Fundamental aspects of waste sewage sludge treatment: Microbial solids biodegradation in an aerobic thermophilic semi-continuous system

The solubilization and biodegradation of whole microbial cells by an aerobic thermophilic microbial population was investigated over a 72 h period. Various parameters were followed including total suspended solids reduction, changes in the dissolved organic carbon, protein and carbohydrate concentrations, and carboxylic acid production and utilisation. From the rates of removal of the various fractions a simple model for the biodegradation processes is proposed and verified with respect to acetic acid production and utilization, and total suspended solids removal. The process is initiated by enzymic degradation of the substrate microbe cell walls followed by growth on the released soluble substrates at low dissolved oxygen concentration with concommitant carboxylic acid production. Subsequent utilization of the unbranched, lower molecular weight carboxylic acids allows additional energy supply following exhaustion of the easily utilisable soluble substrate from microbial cell hydrolysis.List of Symbols Y _Xp/Xs kg/kg yield process microbes on substrate yeast cells - Y _Xp/Ac kg/kg yield process microbes on acetate - Y _Ac/Ss kg/kg yield acetate produced by process microbes growing on substrate yeast cells - Y _Ss/Xs kg/kg yield soluble substrate from lysis of yeast cells - Y _Ss/Xp kg/kg yield soluble substrate from lysis of process microbes - Y _P/Xs kg/kg yield particulates from lysis of yeast cells - Y _P/Xp kg/kg yield particulates from lysis of process microbes - _{max (Ss)} h^–1 maximum specific growth rate constant for growth of process microbes on soluble substrate - _{max (Ac)} h^–1 maximum specific growth rate constant for growth of process microbes on acetate - Ks _Ss kg/m³ saturation coefficient for growth of process microbes on soluble substrate - Ks _Ac kg/m³ saturation coefficient for growth of process microbes on acetate - K _d h^–1 death/lysis rate constant for process microbes - K _i kg/m³ inhibition constant for growth of process microbes on acetate - K _L h^–1 lysis rate constant for whole yeast cells - K _h h^–1 hydrolysis rate constant for particulate biomass     

Direct Image-Based Enumeration of Clostridium phytofermentans Cells on Insoluble Plant Biomass Growth Substrates

A dual-fluorescent-dye protocol to visualize and quantify Clostridium phytofermentans ISDg (ATCC 700394) cells growing on insoluble cellulosic substrates was developed by combining calcofluor white staining of the growth substrate with cell staining using the nucleic acid dye Syto 9. Cell growth, cell substrate attachment, and fermentation product formation were investigated in cultures containing either Whatman no. 1 filter paper, wild-type Sorghum bicolor, or a reduced-lignin S. bicolor double mutant (bmr-6 bmr-12 double mutant) as the growth substrate. After 3 days of growth, cell numbers in cultures grown on filter paper as the substrate were 6.0- and 2.2-fold higher than cell numbers in cultures with wild-type sorghum and double mutant sorghum, respectively. However, cells produced more ethanol per cell when grown with either sorghum substrate than with filter paper as the substrate. Ethanol yields of cultures were significantly higher with double mutant sorghum than with wild-type sorghum or filter paper as the substrate. Moreover, ethanol production correlated with cell attachment in sorghum cultures: 90% of cells were directly attached to the double mutant sorghum substrate, while only 76% of cells were attached to wild-type sorghum substrate. With filter paper as the growth substrate, ethanol production was correlated with cell number; however, with either wild-type or mutant sorghum, ethanol production did not correlate with cell number, suggesting that only a portion of the microbial cell population was active during growth on sorghum. The dual-staining procedure described here may be used to visualize and enumerate cells directly on insoluble cellulosic substrates, enabling in-depth studies of interactions of microbes with plant biomass.     

Aboveground and belowground responses to quality and heterogeneity of organic inputs to the boreal forest

