Hydrogen generation via anaerobic fermentation of paper mill wastes

The objective of this work was to determine the hydrogen production from paper mill wastes using microbial consortia of solid substrate anaerobic digesters. Inocula from mesophilic, continuous solid substrate anaerobic digestion (SSAD) reactors were transferred to small lab scale, batch reactors. Milled paper (used as a surrogate paper waste) was added as substrate and acetylene or 2-bromoethanesulfonate (BES) was spiked for methanogenesis inhibition. In the first phase of experiments it was found that acetylene at 1% v/v in the headspace was as effective as BES in inhibiting methanogenic activity. Hydrogen gas accumulated in the headspace of the bottles, reaching a plateau. Similar final hydrogen concentrations were obtained for reactors spiked with acetylene and BES. In the second phase of tests the headspace of the batch reactors was flushed with nitrogen gas after the first plateau of hydrogen was reached, and subsequently incubated, with no further addition of inhibitor nor substrate. It was found that hydrogen production resumed and reached a second plateau, although somewhat lower than the first one. This procedure was repeated a third time and an additional amount of hydrogen was obtained. The plateaux and initial rates of hydrogen accumulation decreased in each subsequent incubation cycle. The total cumulative hydrogen harvested in the three cycles was much higher (approx. double) than in the first cycle alone. We coined this procedure as IV-SSAH (intermittently vented solid substrate anaerobic hydrogen generation). Our results point out to a feasible strategy for obtaining higher hydrogen yields from the fermentation of industrial solid wastes, and a possible combination of waste treatment processes consisting of a first stage IV-SSAH followed by a second SSAD stage. Useful products of this approach would be hydrogen, organic acids or methane, and anaerobic digestates that could be used as soil amenders after post-treatment.     

Studies on an acetate-fermenting strain of Methanosarcina.

An acetate-fermenting strain of Methanosarcina was isolated from an acetate enrichment culture inoculated with anaerobic sludge from a waste treatment digestor. In pure culture, this organism fermented acetate in the absence of added hydrogen at rates comparable in magnitude to those found in digestor systems. This rate was significantly higher than previously obtained for pure cultures of this genus. Mineral components of yeast extract were highly stimulatory for cultures growing on methanol. Comparable stimulation was not observed for cultures growing on acetate. Labeling studies indicated that acetate was converted to methane and CO2 as predicted by previous studies on mixed cultures. Total oxidation or reduction of acetate was not the mechanism of conversion of acetate to methane by the pure culture. The ability of this strain to form colonies or to produce methane from acetate was apparently influenced by the choice of substrate and conditions used for growing the inoculum.     

Fermentative hydrogen production from low-value substrates

Hydrogen is a promising energy source that is believed to replace the conventional energy sources e.g. fossil fuels over years. Hydrogen production methods can be divided into conventional production methods which depend mainly on fossil fuels and alternative production methods including electrolysis of water, biophotolysis and fermentation hydrogen production from organic waste materials. Compared to the conventional methods, the alternative hydrogen production methods are less energy intensive and negative-value substrates i.e. waste materials can be used to produce hydrogen. Among the alternative methods, fermentation process including dark and photo-fermentation has gained more attention because these processes are simple, waste materials can be utilized, and high hydrogen yields can be achieved. The fermentation process is affected by several parameters such as type of inoculum, pH, temperature, substrate type and concentration, hydraulic retention time, etc. In order to achieve optimum hydrogen yields and maximum substrate degradation, the operating conditions of the fermentation process must be optimized. In this review, two routes for biohydrogen production as dark and photo-fermentation are discussed. Dark/photo-fermentation technology is a new approach that can be used to increase the hydrogen yield and improve the energy recovery from organic wastes.     

