Substrate-to-biomass ratio as a process control parameter in heterogeneous populations growing on complex substrates

The removal of a complex substrate bovine serum albumin (BSA) by activated sludge was examined with respect to the initial level of substrate adsorbed, the rate of substrate removal, and the induction of exoprotease (located both on the sludge matrix and in the cell-free supernatant). The relationship of these various processes, which together make up the overall removal process, was investigated with respect to the level of substrate present expressed both per unit volume and per unit biomass. A higher degree of correlation was observed when the substrate per unit biomass (S/B) ratio was used, thus providing some basis for empirical models of the activated sludge system based on the food-to-microorganisms (F/M) ratio. The interaction of sludge concentration and substrate concentration, plus the fact that there is not a clear distinction, as in pure culture, between substrate and microorganisms, makes the use of models devised for pure culture questionable.     

Enzymatic hydrolysis of lime-pretreated corn stover and investigation of the HCH-1 Model: inhibition pattern, degree of inhibition, validity of simplified HCH-1 Model

The inhibition pattern was identified for a reaction system composed of Trichoderma reesei cellulase enzyme complex and lime-pretreated corn stover. Also, the glucose inhibition effect was quantified for the aforementioned reaction system over a range of enzyme loadings and substrate concentrations. Lastly, the range of substrate concentrations and enzyme loadings were identified in which the linear form of the simplified HCH-1 Model is valid. The HCH-1 Model is a modified Michaelis-Menton Model with non-competitive inhibition and the fraction of insoluble substrate available to bind with enzyme. With a high enzyme loading, the HCH-1 Model can be integrated and simplified in such a way that sugar conversion is linearly proportional to the logarithm of enzyme loading. A wide range of enzyme loadings (0.25-50 FPU/g dry biomass) and substrate concentrations (10-100g/L) were investigated. All experiments were conducted with an excess cellobiase loading to ensure the experimental results were not influenced by cellobiose inhibition. A non-competitive inhibition pattern was identified for the corn stover-cellulase reaction system, thereby validating the assumptions of the HCH-1 Model. At a substrate concentration of 10 g/L, glucose inhibition parameters of 0.986 and 0.979 were measured for enzyme loadings of 2 FPU/g dry biomass and 50 FPU/g dry biomass, respectively. At 5 FPU/g dry biomass, glucose inhibition parameters of 0.985 and 0.853 were measured for substrate concentrations of 10 and 100g/L, respectively. The linear form of the HCH-1 Model predicted biomass digestibility for lime-pretreated corn stover over an enzyme loading range of 0.25-50 FPU/g dry biomass and substrate concentration range of 10-100g/L.     

生物质多糖的高效降解与降解酶（系）的精确定制

自然界中多糖类生物质资源十分丰富,然而其复杂的抗降解屏障限制了生物转化的进程.近年来,随着生物质多糖结构的快速解析以及大量多糖降解酶的鉴定研究,针对不同底物结构或产物需求,仿制高效微生物多糖代谢途径,精确定制多糖降解酶系,促进生物质高效转化已成为可能.本文分析中性多糖(纤维素和木聚糖)、碱性多糖(几丁质和壳聚糖)以及酸性多糖(褐藻胶)的精细结构组成与基团性质,总结3类多糖主要降解酶的活性架构特征及其底物精确结合模式.文章还阐述蛋白质工程设计与定制策略,针对酶分子不同功能区的分析,可为酶分子的功能快速设计与改造提供靶点,以获得适宜于工业应用的高效酶分子,此外,根据微生物胞外降解酶系的降解次序与协同关系,可基于应用需求精确定制复杂多糖降解酶系,实现生物质的高效与高值降解转化.     

High-Throughput Screening Techniques for Biomass Conversion

High-throughput (HTP) screening of biomass or biomass-degrading enzymes, regardless of the desired outcome, is fraught with obstacles and challenges not typically faced in more traditional biotechnology. The enzyme systems are complex and synergistic and the substrate is highly heterogeneous, insoluble, and difficult to dispense. Digestions are often carried out for days at temperatures of 50°C or higher, leading to significant challenges regarding evaporation control in small well volumes. Furthermore, it is often desirable to condition or “pretreat” the biomass at extreme temperatures and/or pH to enhance enzyme digestibility. Once the substrate has been saccharified, evaluation of the extent and efficiency of conversion is made more difficult by time-consuming and tedious techniques used to measure the sugar products. Over the past decade or so, biomass researchers have creatively addressed these challenges by developing techniques to reduce biomass heterogeneity, uniformly distribute biomass samples at the small scale, pretreat the biomass at the small scale, quantitatively load these samples with enzymes, control evaporation of small reaction volumes for multiday incubations, and rapidly quantify the products. Other aspects of these measurements remain problematic and are being addressed. This review will address some of these challenges in detail, but more importantly, we will endeavor to educate the reader about the trials, tribulations, and pitfalls of carrying out HTP screening in biomass conversion research.     

