Genome-Wide and Functional View of Proteolytic and Lipolytic Bacteria for Efficient Biogas Production through Enhanced Sewage Sludge Hydrolysis.
ABSTRACT: In this study, we used a multifaceted approach to select robust bioaugmentation candidates for enhancing biogas production and to demonstrate the usefulness of a genome-centric approach for strain selection for specific bioaugmentation purposes. We also investigated the influence of the isolation source of bacterial strains on their metabolic potential and their efficiency in enhancing anaerobic digestion. Whole genome sequencing, metabolic pathway reconstruction, and physiological analyses, including phenomics, of phylogenetically diverse strains, Rummeliibacillus sp. POC4, Ochrobactrum sp. POC9 (both isolated from sewage sludge) and Brevundimonas sp. LPMIX5 (isolated from an agricultural biogas plant) showed their diverse enzymatic activities, metabolic versatility and ability to survive under varied growth conditions. All tested strains display proteolytic, lipolytic, cellulolytic, amylolytic, and xylanolytic activities and are able to utilize a wide array of single carbon and energy sources, as well as more complex industrial by-products, such as dairy waste and molasses. The specific enzymatic activity expressed by the three strains studied was related to the type of substrate present in the original isolation source. Bioaugmentation with sewage sludge isolates-POC4 and POC9-was more effective for enhancing biogas production from sewage sludge (22% and 28%, respectively) than an approach based on LPMIX5 strain (biogas production boosted by 7%) that had been isolated from an agricultural biogas plant, where other type of substrate is used.
Project description:Bioaugmentation with a mixture of microorganisms (Bacteria and Archaea) was applied to improve the anaerobic digestion of sewage sludge. The study was performed in reactors operating at a temperature of 35 °C in semi-flow mode. Three runs with different doses of bioaugmenting mixture were conducted. Bioaugmentation of sewage sludge improved fermentation and allowed satisfactory biogas/methane yields and a biodegradation efficiency of more than 46%, despite the decrease in hydraulic retention time (HRT) from 20 d to 16.7 d. Moreover, in terms of biogas production, the rate constant k increased from 0.071 h-1 to 0.087 h-1 as doses of the bioaugmenting mixture were increased, as compared to values of 0.066 h-1 and 0.069 h-1 obtained with sewage sludge alone. Next-generation sequencing revealed that Cytophaga sp. predominated among Bacteria in digesters and that the hydrogenotrophic methanogen Methanoculleus sp. was the most abundant genus among Archaea.
Project description:The use of lignocellulosic biomass as a substrate in agricultural biogas plants is very popular and yields good results. However, the efficiency of anaerobic digestion, and thus biogas production, is not always satisfactory due to the slow or incomplete degradation (hydrolysis) of plant matter. To enhance the solubilization of the lignocellulosic biomass various physical, chemical and biological pretreatment methods are used. The aim of this study was to select and characterize cellulose-degrading bacteria, and to construct a microbial consortium, dedicated for degradation of maize silage and enhancing biogas production from this substrate. Over 100 strains of cellulose-degrading bacteria were isolated from: sewage sludge, hydrolyzer from an agricultural biogas plant, cattle slurry and manure. After physiological characterization of the isolates, 16 strains (representatives of Bacillus, Providencia, and Ochrobactrum genera) were chosen for the construction of a Microbial Consortium with High Cellulolytic Activity, called MCHCA. The selected strains had a high endoglucanase activity (exceeding 0.21 IU/mL CMCase activity) and a wide range of tolerance to various physical and chemical conditions. Lab-scale simulation of biogas production using the selected strains for degradation of maize silage was carried out in a two-bioreactor system, similar to those used in agricultural biogas plants. The obtained results showed that the constructed MCHCA consortium is capable of efficient hydrolysis of maize silage, and increases biogas production by even 38%, depending on the inoculum used for methane fermentation. The results in this work indicate that the mesophilic MCHCA has a great potential for application on industrial scale in agricultural biogas plants.
