Endo-exo Synergism in Cellulose Hydrolysis Revisited

Synergistic cooperation of different enzymes is a prerequisite for efficient degradation of cellulose. The conventional mechanistic interpretation of the synergism between randomly acting endoglucanases (EGs) and chain end-specific processive cellobiohydrolases (CBHs) is that EG-generated new chain ends on cellulose surface serve as starting points for CBHs. Here we studied the hydrolysis of bacterial cellulose (BC) by CBH TrCel7A and EG TrCel5A from Trichoderma reesei under both single-turnover and "steady state" conditions. Unaccountable by conventional interpretation, the presence of EG increased the rate constant of TrCel7A-catalyzed hydrolysis of BC in steady state. At optimal enzyme/substrate ratios, the "steady state" rate of synergistic hydrolysis became limited by the velocity of processive movement of TrCel7A on BC. A processivity value of 66 ± 7 cellobiose units measured for TrCel7A on (14)C-labeled BC was close to the leveling off degree of polymerization of BC, suggesting that TrCel7A cannot pass through the amorphous regions on BC and stalls. We propose a mechanism of endo-exo synergism whereby the degradation of amorphous regions by EG avoids the stalling of TrCel7A and leads to its accelerated recruitment. Hydrolysis of pretreated wheat straw suggested that this mechanism of synergism is operative also in the degradation of lignocellulose. Although both mechanisms of synergism are used in parallel, the contribution of conventional mechanism is significant only at high enzyme/substrate ratios.     

Production of lignocellulose-degrading enzymes and changes in soil bacterial communities during the growth ofPleurotus ostreatus in soil with different carbon content

The extracellular enzyme activity and changes in soil bacterial community during the growth of the ligninolytic fungus Pleurotus ostreatus were determined in nonsterile soil with low and high available carbon content. In soil with P. ostreatus, the activity of ligninolytic enzymes laccase and Mn-peroxidase was several orders of magnitude higher than in soil without the fungus. Addition of lignocellulose to soil increased the activity of cellulolytic fungi and the production of Mn-peroxidase by P. ostreatus. The counts of heterotrophic bacteria were more significantly affected by the presence of lignocellulose than by P. ostreatus. The effects of both substrate addition and time (succession) were more significant factors affecting the soil bacterial community than the presence of P. ostreatus. Bacterial community structure was affected by fungal colonization in low carbon soil, where a decrease of diversity and changes in substrate utilization profiles were detected.     

Metabolic load of recombinant protein production: inhibition of cellular capacities for glucose uptake and respiration after induction of a heterologous gene in Escherichia coli

The strong expression of recombinant proteins in bacteria affects the primary carbon and energy metabolism resulting in growth inhibition and acetate formation. By applying glucose pulses to fed-batch fermentations performed for production of a heterologous (alpha-glucosidase in Escherichia coli, we show that the induction of the recombinant gene strongly inhibits the maximum specific uptake capacities for glucose and the respiration capacity. The accumulation of glucose in the fermentation medium promotes the growth of plasmid-free cells. These inhibition effects are well described by including the kinetics of product formation into a recently published dynamic model (Lin et al. [2001] Biotechnol Bioeng 73:349-357). The new model also includes the population characteristics and gives a good fit to the measured data describing growth, production, substrate consumption, by-product formation, and respiration.     

Nutrient Acquisition and Population Growth of a Mixotrophic Alga in Axenic and Bacterized Cultures

Axenic growth of a mixotrophic alga, Ochromonas sp., was compared in several inorganic and organic media, and in the presence of live bacteria under nutrient-replete and low-nutrient conditions. Axenic growth in the light was negligible in inorganic media with or without the addition of glucose. Addition of vitamins increased growth rate, but average cell size declined, resulting in no net increase in biomass. Supplementing axenic cultures with a more complex organic substrate resulted in moderate growth and higher maximal abundance (and biomass) than in the inorganic media with added vitamins. The absence of light did not greatly affect population growth rate in the presence of complex dissolved organic compounds, although cell size was significantly greater in the light than in the dark. The highest growth rates for the alga (up to 2.6 d-1) were measured in treatments containing live bacteria. Increases in cell number of Ochromonas sp. in the presence of bacterial prey were similar in the light and dark, although chloroplast and cell sizes differed. Bacterial abundance was reduced and dissolved phosphorus and ammonia were rapidly released in bacterized cultures in the light and dark, indicating high rates of bacterial ingestion and suggesting an inability of the alga to store or utilize N and P in excess of the quantities required for heterotrophic growth. Low-nutrient conditions in the presence of bacteria were promoted by adding glucose to stimulate bacterial growth and the uptake of N and P released by algal phagotrophy. Subsequent decreases in dissolved N and P following the addition of glucose corresponded to a second period of rapid growth of the alga in both light and dark. This result, combined with evidence for slow axenic growth of this strain, indicated that nutrient acquisition for this species in the presence of bacteria was accomplished primarily via ingestion of bacteria.     