Relation between substrate feeding pattern and development of filamentous bacteria in activated sludge processes: Part III. applications with industrial waste waters

Summary Laboratory scale activated sludge systems were operated under regimes of continuous or intermittent feeding of the waste water. Industrial waste waters from breweries, a dairy plant and a petro-chemical plant were investigated. The systems were started up with sludge from a municipal waste water plant or more often with sludges obtained from the corresponding industrial waste water treatment plants. It was found that intermittently fed systems produced sludges with better settleability characteristics than systems that were continuously supplemented with waste water. Our previous hypothesis that in intermittent systems floc forming bacteria become dominant as a result of higher substrate uptake rates was confirmed and may thus be extended to waste waters containing readily available substrates such as carbohydrates (brewery-and dairy waste water) or acids (petro-chemical waste water). Supplementation of brewery waste water with urea had a negative influence on sludge settleability, especially in continuously operated systems.     

Plug flow simulating and completely mixed reactors with a premixing tank,in the control of filamentous bulking

Summary A sequence of a substrate rich and substrate starvation phase (feast and famine strategy) is created in both compartmentalized reactors and reactors with premixing tanks. This condition is known to be essential in the control of bulking. With a synthetic waste water containing glucose a reactor with 12 compartments was effective in the control of filamentous bulking. With a dairy industrial waste water, containing a slowly biodegradable COD-fraction, this reactor could not suppress the growth of filamentous bacteria. With dairy and brewery waste water, reactors with premixing tanks were used to create a more pronounced exogenous (substrate rich) phase. Using three or more premixing tanks filamentous bulking could be controlled. During the exogenous phase the floc-forming microorganisms, having a higher substrate uptake rate, can take up the largest amount of substrate and can survive better during the endogenous phase.Substrate concentration and respiration rate profiles were studied and the concentration of the reserve materials was monitored.For each type of waste water the sludge loading and the fraction of readily available COD will determine the system's design.     

Biotechnological intensification of biogas production

The importance of syntrophic relationships among microorganisms participating in biogas formation has been emphasized, and the regulatory role of in situ hydrogen production has been recognized. It was assumed that the availability of hydrogen may be a limiting factor for hydrogenotrophic methanogens. This hypothesis was tested under laboratory and field conditions by adding a mesophilic (Enterobacter cloacae) or thermophilic hydrogen-producing (Caldicellulosyruptor saccharolyticus) strain to natural biogas-producing consortia. The substrates were waste water sludge, dried plant biomass from Jerusalem artichoke, and pig manure. In all cases, a significant intensification of biogas production was observed. The composition of the generated biogas did not noticeably change. In addition to being a good hydrogen producer, C. saccharolyticus has cellulolytic activity; hence, it is particularly suitable when cellulose-containing biomass is fermented. The process was tested in a 5-m³ thermophilic biogas digester using pig manure slurry as a substrate. Biogas formation increased at least 160–170% upon addition of the hydrogen-producing bacteria as compared to the biogas production of the spontaneously formed microbial consortium. Using the hydrogenase-minus control strain provided evidence that the observed enhancement was due to interspecies hydrogen transfer. The on-going presence of C. saccharolyticus was demonstrated after several months of semicontinuous operation.     

Technological aspects of the microbial treatment of sulfide-rich wastewaters: A case study

Thiobacillus denitrificans has been shown to be an effective biocatalyst for the treatment of a variety of sulfide-laden waste streams including sour water, sour gases, and refinery spent-sulfidic caustics. The term 'sour' originated in the petroleum industry to describe a waste contaminated with hydrogen sulfide or salts of sulfide and bisulfide. The microbial treatment of sour waste streams resulting from the production or refining of natural gas and crude oil have been investigated in this laboratory for many years. The application of this technology to the treatment of sour wastes on a commercially useful scale has presented several technical barriers including substrate inhibition (sulfide), product inhibition (sulfate), the need for septic operation, biomass recycle and recovery, mixed waste issues, and the need for large-scale cultivation of the organism for process startup. The removal of these barriers through process improvements are discussed in terms of a case study of the full-scale treatment of sulfide-rich wastewater. The ability of T. denitrificans to deodorize and detoxify an oil-field produced water containing sulfides was evaluated under full-scale field conditions at Amoco Production Co. Salt Creek Field in Midwest, WY. More than 800 m³/d of produced water containing 100 mg/L sulfide and total dissolved solids of 4800 mg/L were successfully biotreated in an earthen pit (3000 m³) over a six-month period. Complete removal of sulfides and elimination of associated odors were observed. The system could be upset by severe hydraulic disturbances; however, the system recovered rapidly when normal influent flow rates were restored.     