On the influence of substrate morphology and surface area on phytofauna

The independent effects and interactions between substrate morphology and substrate surface area on invertebrate density or biomass colonizing artificial plant beds were assessed in a clear-water and a turbid playa lake in Castro County, Texas, USA. Total invertebrate density and biomass were consistently greater on filiform substrates than on laminar substrates with equivalent substrate surface areas. The relationship among treatments (substrates with different morphologies and surface areas) and response (invertebrate density or biomass) was assessed with equally spaced surface areas. Few statistically significant interactions between substrate morphology and surface area were detected, indicating that these factors were mostly independent from each other in their effect on colonizing invertebrates. Although infrequently, when substrate morphology and surface area were not independent, the effects of equally spaced changes in substrate surface area on the rate of change of phytofauna density or biomass per unit of substrate surface area were dependent upon substrate morphology. The absence of three-way interactions indicated that effects of substrate morphology and substrate area on phytofauna density or biomass were independent of environmental conditions outside and inside exclosures. Handling editor: D. Harper     

Crystal structures of Clostridium thermocellum xyloglucanase, XGH74A, reveal the structural basis for xyloglucan recognition and degradation

The enzymatic degradation of the plant cell wall is central both to the natural carbon cycle and, increasingly, to environmentally friendly routes to biomass conversion, including the production of biofuels. The plant cell wall is a complex composite of cellulose microfibrils embedded in diverse polysaccharides collectively termed hemicelluloses. Xyloglucan is one such polysaccharide whose hydrolysis is catalyzed by diverse xyloglucanases. Here we present the structure of the Clostridium thermocellum xyloglucanase Xgh74A in both apo and ligand-complexed forms. The structures, in combination with mutagenesis data on the catalytic residues and the kinetics and specificity of xyloglucan hydrolysis reveal a complex subsite specificity accommodating seventeen monosaccharide moieties of the multibranched substrate in an open substrate binding terrain.     

Ecological factors influencing macroinvertebrate standing crop distribution

Influence of substrate, macrophyte growth and detritus on macroinvertebrate standing crop (numbers and biomass) as well as seasonal variations in standing crop were investigated in a trout stream.Ephemeropterans showed no consistent relationship to substrate type, either in numbers or biomass. Numbers of dipteran larvae (primarily chironomids and simuliids) did not show a definite relationship to substrate size. But a larger biomass was associated with larger substrates. Higher numbers and biomass of trichopterans (including Helicopsyche borealis) were associated with larger substrates. Numbers of coleopteran larvae showed no relation to substrate size, but biomass was greater in large substrates. Biomass and numbers of water mites were generally associated with larger substrates, whereas no relationship was observed in plectopterans.Larger numbers and biomass of ephemeropterans, dipterans (chironomids and simuliids but no other dipterans) and trichopterans are associated with macrophyte beds; this is attributed to drifting species and those that have summer generations. Macrophyte growth reduced numbers and biomass of H. borealis, but no differences were observed in coleopterans or plecopterans.A substrate/detritus substitution study was done under natural stream conditions. An area of relatively constant current and depth was selected from which eighteen sites were randomly selected. Gravel and detritus were removed and replaced with two distinct sizes of substrate and three different levels of detritus. This was a 2 × 3 factorial analysis design. Ephemeropteran numbers were significantly related to substrate type and detritus level, whereas biomass was only related significantly to substrate. Numbers of chironomids and other dipterans responded significantly to substrate manipulation. Biomass of dipterans other than chironomids was significantly related to substrate type but not detritus level and chironomid biomass did not respond to either manipulations.Trichopteran numbers responded significantly to substrate and detritus manipulations, whereas biomass was significantly related only to substrate type. Analysis of variance showed that coleopteran numbers did not respond significantly to either manipulation; however, orthogonal contrasts showed that, within large substrate, medium detritus level was significantly different from high detritus level. Oligochaetes did not respond to either substrate or detritus manipulation. The responses of the organisms to manipulations are explained in relation to interstitial spaces and oxygen as well as the interaction between substrate type and amount of detritus.Michigan Agricultural Experiment Station Journal Article No. 5734.     