Influence of environmental factors on endo-β-1,4-glucanase production by Bacillus HR68, isolated from a Zimbabwean hot spring

The production of endo-β-1,4-glucanase by a Bacillus strain isolated from a hot spring in Zimbabwe was studied in batch culture, chemostat culture, and carbon dioxide-regulated auxostat (CO₂-auxostat). The bacteria produced the enzyme in the presence of excess glucose or sucroso, but not under carbon-limited conditions in a chemostat using mineral medium. There was a specific growth rate dependent linear increase in enzyme production in glucose excess, nitrogen-limited chemostat cultures. A high specific growth rate of 2.2 h^-1 and a high rate of enzyme production of 362 nkat (mg dry mass h)^-1 were attained under nutrient rich conditions in the CO₂-auxostat. The bacteria had the highest specific growth rate and endo-β-1,4-glucanase enzyme production at 50° C. The maximum specific growth rate and the rate of enzyme production increased when yeast extract and tryptone were added in increasing amounts to the mineral medium used for cultivation in separate experiments. Increasing the glucose concentration in the CO₂-auxostat cultures increased the rate of enzyme production but did not affect the specific growth rate.     

Improvement of corn stover bioconversion efficiency by using plant glycoside hydrolase

Plant cell wall is the most abundant substrate for bioethanol production, and plants also represent a key resource for glycoside hydrolase (GH). To exploit efficient way for bioethanol production with lower cellulase loading, the potential of plant GH for lignocellulose bioconversion was evaluated. The GH activity for cell wall proteins (CWPs) was detected from fresh corn stover (FCS), and the synergism of which with Trichoderma reesei cellulase was also observed. The properties for the GH of FCS make it a promising enzyme additive for lignocellulose biodegradation. To make use of the plant GH, novel technology for hydrolysis and ethanol fermentation was developed with corn stover as substrate. Taking steam-exploded corn stover as substrate for hydrolysis and ethanol fermentation, compared with T. reesei cellulase loaded alone, the final glucose and ethanol accumulation increased by 60% and 63% respectively with GH of FCS as an addition.     

木质纤维素的微生物降解

木质纤维素广泛存在于自然界中,因结构复杂,其高效降解需要多种微生物的协同互作,由于参与木质纤维素降解的微生物种类繁多,其协同降解机理尚不完全明确。随着微生物分子生物学和组学技术的快速发展,将为微生物协同降解木质纤维素机制的研究提供新的方法和思路。笔者前期研究发现,细菌复合菌系在50℃下表现出强大的木质纤维素降解能力,菌系由可分离培养和暂时不可分离培养细菌组成,但是可分离培养细菌没有降解能力。通过宏基因组和宏转录组研究表明,与木质纤维素降解相关的某些基因表达量发生显著变化,通过组学方法有可能更加深入解释微生物协同降解木质纤维素的微生物学和酶学机理。文中从酶、纯培养菌株和复合菌群三个方面综述了木质纤维素微生物降解研究进展,着重介绍了组学技术在解析复合菌群作用机理方面的现状和应用前景,以期为探索微生物群落协同降解木质纤维素的机理提供借鉴。     

Rheological investigation of synergistic interactions between galactomannans and non-pectic polysaccharide fraction from water soluble yellow mustard mucilage

Synergistic interactions between galactomannans (GMs) and non-pectic polysaccharides (NPP) from yellow mustard mucilage were investigated in the present study. Structural analysis revealed that NPP was mainly composed of β-1,4-d glucosidic linkage. Four types of GMs, namely fenugreek gum (FG), guar gum (GG), tara gum (TG) and locust bean gum (LBG) with mannose to galactose ratios (M/G) of 1.2, 1.7, 3.0 and 3.7, respectively, were blended with NPP at various ratios. The viscoelastic properties of the mixtures were measured in order to evaluate the effects of GM/NPP blending ratio, M/G ratio, total polysaccharide concentration and pH on the synergistic interactions. Results revealed that at a total polysaccharide concentration of 0.5% (w/w), the highest synergism occurred at the GM/NPP blending ratio of 3/7 for all four types of GMs. The interaction between TG and NPP showed the highest synergy, followed by LBG/NPP, FG/NPP and GG/NPP. At a higher total polysaccharide concentration (1.0% w/w), the mixture of TG and NPP still exhibited the highest synergy, however, the order of synergy between NPP and other GMs was changed as FG/NPP, LBG/NPP and GG/NPP. At the total polysaccharide concentration of 0.5% and GM/NPP blending ratio of 3/7, neutral pH (pH 6.5) showed the strongest synergy compared to that at pH 2.0 and pH 12.0. The mechanism of the synergistic effects could be explained by a combination of segregative association model and junction zone model.     