产氢菌Enterobacter sp.和Clostridium sp.的分离鉴定及产氢特性

在高温水体中分离得到2株具有较高产氢活性的微生物菌株Z-16和C-32。根据两菌株的16SrDNA序列分析,初步鉴定菌株Z-16为Enterobactersp.,菌株C-32为Clostridiumsp.。研究了起始pH值、反应温度、碳源等对菌株放氢活性的影响。菌株Z-16的最适产氢条件为:反应系统起始pH7·0,反应温度35℃,以蔗糖为产氢底物。在最适条件下,菌株Z-16的氢转化率为2·68molH2/mol蔗糖。菌株C-32的最适产氢条件为:反应系统起始pH8·0,反应温度35℃,以麦芽糖为产氢底物。在最适条件下,菌株C-32的氢转化率为2·71molH2/mol麦芽糖。以葡萄糖为碳源时,菌株Z-16和菌株C-32的氢转化率分别为2·35和2·48molH2/mol葡萄糖。     

Impacts of sterilization, microwave and ultrasonication pretreatment on hydrogen producing using waste sludge

Hydrogen production by sterilization, microwave and ultrasonication pretreated waste sludge was investigated in this study. A new strain of Pseudomonas sp. GZ1 (EF551040) was inoculated in pretreated waste sludge to produce hydrogen. The experimental results showed that different pretreated sludge had evident differences in the yield of hydrogen production and lag time. Sterilized sludge had the largest yield of hydrogen production, and the maximum yield was 15.02 ml/gTCOD. The lag time of using sterilized sludge was 15 h, longer than other two pretreated sludge. Using the ultrasonicated sludge, the hydrogen production yield was smallest and lag time was shortest in the three pretreated sludge. Protein and carbohydrate could be released from waste sludge by pretreatment. Protein was the main nutrient used for hydrogen production. The concentration of protein, carbohydrate and SCOD increased after pretreatment and fermentation. The impacts of different pretreatments on hydrogen production were also discussed in detail.     

Solid-state cultivation of Bacillus thuringiensis R 176 with shrimp shells and rice straw as a substrate for chitinase production

In this study, shrimp shell powder, prepared by treating shrimp-processing waste by boiling and crushing, was used as a substrate for isolation of chitinase-producing microorganism. These organisms may have an important economic role in the biological control of rice and other fungal pathogens. Two hundred strains of bacteria with the ability to degrade chitin from shrimp shell waste were isolated from paddy soil, and of these, 40 strains showed chitinase activity in a solid state cultivation. One of the most potent isolates (strain R 176) was identified as Bacillus thuringiensis. Identification was carried out using morphological and biochemical properties along with 16S rRNA sequence analysis. This strain was able to produce high levels of extracellular chitinase in solid media containing shrimp shells as sole carbon source [1.36 U/g initial dry substrate (IDS)], which was 0.36-fold higher than the productivity in a liquid culture with colloidal chitin. The effects of medium composition and physical parameters on chitinase production by this organism were studied. The optimal medium contained shrimp shell mixed with rice straw in 1:1 ratio added with ball-milled chitin 0.5 % (w/v) and ammonium sulfate 0.5 % (w/v). The highest enzyme production (3.86 U/g IDS) by B. thuringiensis R 176 was obtained at pH 7, 37 °C after 14 days growth. With respect to the high amount of chitinase production by this strain in a simple medium, this strain could be a suitable candidate for the production of chitinase from chitinous solid substrates, and further investigations into its structure and characteristics are merited.     