Leaf litter and other organic resources returned to the soil are important regulators of ecological processes in forest ecosystems, and their ecological impacts may be strongly influenced both by their quality and by interactions between coexisting resource types. To date, most studies on effects of resource identity and mixing have only involved leaf litter, despite the fact that other resource types constitute a major input to the soil. We investigated how quality and heterogeneity of organic substrates found in boreal forests affects the activity and community structure of soil microbes, and plant growth. Six organic substrates (wood, charcoal, berries, sporocarps, vertebrate faeces and leaf litter) were added singly or in mixtures of two, three and six resource types to pots containing forest soil (with or without tree seedlings of Betula pendula Roth). The largest positive effects of single substrates on microbial basal respiration (BR), substrate-induced respiration (SIR) and microbial metabolic quotient (qCO₂) were found for nutrient-rich substrates (faeces and sporocarps) or substrates with high sugar-content (berries). Mixing of substrates had no effect on BR or SIR, but decreased qCO₂ or altered the microbial community structure for specific combinations of substrates. In contrast to the niche complementarity hypothesis, microbial catabolic diversity was not stimulated by greater diversity of resources. Seedling growth responses to single substrates were neutral or negative; the inhibition of growth probably resulted largely from microbial competition for nutrients. Substrate mixing enhanced seedling nutrient-uptake and growth for all mixtures containing sporocarps and leaf litter. Overall, plants responded more strongly to resource heterogeneity than microbes, and synergistic effects only occurred when nutrient-rich substrates were present within the substrate mixtures. In particular, our results demonstrate a role for complex and non-additive interactions among previously overlooked resource types returned to the soil in influencing ecosystem functions such as nutrient cycling and plant productivity.     

Outlook for cellulase improvement: screening and selection strategies

Cellulose is the most abundant renewable natural biological resource, and the production of biobased products and bioenergy from less costly renewable lignocellulosic materials is important for the sustainable development of human beings. A reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitution of cellulase components. Here, we review quantitative cellulase activity assays using soluble and insoluble substrates, and focus on their advantages and limitations. Because there are no clear relationships between cellulase activities on soluble substrates and those on insoluble substrates, soluble substrates should not be used to screen or select improved cellulases for processing relevant solid substrates, such as plant cell walls. Cellulase improvement strategies based on directed evolution using screening on soluble substrates have been only moderately successful, and have primarily targeted improvement in thermal tolerance. Heterogeneity of insoluble cellulose, unclear dynamic interactions between insoluble substrate and cellulase components, and the complex competitive and/or synergic relationship among cellulase components limit rational design and/or strategies, depending on activity screening approaches. Herein, we hypothesize that continuous culture using insoluble cellulosic substrates could be a powerful selection tool for enriching beneficial cellulase mutants from the large library displayed on the cell surface.     

The cellulosome concept as an efficient microbial strategy for the degradation of insoluble polysaccharides.

The cellulosome is an extracellular supramolecular machine that can efficiently degrade crystalline cellulosic substrates and associated plant cell wall polysaccharides. The cellulosome arrangement can also promote adhesion to the insoluble substrate, thus providing individual microbial cells with a direct competitive advantage in the utilization of the soluble hydrolysis products.     

Extracellular respiration

Although it has long been known that microbes can generate energy using diverse strategies, only recently has it become clear that a growing number involve electron transfer to or from extracellular substrates. The best-known example of what we will term 'extracellular respiration' is electron transfer between microbes and minerals, such as iron and manganese (hydr)oxides. This makes sense, given that these minerals are sparingly soluble. What is perhaps surprising, however, is that a number of substrates that might typically be classified as 'soluble' are also respired at the cell surface. There are several reasons why this might be the case: the substrate, in its ecological context, might be associated with a solid surface and thus effectively insoluble; the substrate, while soluble, might simply be too large to transport inside the cell; or the substrate, while benign in one redox state, might become toxic after it is metabolized. In this review, we discuss various examples of extracellular respiration, paying particular attention to what is known about the molecular mechanisms underlying these processes. As will become clear, much remains to be learned about the biochemistry, cell biology and regulation of extracellular respiration, making it a rich field of study for molecular microbiologists.     

Kinetic model for microbial uptake of insoluble solid-state substrate

A kinetic model for anaerobic digestion of insoluble solid-state substrates was developed. Rate equations for cell growth and substrate consumption were derived based on the assumption that the microorganisms assimilate the substrate mainly at the point of contact where they grow. The model emphasizes effects of substrate particle size, organic loading, and cell concentration on the rates of cell growth and substrate utilization. Batch digestion of a stearic acid emulsion with a mean particle size of 2.0 mum and a biological sludge was conducted at 30 and 37 degrees C to verify the proposed model. Agreement between the experimental and calculated results indicated the validity of the model for describing the microbial degradation of insoluble solid-state substrates. Further examinationof the model revealed that with low cell substrate affinity or at low cell concentration, it coincided with a Michaelis-Menten type kinetics in which the effect of particle size was taken into consideration.     