Synergistic proteins for the enhanced enzymatic hydrolysis of cellulose by cellulase

Reducing the enzyme loadings for enzymatic saccharification of lignocellulose is required for economically feasible production of biofuels and biochemicals. One strategy is addition of small amounts of synergistic proteins to cellulase mixtures. Synergistic proteins increase the activity of cellulase without causing significant hydrolysis of cellulose. Synergistic proteins exert their activity by inducing structural modifications in cellulose. Recently, synergistic proteins from various biological sources, including bacteria, fungi, and plants, were identified based on genomic data, and their synergistic activities were investigated. Currently, an up-to-date overview of several aspects of synergistic proteins, such as their functions, action mechanisms and synergistic activity, are important for future industrial application. In this review, we summarize the current state of research on four synergistic proteins: carbohydrate-binding modules, plant expansins, expansin-like proteins, and Auxiliary Activity family 9 (formerly GH61) proteins. This review provides critical information to aid in promoting research on the development of efficient and industrially feasible synergistic proteins.     

Identification of extracellular proteins from actinomycetes responsible for the solubilisation of lignocellulose

Summary We have utilized strains of three actinomycete species, Actinomadura sp, Streptomyces cyaneus and Thermomonospora mesophila, to study the solubilisation of lignocellulose. The production of extracellular proteins, was measured for each of the organisms during 17 days growth using medium containing either glucose or ball-milled straw. Some of the extracellular proteins (as identified by SDS gel electrophoresis) were present under both growth conditions, but others were specific to the type of medium or the period of incubation. The levels of proteins were compared with the abilities of the extracellular protein preparations to solubilise a substrate of ¹⁴C-labelled lignocellulose. About 6% of the radioactive material were solubilised when the extracellular proteins from the cultures grown on glucose were incubated with the substrate, compared to 20–30% that were solubilised by the extracellular proteins from the cultures grown on ball-milled straw. Partial characterisation of an enzyme from S. cyaneus responsible for the solubilisation of lignocellulose was achieved by gel filtration of the extracellular proteins, using Superose 12. Material that eluted from the column with an apparent molecular weight of about 20 000 accounted for all of the solubilisation of ¹⁴C-labelled (i.e. lignin-derived) moieties. In contrast, when the eluate was tested for the presence of cellulases and xylanases most of the activities were found in fractions containing material with an apparent molecular weight of about 45 000. We conclude that in cultures of S. cyaneus grown on ball-milled straw, a single extracellular enzyme is responsible for the solubilisation of lignin in lignocellulose, and that this enzyme is unlikely to be a cellulase or a xylanase.     

A metabolic approach to study algal–bacterial interactions in changing environments

Increasing evidence exists that bacterial communities interact with and shape the biology of algae and that their evolutionary histories are connected. Despite these findings, physiological studies were and still are generally carried out with axenic or at least antibiotic‐treated cultures. Here, we argue that considering interactions between algae and associated bacteria is key to understanding their biology and evolution. To deal with the complexity of the resulting ‘holobiont’ system, a metabolism‐centred approach that uses combined metabolic models for algae and associated bacteria is proposed. We believe that these models will be valuable tools both to study algal–bacterial interactions and to elucidate processes important for the acclimation of the holobiont to environmental changes.     

Role of planktonic and sessile extracellular metabolic byproducts on <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> intra and interspecies relationships

Bacterial species are found primarily as residents of complex surface-associated communities, known as biofilms. Although these structures prevail in nature, bacteria still exist in planktonic lifestyle and differ from those in morphology, physiology, and metabolism. This study aimed to investigate the influence of physiological states of Pseudomonas aeruginosa and Escherichia coli in cell-to-cell interactions. Filtered supernatants obtained under planktonic and biofilm cultures of each single species were supplemented with tryptic soy broth (TSB) and used as the growth media (conditioned media) to planktonic and sessile growth of both single- and two-species cultures. Planktonic bacterial growth was examined through OD₆₄₀ measurement. One-day-old biofilms were evaluated in terms of biofilm biomass (CV), respiratory activity (XTT), and CFU number. Conditioned media obtained either in biofilm or in planktonic mode of life triggered a synergistic effect on planktonic growth, mainly for E. coli single cultures growing in P. aeruginosa supernatants. Biofilms grown in the presence of P. aeruginosa biofilms-derived metabolites presented less mass and activity. These events highlight that, when developed in biofilm, P. aeruginosa release signals or metabolites able to prejudice single and binary biofilm growth of others species and of their own species. However, products released by their planktonic counterparts did not impair biofilm growth or activity. E. coli, living as planktonic or sessile cultures, released signals and metabolites or removed un-beneficial compounds which promoted the growth and activity of all the species. Our findings revealed that inter and intraspecies behaviors depend on the involved bacteria and their adopted mode of life.     