Project description:Sewage sludge is an abundant source of microorganisms that are metabolically active against numerous contaminants, and thus possibly useful in environmental biotechnologies. However, amongst the sewage sludge isolates, pathogenic bacteria can potentially be found, and such isolates should therefore be carefully tested before their application. A novel bacterial strain, Ochrobactrum sp. POC9, was isolated from a sewage sludge sample collected from a wastewater treatment plant. The strain exhibited lipolytic, proteolytic, cellulolytic, and amylolytic activities, which supports its application in biodegradation of complex organic compounds. We demonstrated that bioaugmentation with this strain substantially improved the overall biogas production and methane content during anaerobic digestion of sewage sludge. The POC9 genome content analysis provided a deeper insight into the biotechnological potential of this bacterium and revealed that it is a metalotolerant and a biofilm-producing strain capable of utilizing various toxic compounds. The strain is resistant to rifampicin, chloramphenicol and ?-lactams. The corresponding antibiotic resistance genes (including blaOCH and cmlA/floR) were identified in the POC9 genome. Nevertheless, as only few genes in the POC9 genome might be linked to pathogenicity, and none of those genes is a critical virulence factor found in severe pathogens, the strain appears safe for application in environmental biotechnologies.
Project description:The metal(loid) and in particular the Arsenic (As) burden of thirteen agricultural biogas plants and two sewage sludge digesters were investigated together with the corresponding microbial consortia. The latter were characterized by ARISA (automated ribosomal intergenetic spacer analysis) and next generation sequencing. The consortia were found to cluster according to digester type rather than substrate or metal(loid) composition. For selected plants, individual As species in the liquid and gaseous phases were quantified, showing that the microorganisms actively metabolize and thereby remove the As from their environment via the formation of (methylated) volatile species. The As metabolites showed some dependency on the microbial consortia, while there was no statistical correlation with the substrate mix. Finally, slurry from one agricultural biogas plant and one sewage sludge digester was transferred into laboratory scale reactors ("satellite reactors") and the response to a defined addition of As (30 and 60 µM sodium arsenite) was studied. The results corroborate the hypothesis of a rapid conversion of dissolved As species into volatile ones. Methanogenesis was reduced during that time, while there was no discernable toxic effect on the microbial population. However, the utilization of the produced biogas as replacement for natural gas, e.g. as fuel, may be problematic, as catalysts and machinery are known to suffer from prolonged exposure even to low As concentrations.
Project description:Acetate production from food waste or sewage sludge was evaluated in four semi-continuous anaerobic digestion processes. To examine the importance of inoculum and substrate for acid production, two different inoculum sources (a wastewater treatment plant (WWTP) and a co-digestion plant treating food and industry waste) and two common substrates (sewage sludge and food waste) were used in process operations. The processes were evaluated with regard to the efficiency of hydrolysis, acidogenesis, acetogenesis, and methanogenesis and the microbial community structure was determined. Feeding sewage sludge led to mixed acid fermentation and low total acid yield, whereas feeding food waste resulted in the production of high acetate and lactate yields. Inoculum from WWTP with sewage sludge substrate resulted in maintained methane production, despite a low hydraulic retention time. For food waste, the process using inoculum from WWTP produced high levels of lactate (30 g/L) and acetate (10 g/L), while the process initiated with inoculum from the co-digestion plant had higher acetate (25 g/L) and lower lactate (15 g/L) levels. The microbial communities developed during acid production consisted of the major genera Lactobacillus (92-100%) with food waste substrate, and Roseburia (44-45%) and Fastidiosipila (16-36%) with sewage sludge substrate. Use of the outgoing material (hydrolysates) in a biogas production system resulted in a non-significant increase in bio-methane production (+5-20%) compared with direct biogas production from food waste and sewage sludge.
Project description:The study presents a comparison of the influence of a clinoptilolite-rich rock-zeolite (commonly used for improving anaerobic digestion processes)-and a highly porous clay mineral, halloysite (mainly used for gas purification), on the biogas production process. Batch experiments showed that the addition of each mineral increased the efficiency of mesophilic anaerobic digestion of both sewage sludge and maize silage. However, halloysite generated 15% higher biogas production during maize silage transformation. Halloysite also contributed to a much higher reduction of chemical oxygen demand for both substrates (by ~8% for maize silage and ~14% for sewage sludge) and a higher reduction of volatile solids and total ammonia for maize silage (by ~8% and ~4%, respectively). Metagenomic analysis of the microbial community structure showed that the addition of both mineral sorbents influenced the presence of key members of archaea and bacteria occurring in a well-operated biogas reactor. The significant difference between zeolite and halloysite is that the latter promoted the immobilization of key methanogenic archaea Methanolinea (belong to Methanomicrobia class). Based on this result, we postulate that halloysite could be useful not only as a sorbent for (bio)gas treatment methodologies but also as an agent for improving biogas production.