Identification of by-products in hydrogen producing bacteria; Rhodobacter sphaeroides O.U. 001 grown in the waste water of a sugar refinery

Rhodobacter sphaeroides O.U. 001 is able to produce hydrogen anaerobically upon illumination. The cells were screened for the presence of valuable by-products such as poly-β-hydroxy (PHB) butyric acid aiming to improve the feasibility of the system. Also waste water from a sugar refinery was used for bacterial growth to further increase the feasibility. Under aerobic conditions the standard growth media containing -malic acid and sodium glutamate in 7.5/10 and 15/2 molar ratios and a medium containing 30% waste water from sugar refinery were used. In this case the maximum concentration of PHB produced were approximately 0.2 g l⁻¹ in both of the standard media whereas it was 0.3 g l⁻¹ in medium containing 30% waste water. By using the medium containing 30% waste water, PHB and hydrogen productions were determined under anaerobic conditions. The maximum concentration of PHB produced was around 0.5 g l⁻¹ and the amount of gas collected was 35 ml in 108 h. From these results it can be concluded that PHB can be collected during hydrogen production. The use of waste water from sugar refinery increased the yield.     

Isolation and characterization of a copper-resistant methanogen from a copper-mining soil sample.

A copper-resistant methanogen for which the CuSO4 MICs were approximately 2- to 36-fold higher than those for other methanogens tested was isolated from a copper-mining area in the upper peninsula of Michigan. The rod-shaped methanogen used H2-CO2 or formate, but not acetate or methanol, as a growth substrate. Standing incubation with H2-CO2 medium resulted in a mat-like surface growth, dependent on the presence of hydrogen. The presence of 1 mM cupric salt resulted in longer filamentous and intertwined cells. Antigenic fingerprinting, 16S rRNA gene analysis, morphology, and substrate use suggest that the new isolate is a novel strain of Methanobacterium bryantii that is able to use formate.     

Evolutionary variation and adaptation in a conserved protein kinase allosteric network: Implications for inhibitor design

The activation of protein kinases involves conformational changes in key functional regions of the kinase domain, a detailed understanding of which is essential for the design of selective protein kinase inhibitors. Through statistical analysis of protein kinase sequences and crystal structures from diverse organisms, we recently proposed that the activation of protein kinases involves a hidden strain switch in the catalytic loop. Specifically, we demonstrated that the backbone torsion-angles of residues in the catalytic loop switch from a “relaxed” to “strained” conformation upon kinase activation and the strained geometry results in a network of hydrogen bonds involving conserved non-catalytic residues in the ATP and substrate binding lobes. Here, we further explore this activation mechanism by analyzing families that lack the canonical hydrogen bonding interactions with the strained backbone. We find that alternative mechanisms have evolved to maintain catalytic loop strain. In PIM kinase, for example, two water molecules account for the lack of a conserved aspartate in the substrate binding by hydrogen bonds to the strained backbone. We discuss the relevance of these findings in the design of family-specific allosteric inhibitors, and in predicting the structural and functional impact of cancer mutations that alter the strain associated hydrogen bonding network. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).     

Validation of thermal composting process using olive mill solid waste for industrial scale cultivation of Agaricus bisporus

The production of a substrate containing destoned olive mill solid waste for the cultivation of Agaricus bisporus (Lange) Imbach on an industrial scale was studied. A standard mushroom compost (C) mainly made from straw and poultry manure was compared with the experimental compost (EC) containing the same ingredients as (C) but with added olive mill solid waste (10.6% w/w). Microbial indicators such as counts of heterotrophic microbes and actinomycetes were higher in EC than in C. In addition, compost selectivity as indicated by higher mushroom yield and biological efficiency of EC was higher than that of C. Market quality of the mushrooms produced in both C and EC were comparable. These findings support our work that olive mill solid waste can be used safely in thermal composting process to produce a selective substrate for industrial-scale cultivation of A. bisporus. This study also demonstrates an environmentally sustainable system to manage solid waste from olive oil extraction processes thus overcoming environmental pollution brought about by irrational disposal of the waste on farm lands.     