Towards predicting basin-wide invertebrate organic biomass and production in marine sediments from a coastal sea

Detailed knowledge of environmental conditions is required to understand faunal production in coastal seas with topographic and hydrographic complexity. We test the hypothesis that organic biomass and production of subtidal sediment invertebrates throughout the Strait of Georgia, west coast of Canada, can be predicted by depth, substrate type and organic flux modified to reflect lability and age of material. A basin-wide database of biological, geochemical and flux data was analysed using an empirical production/biomass (P/B) model to test this hypothesis. This analysis is unique in the spatial extent and detail of P/B and concurrent environmental measurements over a temperate coastal region. Modified organic flux was the most important predictor of organic biomass and production. Depth and substrate type were secondary modifiers. Between 69-74% of variability in biomass and production could be explained by the combined environmental factors. Organisms <1 mm were important contributors to biomass and production primarily in shallow, sandy sediments, where high P/B values were found despite low organic flux. Low biomass, production, and P/B values were found in the deep, northern basin and mainland fjords, which had silty sediments, low organic flux, low biomass of organisms <1 mm, and dominance by large, slow-growing macrofauna. In the highest organic flux and biomass areas near the Fraser River discharge, production did not increase beyond moderate flux levels. Although highly productive, this area had low P/B. Clearly, food input is insufficient to explain the complex patterns in faunal production revealed here. Additional environmental factors (depth, substrate type and unmeasured factors) are important modifiers of these patterns. Potential reasons for the above patterns are explored, along with a discussion of unmeasured factors possibly responsible for unexplained (30%) variance in biomass and production. We now have the tools for basin-wide first-order estimates of sediment invertebrate production.     

Structured modeling and state estimation in a fermentation process: Lipase production by Candida rugosa

A simple structured mathematical model coupled with a methodology of state and parameter estimation is developed for lipase production by Candida rugosa in batch fermentation. The model describes the system according to the following qualitative observations and hypothesis: Lipase production is induced by extracellular oleic acid present in the medium. The acid is transported into the cell where it is consumed, transformed, and stored. Lipase is excreted to the medium where it is distributed between the available oil-water interphase and aqueous phase. Cell growth is modulated by the intracellular substrate concentration. Model parameters have been determined and the whole model validated against experiments not used in their determination. The estimation problem consists in the estimation of three state variables (biomass, intra- and extracellular substrate) and two kinetic parameters by using only the on-line measurement provided by exhaust gas analysis. The presented estimation strategy divides the complex problem into three subproblems that can be solved by stable algorithms. The estimation of biomass (X) and the specific growth rate (mu), is achieved by a recursive prediction error algorithm using the on-line measurement of the carbon dioxide evolution rate. mu is then used to perform an estimation of intracellular substrate and the other kinetic parameter related to substrate transport (A) by an adaptive observer. Extracellular substrate is then evaluated by means of the estimated values of intracellular substrate and biomass through the material balance of the reactor. Simulation and experimental tests showed good performance of the developed estimator, which appears suitable to be used for process control and monitoring. (c) 1995 John Wiley & Sons, Inc.     

Measurements of the specific methanogenic activity of anaerobic digestor biomass

A specific methanogenic activity test was tested for its use as a simple procedure suitable for measurement of the activity of the various physiological groups of microorganisms involved in the terminal processes of methanogenesis from complex organic matter. Activity was estimated by supplying sufficient substrate (acetate, propionate, butyrate, H₂ or none) to saturate the catabolic systems of the various physiological groups, whereafter the specific methane production rate was determined. Activity was defined as the substrate-dependent methane production rate per unit mass of volatile solids biomass, i.e. the rate with a saturating concentration of substrate present when the background methane production rate had been diluted to an insignificant level. When the digestor was perturbed, the concentration of unused substrate in the biomass obscured the effect of added substrates on the test batches. It was generally found that if the background level of substrates could not be sufficiently lowered by dilution, substrate specific activity tests, as commonly described in the literature, were useless. Correspondence to: B. K. Ahring     

Indirect Effects of Nitrogen Amendments on Organic Substrate Quality Increase Enzymatic Activity Driving Decomposition in a Mesic Grassland

