In addition, a graphical method existing in enzyme kinetics is successfully adapted and further developed to determine the number of inhibition sites of a basic functional unit of a bacterial cell. (c) 1996 John Wiley & Sons, Inc.     

Determination of the Monod substrate saturation constant for microbial growth

Abstract Methods used to determine the Monod substrate saturation constant for microbial growth are surveyed. The preferred and most accurate method is to assay the concentrations of growth rate-limiting nutrients in steady-state continuous cultures. But, this is not always possible due to the lack of sufficiently sensitive assay methods or due to high nutrient fluxes in rapidly growing cultures. It is suggested that an acceptable and simple alternative method for aerobic microorganisms is to measure initial oxygen uptake rates during growth in the presence of different initial concentrations of growth rate-limiting nutrient. It is important in this method that the microbial cells are taken from rapidly growing cultures and are suspended in a medium permitting growth.     

Solid substrate fermentation of Monascus purpureus: growth, carbon balance, and consistency analysis

Solid substrate fermentation (SSF) of Monascus purpureus on rice is a promising new technology for obtaining natural pigments. However, before attempts can be made at maximizing pigment yield, all significant macroscopic compounds should be assayed. Here, Monascus purpureus has been grown on rice in batch mode, and the evolution of the main components, biomass, residual rice, O(2), CO(2), ethanol, acetic acid, and pigments, have been followed. This set of data, never previously studied for Monascus SSF, allowed both the performance of a macroscopic elemental balance, which accounted for 83-94% of the initial substrate carbon, and a check of data consistency. Standard consistency analysis showed a significant underestimation of the nitrogen fraction of biomass, but it was unable to discriminate the errors in the carbon balance as a result of the simultaneous presence of two gross errors in the system. A simple stoichiometric model in tandem with consistency analysis explained unaccounted carbon as an underestimation of CO(2) and ethanol. Using the simplified method to estimate ethanol, the macroscopic balance accounted for 87-99% of the initial carbon.     

Methane as a carbon substrate for the production of microbial cells

The cultivation of aerobic, methane-utilizing, microbial cells by submerged culture techniques, in an entirely mineral salts medium, with a view to their use as an edible protein source is discussed. Particular emphasis is placed on the potentially explosive nature of gaseous mixtures containing methane and oxygen. The experiments described investigate if fully safe operation at all times, by oxygen concentration control, is possible in agitated and sparged batch fermentors. Appreciable wastage of methane is prevented by gaseous-phase recirculation. It is concluded that fully safe operation is possible, cultures being able to grow exponentially without substrate limitation by the gaseous-phase nutrients.     

土壤微生物碳素利用效率研究进展

土壤微生物碳素利用效率(CUE)是指微生物将吸收的碳(C)转化为自身生物量C的效率,也称为微生物的生长效率。土壤微生物CUE是生态系统C循环中的重要生理生态学参数,影响着生态系统的C固持、周转、土壤矿化以及温室气体排放等过程。在全球环境变化背景下,认识土壤微生物CUE的变异及其影响机制,对于更好的认识生态系统C循环过程及其对全球变化的响应具有重要意义。概述了CUE的定义及其测定方法,重点综述和分析土壤微生物CUE的变异及影响因素取得的研究进展。基于现有研究的分析得出,土壤微生物CUE通常表示为微生物的生长与吸收的比值,分为基于微生物生长速率、微生物生物量、底物吸收速率和底物浓度变化等方法进行测定。土壤微生物CUE在0.2—0.8的范围内变化,这种变异主要受到来自热力学、生态环境因子、底物养分质量和有效性、化学计量平衡以及微生物群落组成的影响。今后土壤微生物CUE的研究应加强对微量代谢组分的定量分析,生物和环境要素交互影响的调控机理解析,以及微生物动态生理响应过程的碳循环模型优化。     

Plant influences on soil microbial carbon pump efficiency

Microbe-mediated carbon transformation plays an important role in soil carbon sequestration, which is considered to be one of the key strategies to achieve carbon neutrality in the long term. Assessing the efficiency of microbial necromass accumulation relative to plant carbon input or microbial respiration will help to identify ways to promote soil carbon sequestration from an ecosystem perspective.     

Correlation between cell composition and carbon conversion efficiency in microbial growth: a theoretical study

Summary As the macromolecular composition of microorganisms varies during their life cycle it was asked whether, and to what extent such changes exert any influence on substrate consumption, i.e. growth yield and carbon conversion efficiency, respectively. This question was dealt with in a theoretical study by use of the Y _APT ^max -concept. The growth substrates considered were methanol, acetate and glucose; the latter was assumed to be assimilated via both the glycolytic and the oxidative hexosemonophosphate pathway. Five fictitious biomasses were used which were altered in their proportion of polysaccharides, proteins, lipids, RNA and DNA. As a result, only small variations in the individual biomass formulae were obtained. On the basis of the energy balances for the syntheses of all cell constituents it was found that variations in the macromolecular composition of microbial biomass have only a slight effect on carbon conversion efficiency, amounting to maximally 3%. From the material balances it could be calculated that the upper, solely metabolism-determined limit of carbon conversion efficiency is 85% for substrates assimilated glycolytically via phosphoglycerate; for gluconeogenetic substrates, the upper limit was 75%. These limits are essentially determined by carbon loss on the way to amino acid syntheses.Abbreviations Ac acetate - CCE carbon conversion efficiency (%) - EMP Embden-Meyerhof-Parnas (glycolytic) pathway - Gluc glucose - HMP oxidative hexosemonophosphate pathway - m _e maintenance coefficient (mmol g^-1 h^-1) - MeOH methanol - PGA phosphoglycerate, Y, growth yield (g dry weight per g substrate) - Y _ATP growth yield (g dry weight per mole ATP) - specific growth rate (h^-1)     