Estimation of biological kinetic parameters from an analysis of the BOD curve of waste waters–effects of a chemical preoxidation

Urban waste waters were treated with pure ozone or combinations of ozone, hydrogen peroxide and/or UV radiation to study the course of resulting BOD (biological oxygen demand)-time profiles and to propose a kinetic model. BOD-time profiles of chemically treated waste waters show an initial lag period that first order kinetic models cannot describe. A second order kinetic model is then proposed that satisfactorily fits experimental BOD-time profiles, except when hydrogen peroxide has been used. In these cases, BOD-time profiles present the highest lag periods observed. By applying this model, three parameters are determined: the biokinetic constant (k) which is an index of the biological removal rate; the potential amount of biodegradable matter (BOD_T), and the measure of the size of inocula and microbial activities of microoganisms (λ). The model was checked with experimental results of BOD-time profiles corresponding to both untreated and chemically ozonated urban waste waters. Ozonated waste waters showed the highest values of k and BOD_T, which implies an improvement of waste water biodegradability after ozonation. However, values of λ corresponding to ozonated waste waters presented lower values than those of untreated waste waters. This was due to the lag period observed in the BOD-time profile, which was a consequence of a lack of microorganism acclimation to ozonated waste waters. The effect of the ozone does, pH and carbonates during ozonation on COD (chemical oxygen demand) and the above indicated parameters was also studied. There was an optimum ozone dose which was 138 mg/l for this specific system. This led to the highest biodegradable fraction (φ) and the highest biokinetic constant (39% increase in φ and 4.7- fold increase in the value of k, respectively, compared to untreated waste waters.). Another significant fact was that a higher COD reduction was observed in the absence of carbonate during ozonation at basic pH values. In addition, the percentage of variation in the biodegradable fraction (Δφ) of ozonated waste water increased compared to the untreated waste water at acid pH. The results suggest that ozonolysis, the direct molecular ozone way of reaction, due to its selective character, increases the biodegradability of waste water more than other chemically advanced oxidation processes based on hydroxyl radical reactions.     

Hydrogen production by Rhodobacter sphaeroides strain O.U.001 using spent media of Enterobacter cloacae strain DM11

Combined dark and photo-fermentation was carried out to study the feasibility of biological hydrogen production. In dark fermentation, hydrogen was produced by Enterobacter cloacae strain DM11 using glucose as substrate. This was followed by a photo-fermentation process. Here, the spent medium from the dark process (containing unconverted metabolites, mainly acetic acid etc.) underwent photo-fermentation by Rhodobacter sphaeroides strain O.U.001 in a column photo-bioreactor. This combination could achieve higher yields of hydrogen by complete utilization of the chemical energy stored in the substrate. Dark fermentation was studied in terms of several process parameters, such as initial substrate concentration, initial pH of the medium and temperature, to establish favorable conditions for maximum hydrogen production. Also, the effects of the threshold concentration of acetic acid, light intensity and the presence of additional nitrogen sources in the spent effluent on the amount of hydrogen produced during photo-fermentation were investigated. The light conversion efficiency of hydrogen was found to be inversely proportional to the incident light intensity. In a batch system, the yield of hydrogen in the dark fermentation was about 1.86 mol H₂ mol⁻¹ glucose; and the yield in the photo-fermentation was about 1.5–1.72 mol H₂ mol⁻¹ acetic acid. The overall yield of hydrogen in the combined process, considering glucose as the preliminary substrate, was found to be higher than that in a single process.     

Growth dynamics of Salmonella enterica strains on alfalfa sprouts and in waste seed irrigation water

Alfalfa sprouts and other seed sprouts have been implicated in numerous outbreaks of salmonellosis. The source of these epidemics appears to have been low-level contamination of seeds by Salmonella bacteria that developed into clinically significant populations during the seed germination process. To test the possibility that Salmonella enterica strains carry host range determinants that allow them to grow on alfalfa, strains isolated from alfalfa or other sources were surveyed for their ability to grow on germinating alfalfa seeds. An S. enterica serovar Cubana strain originally isolated from contaminated alfalfa sprouts multiplied most rapidly during the initial 24 h of the seed germination process. Germinating alfalfa seeds supported the multiplication of S. enterica cells prior to the emergence of the root radicle at 72 h. Thereafter, much lower rates of multiplication were apparent. The ability of S. enterica to grow on germinating alfalfa seeds was independent of the serovar, isolation source, or virulence of the strain. Isolates obtained from alfalfa attained population levels similar to those observed for strains isolated from contaminated meat products or stools. Each of the strains could be detected in the waste irrigation water, with populations being strongly correlated with those detected on the germinating alfalfa seeds. The S. enterica strains were capable of utilizing the waste irrigation water as a sole carbon and nitrogen source. S. enterica strains thus appear to grow saprophytically on soluble organics released from seeds during early phases of germination. The ability to detect S. enterica in the waste irrigation water early in the germination process indicates that this method may be used as a simple way to monitor the contamination of sprouts during commercial operations.     