Behavior of microbial communities developed in the presence/reduced level of soluble microbial products

Soluble microbial products (SMP) are organics produced by microorganisms as they degrade substrates. The available literature does not reveal how SMP affect and regulate microbial activities. In this study, we monitored variations in pH, dissolved oxygen concentration, soluble biological and chemical oxygen demands (sBOD₅ and sCOD) as a measure of microbial activity in synthetic wastewater. Aerobic degradation tests were carried out under the following conditions: aeration, 1,500 cm³ /min; initial sBOD₅, 515±5 mg/l; initial sCOD, 859±6 mg/l; initial biomass concentration (defined as mixed liquor suspended solids), 1,200±25 mg/l; sludge retention time, 24 h; and temperature, 20±1°C. The study involved non-acclimated biomass (R0 flora), biomass developed in the presence of SMP (R1 flora), and biomass developed in reduced level of SMP (R2 flora). We also determined which of these flora produced more refractory SMP. The results showed that R2 flora utilized the synthetic feed more quickly, and produced less refractory organic matter than R0 and R1 flora. The production of more refractory organics by R0 and R1 flora shows that not all the biomass was active. R1 flora degraded the substrates irregularly, suggesting that some microbes were dependent on the metabolic products of those that could utilize the feed components. These results show that production of SMP also depends on the prior substrates and on the ability of the flora to respond to changes in substrate composition.     

Microbial community development on model particles in the deep sulfidic waters of the Black Sea

Microorganisms attached to particles have been shown to be different from free-living microbes and to display diverse metabolic activities. However, little is known about the ecotypes associated with particles and their substrate preference in anoxic marine waters. Here, we investigate the microbial community colonizing particles in the anoxic and sulfide-rich waters of the Black Sea. We incubated beads coated with different substrates in situ at 1000 and 2000 m depth. After 6 h, the particle-attached microbes were dominated by Gamma- and Alpha-proteobacteria, and groups related to the phyla Latescibacteria, Bacteroidetes, Planctomycetes and Firmicutes, with substantial variation across the bead types, indicating that the attaching communities were selected by the substrate. Further laboratory incubations for 7 days suggested the presence of a community of highly specialized taxa. After incubation for 35 days, the microbial composition across all beads and depths was similar and primarily composed of putative sulfur cycling microbes. In addition to the major shared microbial groups, subdominant taxa on chitin and protein-coated beads were detected pointing to specialized microbial degraders. These results highlight the role of particles as sites for attachment and biofilm formation, while the composition of organic matter defined a secondary part of the microbial community.     

Synergetic effect of pH and biochemical components on bacterial diversity during mesophilic anaerobic fermentation of biomass-origin waste

Aims: To investigate the synergetic effect of pH and biochemical components on bacterial community structure during mesophilic anaerobic degradation of solid wastes with different origins, and under acidic or neutral conditions. Methods and Results: The bacterial community in 16 samples of solid wastes with different biochemical compositions and origins was evaluated during mesophilic anaerobic degradation at acidic and neutral pH. Denaturing gradient gel electrophoresis (DGGE) and single‐strand conformation polymorphism (SSCP) were used to compare the communities. Multivariate analysis of the DGGE and SSCP results revealed that most of the dominant microbes were dependent on the content of easily degradable carbohydrates in the samples. Furthermore, the dominant microbes were divided into two types, those that preferred an acid environment and those that preferred a neutral environment. A shift in pH was found to change their preference for medium substrates. Although most of the substrates with similar origin and biochemical composition had similar microbial diversity during fermentation, some microbes were found only in substrates with specific origins. For example, two microbes were only found in substrate that contained lignocellulose and animal protein without starch. These microbes were related to micro‐organisms that are found in swine manure, as well as in other intestinal or oral niches. In addition, the distribution of fermentation products was less sensitive to the changes in pH and biochemical components than the microbial community. Conclusions: Bacterial diversity during anaerobic degradation of organic wastes was affected by both pH and biochemical components; however, pH exerted a greater effect. Significance and Impact of the Study: The results of this study reveal that control of pH may be an effective method to produce a stable bacterial community and relatively similar product distribution during anaerobic digestion of waste, regardless of variation in the waste feedstocks.     