Regulation of Bacterial Growth Rates by Dissolved Organic Carbon and Temperature in the Equatorial Pacific Ocean

Abstract The effect of dissolved organic matter (DOM) and temperature on bacterial production was examined in the equatorial Pacific Ocean. Addition of glucose, glucose plus ammonium, or free amino acids stimulated bacterial production ([³H]thymidine incorporation), whereas changes in bacterial abundance were either negligible or much less than changes in bacterial production. The average bacterial growth rate also greatly increased following DOM additions, whereas in contrast, addition of ammonium alone never affected production, bacterial abundance, or growth rates. Since the large glucose effect was not observed in previous studies of cold oceanic waters, several experiments were conducted to examine DOM-temperature interactions. These experiments suggest that bacteria respond more quickly and to a greater extent to DOM additions at higher temperatures, which may explain apparently conflicting results from previous studies. We also examined how temperate affects the kinetic parameters of sugar uptake. Maximum uptake rates (V_max) of glucose and mannose increased with temperature (Q₁₀= 2.4), although the half-saturation constant (K_m) was unaffected; K_m+ S was roughly equal to glucose concentrations (S) measured by a high pressure liquid chromographic technique. Bacterial production and growth rates appear to be limited by DOM in the equatorial Pacific, and thus bacterial production follows primary production over large spatial and temporal scales in this oceanic regime, as has been observed in other aquatic systems. Although temperature may not limit bacterial growth rates in the equatorial Pacific and similar warm waters, it could still affect how bacteria respond to changes in DOM supply and help set steady-state DOM concentrations. Received: 26 July 1995; Revised: 19 January 1996     

细菌降解木质素的研究进展

木质素是自然界最丰富的芳香化合物,其分解与陆地上碳循环密切相关。提取木质纤维素中的葡萄糖使其转化成乙醇,是生产第二代生物能源的关键步骤。但是由于木质素是一种非常稳定的化合物,难以降解是实现生物乙醇转化的主要屏障,因此关于木质素的生物降解研究具有非常重要的意义。真菌降解木质素的研究已经深入的进行了多年,并取得丰富的成果,但是关于细菌降解木质素的研究还处在初级阶段。由于广泛的生长条件和良好的环境适应能力,细菌在木质素降解方面深受研究人员的关注。本文通过总结前人的研究成果,讨论了木质素的降解机制、代谢途径及细菌降解木质素的工业应用前景,同时还展望了分子生物学及生物信息学在木质素降解方面的应用前景。     

Solvent production from xylose

Xylose is the second most abundant sugar derived from lignocellulose; it is considered less desirable than glucose for fermentation, and strategies that specifically increase xylose utilization in wild type or engineered cells are goals for biofuel production. Issues arise with xylose utilization because of carbohydrate catabolite repression, which is the preferential utilization of glucose relative to xylose in fermentations with both pure and mixed cultures. Taken together the low substrate utilization rates and solvent yields with xylose compared to glucose, many industrial fermentations ignore the xylolytic portion of the reaction in lieu of methods to maintain high glucose. This is shortsighted given the massive potential for xylose generation from a number of sustainable biomass feedstocks, based on utilization of the hemicellulose fraction(s) that enter pretreatment. A number of strategies have been developed in recent years to address xylose utilization and solvent production from xylose in systems with just xylose, or in systems with mixtures of glucose plus xylose, which are more typical of pretreated lignocellulose. The approaches vary in terms of complexity, stability, and ease of introduction to existing fermentation infrastructure (i.e., so-called drop-in fermentation strategies). Some approaches can be considered traditional engineering approaches (e.g., change the reaction conditions), while others are more subtle cellular approaches to eliminate the impacts of catabolite repression. Finally, genetic engineering has been used to increase xylose utilization, although this can be considered a relatively nascent approach compared to manipulations completed to date for glucose utilization.

     

Detection of interactions between yeasts and lactic acid bacteria isolated from sugary kefir grains

Different techniques were tested for studying the synergism between the micro-organisms of sugary kefir grains. Agar cultures in Petri dishes did not give reproducible results. In sequential cultures, i.e. growing one organism, sterile filtering and then inoculating the other, 10 of 18 selected lactic acid bacteria/yeast pairs revealed stimulation of bacterial growth in a poor glucose medium. In mixed culture, Saccharomyces florentinus supported better survival of Lactobacillus hilgardii and a significant increase in lactic acid production; at the same time, the growth and alcoholic fermentation of S. florentinus were drastically reduced. The inter-relationships between these two strains were the same when immobilized in calcium alginate beads, even though total metabolite production was always lower than with free cells. The stimulation of Lact. hilgardii by Candida lambica in sequential culture was not confirmed in mixed culture, where the two organisms grew as in pure culture, and bacterial growth and lactic acid production were inhibited in the immobilized system.