Fermentative biohydrogen production: trends and perspectives

Biologically produced hydrogen (biohydrogen) is a valuable gas that is seen as a future energy carrier, since its utilization via combustion or fuel cells produces pure water. Heterotrophic fermentations for biohydrogen production are driven by a wide variety of microorganisms such as strict anaerobes, facultative anaerobes and aerobes kept under anoxic conditions. Substrates such as simple sugars, starch, cellulose, as well as diverse organic waste materials can be used for biohydrogen production. Various bioreactor types have been used and operated under batch and continuous conditions; substantial increases in hydrogen yields have been achieved through optimum design of the bioreactor and fermentation conditions. This review explores the research work carried out in fermentative hydrogen production using organic compounds as substrates. The review also presents the state of the art in novel molecular strategies to improve the hydrogen production.     

Modeling alginate encapsulation system for biological hydrogen production

Wastewater treatment using encapsulated biomass is a promising approach for high-rate resource recovery. Encapsulation matrices can be customized to achieve desired biomass retention and mass transport performance. This, in turn, facilitates treatment of different waste streams. In this study, a model was developed to describe calcium-alginate beads encapsulating hydrogen-producing biomass, with the goal of enabling appropriate a priori customization of the system. The model was based on a classic diffusion-reaction model, but also included the growth of encapsulated biomass and product inhibition. Experimental data were used to verify the model, which accurately described the effect of hydraulic retention time, bead size, and feed concentration on resource (hydrogen) recovery from brewery wastewater. Sensitivity analyses revealed that the hydrogen production rate was insensitive to substrate diffusivity and bead size, but sensitive to the substrate partition coefficient, initial encapsulated biomass concentration, and the total volume of beads in the reactor, demonstrating that this system was growth-limited rather than diffusion-limited under the tested conditions. Because the model quantifies the relationship between the hydrogen production rate and various input and operating parameters, it should be possible to extend the model to determine the most cost-effective system for optimal performance with a given waste stream.     

Heat, hydrogen peroxide, and UV resistance of Bacillus subtilis spores with increased core water content and with or without major DNA-binding proteins.

Spores of a Bacillus subtilis strain with an insertion mutation in the dacB gene, which codes for an enzyme involved in spore cortex biosynthesis, have a higher core water content than wild-type spores. Spores lacking the two major alpha/beta-type small, acid-soluble proteins (SASP) (termed alpha-beta- spores) have the same core water content as do wild-type spores, but alpha-beta- dacB spores had more core water than did dacB spores. The resistance of alpha-beta-, alpha-beta- dacB, dacB, and wild-type spores to dry and moist heat, hydrogen peroxide, and UV radiation has been determined, as has the role of DNA damage in spore killing by moist heat and hydrogen peroxide. These data (i) suggest that core water content has little if any role in spore UV resistance and are consistent with binding of alpha/beta-type SASP to DNA being the major mechanism providing protection to spores from UV radiation; (ii) suggest that binding of alpha/beta-type SASP to DNA is the major mechanism unique to spores providing protection from dry heat; (iii) suggest that spore resistance to moist heat and hydrogen peroxide is affected to a large degree by the core water content, as increased core water resulted in large decreases in spore resistance to these agents; and (iv) indicate that since this decreased resistance (i.e., in dacB spores) is not associated with increased spore killing by DNA damage, spore DNA must normally be extremely well protected against such damage, presumably by the saturation of spore DNA by alpha/beta-type SASP.