Microbial Activity in Caves−A Geological Perspective

Caves are commonly the home of diverse microbial biotas, the sites of active mineral precipitation, and/or receptacles for the deposition of sediment. Mineral precipitation is commonly considered to be abiogenic despite the fact that microbes are present in caves, especially in the twilight zone. Detailed analysis of cave substrates from a geological perspective shows that microbes can mediate constructive (microbe calcification, trapping and binding, mediation of crystal growth) and destructive (substrate etching and breakdown) processes. Potentially these processes can significantly influence the formation and preservation of any cave deposit. Preservation of microbes is possible if mineralization takes place while the microbe is alive or shortly after its demise. If not, all record of the microbe will be lost to decay. Even if the microbes are preserved, it may be difficult to determine if they played an active or passive role in the formation of the deposits in which they are entombed. For old cave deposits, such an assessment must rely on spatial relationships and comparison of textures with those known to form as a result of microbial activity. Nevertheless, available evidence indicates that microbes can play a major role in the formation and modification of cave deposits. Equally, however, it is apparent that the full scope and impact of microbial activity on cave deposits has yet to be realized. Recognition of microbial activity in old CaCO 3 cave deposits relies on (1) documentation and recognition of mineralized microbes, (2) recognition of stromatolitic structures that formed through microbial activity, and/or (3) the identification of fabrics/textures that are known to be indicative of microbial activity. All of these criteria fundamentally rely on the interpretation of fabrics preserved in the cave deposits. Virtually all of these interpretations are open to debate.     

Charcoal as a habitat for microbes and its effect on the microbial community of the underlying humus

Wildfires produce a charcoal layer, which has an adsorbing capacity resembling activated carbon. After the fire a new litter layer starts to accumulate on top of the charcoal layer, which liberates water‐soluble compounds that percolate through the charcoal and the unburned humus layer. We first hypothesized that since charcoal has the capacity to adsorb organic compounds it may form a new habitat for microbes, which decompose the adsorbed compounds. Secondly, we hypothesized that the charcoal may cause depletion of decomposable organic carbon in the underlying humus and thus reduce the microbial biomass. To test our hypotheses we prepared microcosms, where we placed non‐heated humus and on top one of the adsorbents: non‐adsorptive pumice (Pum), charcoal from Empetrum nigrum (EmpCh), charcoal from humus (HuCh) or activated carbon (ActC). We watered them with birch leaf litter extract. The adsorbing capacity increased in the order Pumorg in the litter extract, respectively. After one month, all adsorbents harboured microbes, but their amount and basal respiration was largest in EmpCh and HuCh, and smallest in Pum. In addition, different kinds of microbial communities with respect to their phospholipid fatty acid and substrate utilization patterns were formed in the adsorbents. The amount of microbial biomass and number of bacteria did not differ between humus under different adsorbents, although different microbial communities developed in humus under EmpCh compared with Pum, which is obviously related to the increased pH of the humus under EmpCh, and also ActC. We suggest that charcoal from burning can support microbial communities, which are small in size but have a higher specific growth rate than those of the humus. Although the charcoal layer induces changes in the microbial community of the humus, it does not reduce the amount of humus microbes.     

Simple model for the energetics of growth on substrates with different degrees of reduction

A simple model is developed for the energy transformation in growing microbial systems. The model is based on a linear equation for ATP consumption in the processes of growth and maintenance. A combination of this equation with macroscopic balances for the various components, the systems exchanges with the environment, and application of the concepts of the elementary balance allow the derivation of linear equations for the exchange of substrate, oxygen, and carbon dioxide with the environment. For growth on one sole carbon and energy source the model allows the definition of a critical substrate yield are expected and below which is decreasing substrate yield and energy supply growth limitation are expected. This restriction can be interpreted in a variety of other ways. It supplies a rationale for non-energy-production-coupled transfer of hydrogen to oxygen or wasteful expenditure of ATP in growth on highly reduced substrates. It also allows the formulation of a limit to the maximum yield on oxygen that can never be exceeded in growth on highly reduced substrates.     

Effect of monensin on rumen metabolism in vitro.

The effect of Monensin (Rumensin, Eli Lilly & Co.) in incubations with mixed rumen microorganisms metabolizing carbohydrate or protein substrates was investigated. Monensin partly inhibited methanogenesis and increased propionate production, although the effect was not always statistically significant. Incubations with substrates specific for methane bacteria suggest that inhibition of methanogenesis by Monensin was not due to a specific toxic action on the methanogenic flora, but rather to an inhibition of hydrogen production from formate. Total and net microbial growth were considerably decreased by addition of Monensin, although the amount of substrate fermented was not altered, resulting in lowered values of microbial growth efficiency. In incubations with casein, Monensin lowered protein degradation in line with a lowered ammonia production, whereas a slight accumulation of alpha-amino nitrogen was observed. The results suggest that besides an influence of Monensin on the rumen carbohydrate fermentation pattern, another reason for the beneficial effects observed in vivo might be decreased food protein degradation in the rumen, altering the final site of protein digestion in the animal. Also, the possibility of a decrease in rumen microbial growth efficiency has to be considered when using Monensin as a food additive.     