The fate of soil organic carbon (SOC) is determined, in part, by complex interactions between the quality of plant litter inputs, nutrient availability, and the microbial communities that control decomposition rates. This study explores these interactions in a mesic grassland where C and nitrogen (N) availability and plant litter quality have been manipulated using both fertilization and haying for 7 years. We measured a suite of soil parameters including inorganic N, extractable organic C and N (EOC and EON), soil moisture, extracellular enzyme activity (EEA), and the isotopic composition of C and N in the microbial biomass and substrate sources. We use these data to determine how the activity of microbial decomposers was influenced by varying levels of substrate C and N quality and quantity and to explore potential mechanisms explaining the fate of enhanced plant biomass inputs with fertilization. Oxidative EEA targeting relatively recalcitrant C pools was not affected by fertilization. EEA linked to the breakdown of relatively labile C rich substrates exhibited no relationship with inorganic N availability but was significantly greater with fertilization and associated increases in substrate quality. These increases in EEA were not related to an increase in microbial biomass C. The ratio of hydrolytic C:N acquisition enzymes and δ¹³C and δ¹⁵N values of microbial biomass relative to bulk soil C and N, or EOC and EON suggest that microbial communities in fertilized plots were relatively C limited, a feature likely driving enhanced microbial efforts to acquire C from labile sources. These data suggest that in mesic grasslands, enhancements in biomass inputs and quality with fertilization can prompt an increase in EEA within the mineral soil profile with no significant increases in microbial biomass. Our work helps elucidate the microbially mediated fate of enhanced biomass inputs that are greater in magnitude than the associated increases in mineral soil organic matter.     

Ecosystem development on Hawaiian lava flows: biomass and species composition

Abstract. The strong environmental gradients and ‘natural experimental design’ of Mauna Loa volcano, Hawaii, provide an outstanding opportunity to study controls on ecosystem development. We measured above-ground vascular plant biomass and species composition on 42 sites on which precipitation, temperature, substrate texture, and substrate age varied substantially and largely independently. Biomass and species richness of live plants were strongly correlated with precipitation and lava flow age, but not with temperature or lava flow texture. Species composition, as measured by correspondence analysis, was likewise correlated with precipitation and flow age, but composition was also strongly influenced by temperature. Lava texture had a complex effect on vegetation, with ‘a’ a lava favoring vegetation development on wet sites and pāhoehoe favoring development on dry sites. Many locations remain virtually free of invasion by alien species; aliens appear where disturbance has facilitated invasion, either from stand-level dieback in rainforest or a grass-fire cycle on the dry, leeward side of the mountain. All four of the environmental factors studied here (precipitation, temperature, substrate texture, and substrate age) exert significant and independent control over vegetation biomass and/or species composition on Mauna Loa.     

Modeling and static optimization of the ethanol production in a cascade reactor. I. Modeling

Let us consider the modeling of a cascade reactor for the production of ethanol. The rates of reaction in alcoholic fermentation are modeled so that it can assume both ethanol and substrate inhibition, in relation to the observed results.A nonstructured model, based on biomass, substrate, and ethanol concentrations, is developed and identified. It is a complex model, this being due to the nonlinearity between the specific rate of ethanol production and the growth rate and, on the other hand, the study of the static optimization of ethanol fermentation is performed.     

Evaluation of growth yield of Spirulina (Arthrospira) sp. in photoautotrophic, heterotrophic and mixotrophic cultures

In microbial cultures, both cellular growth rate and yield (defined as the degree of substrate conversion into the biomass) are important. Although effect of culture conditions on growth kinetics has been well documented for various microbial strains, there is almost no literature concerning the effect of environmental conditions on growth equilibrium, expressed as biomass yield coefficients from substrate. The present paper discusses the effect of culture conditions: irradiance (physical substrate) and glucose concentration (chemical substrate) on biomass yield coefficients from two chemical substrates: glucose and nitrate-nitrogen in photoautotrophic, heterotrophic and mixotrophic culture of blue-green alga Spirulina (Arthrospira) sp. The efficiency of substrates incorporation into the biomass can be precisely determined only if the elemental composition of the biomass is known. The experimental results showed that culture conditions had a substantial influence on biomass yield coefficients (biomass yield from glucose and nitrate-nitrogen). It was found that, the increase of irradiance favoured increase of biomass yield coefficient from both, glucose and nitrate-nitrogen. However, in the case of yield from nitrogen in mixotrophic culture, the effect was opposite. The effect of glucose concentration was different: the higher the initial glucose concentration, the lower the biomass yield coefficients from chemical substrates.     