Linear growth in microbial cultures limited by accumulated substrate

Summary The linear growth phase in cultures limited by intracellular (conservative) substrate is represented by a flat exponential curve. Within the range of experimental errors, the presented model fits well the data from both batch and continuous cultures ofEscherichia coli, whose growth is limited in that way.List of symbols D dilution rate, h^–1 - K_S saturation constant, g.L^–1 - S concentration of the limiting substrate, g.L^–1 - S_i concentration of the limiting substrate accumulated in the cells, g.g^–1 - S_o initial concentration of the limiting substrate, g.L^–1 - t time of cultivation, h - t₁ time of exhaustion of the limiting substrate from medium, h - t_o beginning of exponential phase, h - X biomass concentration, g.L^–1 - X₁ biomass concentration at the time of exhaustion of the limiting substrate from the medium, g.L^–1 - X_o biomass concn. at the beginning of exponential phase, g.L^–1 - biomass concn. at steady-state, g.L^–1 - Y growth yield coefficient (biomass/substrate) - specific growth rate, h^–1 - _m maximum specific growth rate, h^–1     

Calculation of microbial growth efficiency from15N immobilization

Microbial growth rate was estimated by multiplying¹⁵N immobilization by an estimated microbial C:N ratio. This growth rate, in combination with measurements of respiration, was used to calculate growth efficiency. Growth rates and efficiencies were calculated for grassland and cultivated soils of three textures. Calculated efficiencies (Y_c), assuming a microbial C: N ratio of 7, ranged from 32 to 54. Cultivated soils tended to have higher Y_c values than did grassland soils. This calculation depends on several hard-to-verify assumptions, but yields numbers that should be of great interest in comparative studies.     

Quantifying microbial growth and carbon use efficiency in dry soil environments via 18O water vapor equilibration

Soil microbial physiology controls large fluxes of C to the atmosphere, thus, improving our ability to accurately quantify microbial physiology in soil is essential. However, current methods to determine microbial C metabolism require liquid water addition, which makes it practically impossible to measure microbial physiology in dry soil samples without stimulating microbial growth and respiration (namely, the “Birch effect”). We developed a new method based on in vivo ¹⁸O‐water vapor equilibration to minimize soil rewetting effects. This method allows the isotopic labeling of soil water without direct liquid water addition. This was compared to the main current method (direct ¹⁸O‐liquid water addition) in moist and air‐dry soils. We determined the time kinetics and calculated the average ¹⁸O enrichment of soil water over incubation time, which is necessary to calculate microbial growth from ¹⁸O incorporation in genomic DNA. We tested isotopic equilibration patterns in three natural and six artificially constructed soils covering a wide range of soil texture and soil organic matter content. We then measured microbial growth, respiration and carbon use efficiency (CUE) in three natural soils (either air‐dry or moist). The proposed ¹⁸O‐vapor equilibration method provided similar results as the current method of liquid ¹⁸O‐water addition when used for moist soils. However, when applied to air‐dry soils the liquid ¹⁸O‐water addition method overestimated growth by up to 250%, respiration by up to 500%, and underestimated CUE by up to 40%. We finally describe the new insights into biogeochemical cycling of C that the new method can help uncover, and we consider a range of questions regarding microbial physiology and its response to global change that can now be addressed.     

氮添加土壤微生物群落结构影响微生物碳利用效率

尽管近年来中国氮(N)沉降水平逐渐趋于稳定,但中国东南地区N沉降相比于其他地区仍处于较高水平。N沉降对陆地生态系统碳循环过程的影响不容忽视。微生物碳利用效率(CUE)是指微生物将吸收的碳转化为生物量碳的效率,高微生物CUE意味着高土壤有机碳存储潜力。因此,探究N沉降背景下微生物CUE的变化将有助于进一步认识陆地生态系统土壤碳存储的变化。然而,目前关于N沉降下微生物群落结构的变化如何影响微生物CUE鲜有报道。在福建省泉州市戴云山国家级自然保护区的罗浮栲林通过N添加模拟N沉降。实验共包括三个N添加处理：对照(CT,+0 kg hm^-2 a^-1)、低氮(LN,+40 kg hm^-2 a^-1)和高氮(HN,+80 kg hm^-2 a^-1)。测定不同处理土壤基本理化性质、微生物生物量、酶活性和CUE,并使用高通量测序对微生物群落结构和多样性进行测定。结果表明,N添加显著影响微生物CUE,随着N添加水平的增加,CUE逐渐增加;相反,土壤pH、可提取有机碳(EOC)和微...     