Project description:The application of sewage sludge to agricultural soil induces co-exposure of prokaryotic populations to antibiotics and heavy metals, thus exerting a selection pressure that may lead to the development of antibiotic resistance. Here, soil samples from a long-term factorial field experiment in which sewage sludge was applied to agricultural soil, at different rates (40 and 80?t?ha<sup>-1</sup>) and frequencies (every 1, 2 and 4 years) of application, were studied to assess: (i) the effect of sewage sludge application on prokaryotic community composition, (ii) the links between prokaryotic community composition and antibiotic resistance profiles, and (iii) the links between antibiotic resistance and metal(oid) concentrations in amended soil. We found no significant impact of sewage sludge on prokaryotic community composition. Some antibiotic resistance genes (ARGs) correlated positively with particular prokaryotic taxa, being <i>Gemmatimonadetes</i> the taxon with the greatest number of positive correlations at phylum level. No positive correlation was found between prokaryotic taxa and genes encoding resistance to sulfonamides and FCA. All metal(oid)s showed positive correlations with, at least, one ARG. Metal(oid) concentrations in soil also showed positive correlations with mobile genetic element genes, particularly with the gene tnpA-07. These data provide useful information on the links between soil prokaryotic composition and resistome profiles, and between antibiotic resistance and metal(oid) concentrations, in agricultural soils amended with sewage sludge.
Project description:Anaerobic digestion (AD), being the most effective treatment method of waste activated sludge (WAS), allows for safe disposal. The present study deals with the electro-Fenton (EF) pretreatment for enhancing the WAS biogas potential with low-cost iron electrodes. The effect of pretreatment on the physicochemical characteristics of sludge was assessed. Following EF pretreatment, the pH, conductivity, soluble chemical oxygen demand (SCOD), and volatile fatty acids (VFA) increased to 7.5, 13.72 mS/cm, 4.1 g/L, and 925 mg/L, respectively. Capillary suction time (CST) analysis highlighted the dewaterability effect of EF on WAS, as demonstrated by the decrease in CST from 429 to 180 s following 30 min of pretreatment. Batch digestion assays presented an increase in the biogas yield to 0.135 L/g volatile solids (VS) after 60 min of EF pretreatment in comparison to raw sludge (0.08 L/g VS). Production of biogas was also found to improve during semi-continuous fermentation of EF-pretreated sludge conducted in a lab-scale reactor. In comparison to raw sludge, EF-pretreated sludge produced the highest biogas yield (0.81 L biogas/g VS) with a high COD removal rate, reaching 96.6% at an organic loading rate (OLR) of 2.5 g VS/L. d. Results revealed that the EF process could be an effective WAS disintegration method with maximum recovery of bioenergy during AD.
Project description:Application of fish sludge as fertiliser to agricultural land can contribute to closing nutrient cycles in fish farming. The effect of different treatment technologies on the nitrogen fertilisation effects of fish sludge was studied by a bioassay with barley (Hordeum vulgare), an incubation and a field experiment. Dried fish sludge resulted in relative agronomic efficiency of 50-80% compared with mineral fertiliser. The anaerobic digestate based on fish sludge (20 vol%) and dairy manure did not increase nitrogen uptake in barley. Increasing the ratio of fish sludge in the digestate increased the fertilisation effect, but requires optimisation of the biogas process. A simple logistics analysis conducted for a case hatchery showed that on-site drying and co-digestion of fish sludge in a central biogas plant can be regarded as equal in terms of costs. Norway can become an exporter of fish sludge-based recycling fertilisers if current regulations are modified to facilitate nutrient recycling.
Project description:Methanoculleus bourgensis, of the order Methanomicrobiales, is a dominant methanogenic archaeon in many biogas-producing reactor systems fed with renewable primary products. It is capable of synthesizing methane via the hydrogenotrophic pathway utilizing hydrogen and carbon dioxide or formate as the substrates. Here we report the complete and finished genome sequence of M. bourgensis strain MS2(T), isolated from a sewage sludge digester.