Hydraulic parameters and benthic invertebrate distributions in two gravel-bed New Zealand rivers

1. Benthic invertebrates were sampled over a matrix of about eighty combinations of mean velocity (10–150cms^?1) and depth (10–150cm) in two rivers that differed in substrate size variability. Vertical velocity profiles were measured at each sample site and substratum roughness was measured and estimated from percentage cover by stone size classes. The influence of depth on periphyton biomass was also measured. 2. The hydraulic and substrate data were used to investigate the correlations between conventional (mean velocity, depth, substrate size) and complex hydraulic variables (Froude number, shear velocity, and water column and boundary Reynolds number) that were either calculated from direct measurements or inferred from mean velocity, depth, kinematic viscosity and substrate roughness. The ecological relevance of these hydraulic variables was investigated by comparing their degree of correlation with invertebrate densities and community metrics. 3. The invertebrate variables had similar correlations with mean velocity and the complex near-bed hydraulic variables in the river with uniform cobble substrates. In the river with diverse substrates, however, average correlations with Froude number, and inferred shear velocity and boundary Reynolds number were 25–45% higher than with velocity. Of all the individual hydraulic parameters, the boundary Reynolds number, calculated from simple measures, was most strongly correlated with benthic invertebrate distributions and taxa richness. However, invertebrate distributions were more strongly correlated with predictions of multiple regression models, incorporating substrate size, depth and mean velocity, than with any single hydraulic variable. 4. Hydraulic influences on food availability and oxygen concentration in the benthos are likely mechanisms affecting the hydraulic preferences of several taxa. Lower periphyton biomass with depth, partly attributable to light attenuation, appeared to have a non-hydraulic influence on a collector-browser species.     

Pressure drop increase by biofilm accumulation in spiral wound RO and NF membrane systems: role of substrate concentration,flow velocity,substrate load and flow direction

In an earlier study, it was shown that biofouling predominantly is a feed spacer channel problem. In this article, pressure drop development and biofilm accumulation in membrane fouling simulators have been studied without permeate production as a function of the process parameters substrate concentration, linear flow velocity, substrate load and flow direction. At the applied substrate concentration range, 100–400 μg l^?1 as acetate carbon, a higher concentration caused a faster and greater pressure drop increase and a greater accumulation of biomass. Within the range of linear flow velocities as applied in practice, a higher linear flow velocity resulted in a higher initial pressure drop in addition to a more rapid and greater pressure drop increase and biomass accumulation. Reduction of the linear flow velocity resulted in an instantaneous reduction of the pressure drop caused by the accumulated biomass, without changing the biofilm concentration. A higher substrate load (product of substrate concentration and flow velocity) was related to biomass accumulation. The effect of the same amount of accumulated biomass on the pressure drop increase was related to the linear flow velocity. A decrease of substrate load caused a gradual decline in time of both biomass concentration and pressure drop increase. It was concluded that the pressure drop increase over spiral wound reverse osmosis (RO) and nanofiltration (NF) membrane systems can be reduced by lowering both substrate load and linear flow velocity. There is a need for RO and NF systems with a low pressure drop increase irrespective of the biomass formation. Current efforts to control biofouling of spiral wound membranes focus in addition to pretreatment on membrane improvement. According to these authors, adaptation of the hydrodynamics, spacers and pressure vessel configuration offer promising alternatives. Additional approaches may be replacing heavily biofouled elements and flow direction reversal.     

Morphologically structured model for antitumoral retamycin production during batch and fed-batch cultivations of Streptomyces olindensis

A morphologically structured model is proposed to describe trends in biomass growth, substrate consumption, and antitumoral retamycin production during batch and fed-batch cultivations of Streptomyces olindensis. Filamentous biomass is structured into three morphological compartments (apical, subapical, and hyphal), and the production of retamycin, a secondary metabolite, is assumed to take place in the subapical cell compartment. Model accounts for the effect of glucose as well as complex nitrogen source on both the biomass growth and retamycin production. Laboratory data from bench-scale batch and fed-batch fermentations were used to estimate some model parameters by nonlinear regression. The predictive capability of the model was then tested for additional fed-batch and continuous experiments not used in the previous fitting procedure. The model predictions show fair agreement to the experimental data. The proposed model can be useful for further studies on process optimization and control.     

Biosorption of milk substrates onto anaerobic flocculent and granular sludge

Experiments were performed for adsorption of milk-based substrates onto anaerobic biomass at 35 degrees C. The influence of two parameters was studied, namely, the type of biomass (flocculent or granular) and the sludge adaptation to the substrate. It was found that flocculent sludge presented an adsorption capacity roughly 3 times higher than that of granular sludge. The adsorption data fit well with the Freundlich and Langmuir isotherms. Apparently, short-term sludge adaptation is not influential on the adsorption behavior. On the other hand, long-term adapted sludge showed a higher adsorption capacity than nonadapted sludge, which probably is an indirect effect of different microbial populations. These results suggest that the role of adsorption in the anaerobic treatment of complex substrates containing fat cannot be overlooked, especially for flocculent sludge systems, since organic matter accumulation could cause process failure due to biomass washout.