Reduced carbon use efficiency and increased microbial turnover with soil warming

Global soil carbon (C) stocks are expected to decline with warming, and changes in microbial processes are key to this projection. However, warming responses of critical microbial parameters such as carbon use efficiency (CUE) and biomass turnover (rB) are not well understood. Here, we determine these parameters using a probabilistic inversion approach that integrates a microbial‐enzyme model with 22 years of carbon cycling measurements at Harvard Forest. We find that increasing temperature reduces CUE but increases rB, and that two decades of soil warming increases the temperature sensitivities of CUE and rB. These temperature sensitivities, which are derived from decades‐long field observations, contrast with values obtained from short‐term laboratory experiments. We also show that long‐term soil C flux and pool changes in response to warming are more dependent on the temperature sensitivity of CUE than that of rB. Using the inversion‐derived parameters, we project that chronic soil warming at Harvard Forest over six decades will result in soil C gain of <1.0% on average (1st and 3rd quartiles: 3.0% loss and 10.5% gain) in the surface mineral horizon. Our results demonstrate that estimates of temperature sensitivity of microbial CUE and rB can be obtained and evaluated rigorously by integrating multidecadal datasets. This approach can potentially be applied in broader spatiotemporal scales to improve long‐term projections of soil C feedbacks to climate warming.     

Preparation and testing of an autolysate of fish viscera as growth substrate for bacteria

The aqueous soluble phase of acidified and autolyzed fish viscera was used as the nitrogen source in a growth medium for bacteria. The bacteria tested grew faster and produced higher yields of cell mass on this growth medium than on corresponding media with standard tryptone preparations as the nitrogen source.     

Estimation of available efficiency of microbial growth on methanol and ethanol

The balances of reductivity and high-energy bonds (HEB) during microbial growth on glucose (a standard substrate), methanol, and ethanol are reported. Also, numerical values for the quantities of HEB formation in the respiratory metabolism, HEB consumption in the constructive metabolism, as well as in a number of the other intracellular processes are evaluated. Estimations of maximum cell yields by mass and energy are made during growth on methanol and ethanol with regard to peculiar features of different microbe metabolism.     

Simple absolute quantification method correcting for quantitative PCR efficiency variations for microbial community samples

Real-time quantitative PCR (qPCR) is a widely used technique in microbial community analysis, allowing the quantification of the number of target genes in a community sample. Currently, the standard-curve (SC) method of absolute quantification is widely employed for these kinds of analysis. However, the SC method assumes that the amplification efficiency (E) is the same for both the standard and the sample target template. We analyzed 19 bacterial strains and nine environmental samples in qPCR assays, targeting the nifH and 16S rRNA genes. The E values of the qPCRs differed significantly, depending on the template. This has major implications for the quantification. If the sample and standard differ in their E values, quantification errors of up to orders of magnitude are possible. To address this problem, we propose and test the one-point calibration (OPC) method for absolute quantification. The OPC method corrects for differences in E and was derived from the ΔΔC(T) method with correction for E, which is commonly used for relative quantification in gene expression studies. The SC and OPC methods were compared by quantifying artificial template mixtures from Geobacter sulfurreducens (DSM 12127) and Nostoc commune (Culture Collection of Algae and Protozoa [CCAP] 1453/33), which differ in their E values. While the SC method deviated from the expected nifH gene copy number by 3- to 5-fold, the OPC method quantified the template mixtures with high accuracy. Moreover, analyzing environmental samples, we show that even small differences in E between the standard and the sample can cause significant differences between the copy numbers calculated by the SC and the OPC methods.     

Increasing microbial carbon use efficiency with warming predicts soil heterotrophic respiration globally

The degree to which climate warming will stimulate soil organic carbon (SOC) losses via heterotrophic respiration remains uncertain, in part because different or even opposite microbial physiology and temperature relationships have been proposed in SOC models. We incorporated competing microbial carbon use efficiency (CUE)–mean annual temperature (MAT) and enzyme kinetic–MAT relationships into SOC models, and compared the simulated mass‐specific soil heterotrophic respiration rates with multiple published datasets of measured respiration. The measured data included 110 dryland soils globally distributed and two continental to global‐scale cross‐biome datasets. Model–data comparisons suggested that a positive CUE–MAT relationship best predicts the measured mass‐specific soil heterotrophic respiration rates in soils distributed globally. These results are robust when considering models of increasing complexity and competing mechanisms driving soil heterotrophic respiration–MAT relationships (e.g., carbon substrate availability). Our findings suggest that a warmer climate selects for microbial communities with higher CUE, as opposed to the often hypothesized reductions in CUE by warming based on soil laboratory assays. Our results help to build the impetus for, and confidence in, including microbial mechanisms in soil biogeochemical models used to forecast changes in global soil carbon stocks in response to warming.