Influence of decomposer food web structure and nitrogen availability on plant growth

We studied the sensitivity of soil microbial communities and ecosystem processes to variation in the vertical and horizontal structure of decomposer food web under nitrogen poor and N-enriched conditions. Microcosms with humus and litter layer of boreal forest floor, birch seedlings infected with mycorrhizal fungi, and decomposer food webs with differing trophic group and species composition of soil fauna were constructed. During the second growing period for the birch, we irrigated half of the microcosms with urea solution, and the other half with de-ionised water to create two levels of N concentration in the substrate. During the experiment night time respirations of the microcosms were measured, and the water leached through the microcosms was analysed for concentration of mineral N, and nematode numbers. The microcosms were destructively sampled after 37 weeks for plant biomass and N uptake, structure of soil animal and microbial community (indicated by PLFA profiles), and physical and chemical properties of the humus and litter materials. Predatory mites and nematodes had a negative influence on the biomass of their microbivorous and microbi-detritivorous prey, and microbi-detritivores affected the biomass and community structure of microbes (indicated by PLFA-analysis). Moreover, predatory mites and nematodes increased microbial biomass and changed the microbial community structure. The decomposer food web structure affected also N uptake and growth of plants. Microbi-detritivorous fauna had a positive effect, whereas predators of microbial and detritus feeding fauna exerted a negative influence on plant N uptake and biomass production. The impact of a trophic group on the microbes and plant was also strongly dependent on species composition within the group. Nitrogen addition magnified the influence of food web structure on microbial biomass and plant N uptake. We suggest that addition of urea-N to the soil modified the animal-microbe interaction by increasing microbial growth and altering community structure of microbes. The presence of microbi-detritivores and predators reduced loss of carbon from the microcosms, and the food web structure influenced also water holding capacity of the materials. The changes in plant growth, nutrient cycling, size of N and C pools, and in the physical properties of the soil emphasize the importance and diversity of indirect consequences of decomposer food web structure. This revised version was published online in June 2006 with corrections to the Cover Date.     

Changes in soil heterotrophic respiration,carbon availability,and microbial function in seven forests along a climate gradient

Soil microbial communities play an essential role in soil carbon (C) emission and C sequestration in forest ecosystems. However, little information is available regarding the relationship between soil C dynamics and microbial substrate utilization at large scales. Along the North–South Transect of Eastern China (NSTEC), seven forests representative of boreal, temperate and tropical biomes were examined. Soil heterotrophic respiration (R_h), soil dissolved organic C (DOC), microbial biomass C (MBC), and microbial community-level physiological profiles (CLPPs) were investigated using biochemical measurements, static chamber-gas chromatography analysis, and Biolog-Eco microplates, respectively. We found that soil R_h rates were significantly higher in subtropical and boreal forests than in temperate forests. Conversely, the concentrations of soil DOC and MBC, as well as microbial metabolic activity and functional diversity, were consistently higher in temperate forests than in subtropical forests. There were considerably different substrate utilization profiles among the boreal, temperate, and subtropical forests. Soil microorganisms from the temperate and boreal forests mainly metabolized high-energy substrates, while those from the subtropical forests used all substrates equally. In addition, soil R_h rates were significantly and negatively related to soil labile C concentrations, total metabolic activity, and the intensity of individual substrate utilization, indicating that soil microbes assimilated more soil substrates, thereby reducing CO₂ emissions. Overall, our study suggests that climate factors, as well as substrate availability, dominate the activities and functions of soil microbes at large scales.     

微生物胞外呼吸电子传递机制研究进展

胞外呼吸是近年来发现的新型微生物厌氧能量代谢方式,主要包括铁呼吸、腐殖质呼吸与产电呼吸3种形式。微生物胞外呼吸与传统的有氧呼吸、胞内厌氧呼吸存在显著差异。其电子受体多以固态形式存在于胞外;氧化产生的电子必须通过电子传递链从胞内转移到细胞周质和外膜,并通过外膜上的细胞色素c、纳米导线或自身产生的电子穿梭体等方式,最终将电子传递至胞外的末端受体。胞外呼吸的本质问题是微生物与胞外电子受体(铁/锰氧化物、固态电极或腐殖质等)的相互作用,即微生物如何将胞内电子传递至胞外受体。胞外呼吸的研究丰富了人们对微生物呼吸多样性的认识,同时在污染物原位修复及清洁生物能源提取方面具有重要应用前景,是当前研究的热点问题。总结了胞外呼吸类型和胞外呼吸菌的多样性,重点阐述了胞外呼吸的电子传递过程,并提出了其应用前景及今后的研究方向。