Bioaugmentation of anaerobic digesters with the enriched lignin-degrading microbial consortia through a metagenomic approach.

The recalcitrance of lignin impedes the efficient utilization of lignocellulosic biomass, hindering the efficient production of biogas and value-added materials. Despite the emergence of anaerobic digestion as a superior alternative to the aerobic method for lignin processing, achieving its feasibility requires thorough characterization of lignin-degrading anaerobic microorganisms, assessment of their biomethane production potential, and a comprehensive understanding of the degradation pathway. This study aimed to address the aforementioned necessities by bioaugmenting seed sludge with three distinct enriched lignin-degrading microbial consortia at both 25 °C and 37 °C. Enhanced biomethane yields was detected in the bioaugmented digesters, while the highest production was observed as 188 mLN CH4 gVS-1 in digesters operated at 37 °C. Moreover, methane yield showed a significant improvement in the samples at 37 °C ranging from 110% to 141% compared to the control, demonstrating the efficiency of the enriched lignin-degrading microbial community. Temperature and substrate were identified as key factors influencing microbial community dynamics. The observation that microbial communities tended to revert to the initial state after lignin depletion, indicating the stability of the overall microbiota composition in the digesters, is a promising finding for large-scale studies. Noteworthy candidates for lignin degradation, including Sporosarcina psychrophila, Comamonas aquatica, Shewanella baltica, Pseudomonas sp. C27, and Brevefilum fermentans were identified in the bioaugmented samples. PICRUSt2 predictions suggest that the pathway and specific proteins involved in anaerobic lignin degradation might share similarities with those engaged in the degradation of aromatic compounds.

Is a floodplain forest a valuable source for lignin-degrading anaerobic microbial communities: A metagenomic approach.

Lignin is one of the most substantial obstacles in the evaluation of lignocellulosic compounds. Although there are numerous approaches for the enhancement of lignin digestion in the literature, there has yet to be an optimized system to date. In this study, samples taken from Igneada floodplain forests were enriched anaerobically at 25 °C and 37 °C, with alkali lignin as the sole carbon source. The activity of the anaerobic lignin-degrading microbial consortium was detected more efficiently at 37 °C, where biogas production exceeded 3.5 mLgas/mLmedium. It was observed that the microbial community initially dominated by Proteobacteria (around 60%) changed completely after enrichment and was led by members of the Firmicutes phylum (up to 90%). The dominant species (Sporomusa termitida, Desulfitobacterium hafniense, Citrobacter freundii, Citrobacter portucalensis, Alkalibacter rhizosphaerae, and Gudongella oleilytica) occupying more than 50% in the final enrichment culture were only around 2% in the raw samples. Therefore, this study, one of the few in which enriched environmental samples were sequenced using MinION, demonstrated that longoses are exceptional reservoirs for lignin-digesting anaerobic microorganisms.

Monitoring of cyanobacterial blooms and assessing polymer-enhanced microfiltration and ultrafiltration for microcystin removal in an Italian drinking water treatment plant.

The water intake of a drinking water treatment plant (DWTP) in Central Italy was monitored over six bloom seasons for cyanotoxin severity, which supplies drinking water from an oligo-mesotrophic lake with microcystin levels up to 10.3 µg/L. The historical data showed that the water temperature did not show extreme/large seasonal variation and it was not correlated either with cyanobacterial growth or microcystin concentration. Among all parameters, the cyanobacteria growth was negatively correlated with humidity and manganese and positively correlated with atmospheric temperature. No significant correlation was found between microcystin concentration and the climatic parameters. Polymer(chitosan)-enhanced microfiltration (PEMF) and ultrafiltration (PEUF) were further tested as an alternative microcystin removal approach from dense cyanobacteria-rich flows. The dominant cyanobacteria in the water intake, Planktothrix rubescens, was isolated and enriched to simulate cyanobacterial blooms in the lake. The PEMF and PEUF were separately applied to enriched P. rubescens culture (PC) (microcystin = 1.236 µg/L) as well as to the sand filter backwash water (SFBW) of the DWTP where microcystin concentration was higher than 12 µg/L. The overall microcystin removal rates from the final effluent of PC (always <0.15 µg/L) were between 90.1-94.7% and 89.5-95.4% using 4 and 20 mg chitosan/L, respectively. Meanwhile, after the PEMF and PEUF of SFBW, the final effluent contained only 0.099 and 0.057 µg microcystin/L with an overall removal >99%. The presented results are the first from the application of chitosan to remove P. rubescens as well as the implementation of PEMF and PEUF on SFBW to remove cyanobacterial cells and associated toxins.

Contrasting the Water Quality and Bacterial Community Patterns in Shallow and Deep Lakes: Manyas vs. Iznik.

The objectives of this study are to monitor the physicochemical properties of two freshwater lakes with different chemical characteristics and trophic status over a year (2019) and assess the bacterial diversity by a high-throughput sequencing method for a certain time. Carlson Trophic Index analysis revealed that, whereas the deep lake, Iznik Lake, (TSImean = 48.9) has mesotrophic characteristics, the shallow lake Manyas Lake (TSImean = 74.2) was found at a hypertrophic status. The most important parameters controlling water qualities in the lakes were temperature, alkalinity, and phosphate levels. Although the bacterial communities were dominated by the same phyla (Cyanobacteria, Bacteroidetes, Actinomicrobia, Proteobacteria, and Verrucomicrobia) in both lakes, the communities differed distinctly at the lower levels. Whereas Sporichthyaceae in Manyas Lake accounted for 10% of the total reads, the major share of the sequences was assigned to Cyanobacteria Family I (8%) in Iznik Lake. The hypertrophic Manyas Lake had a more diverse bacterial community rather than Iznik Lake and contained higher numbers of unique Operational Taxonomic Units.

Microbial community shifts in the oxic-settling-anoxic process in response to changes to sludge interchange ratio.

This particular study set out to demonstrate alterations on the microbial community of the oxic-settling-anaerobic/anoxic (OSA) process treating real domestic wastewater by changing interchange ratios (IRs). The sludge yield of systems operated at different IRs (1/13, 1/17 and 1/20) to assess sludge reduction was used to analyze microbial community composition variations. The highest IR (1/13) resulted in the highest sludge reduction (52.1%), while the OSA systems with IR of 1/17 and 1/20 reduced sludge production by 37.4% and 35.5%, respectively, in comparison to conventional systems. 16S rRNA gene amplicon sequencing analysis showed that the bacterial communities were composed of similar phylogenetic groups, Proteobacteria, Acidobacteria, and Bacteroidetes being dominant. The relative abundances differed due to the applied IRs. The highest abundance of Actinobacteria was determined at the highest IR (1/13) and increasing of the HRT to 1/20 caused a significant reduction in Actinobacteria species and the lowest abundance (6%) was determined in the OSA systems. The abundant of Thiothrix species that are boosted in the OSA trials may have a vital role in OSA systems, where its abundance was below the detection limits in the seed sludge sample. Therefore, they could be used as bioindicators in the OSA system.

Fungal bioaugmentation of anaerobic digesters fed with lignocellulosic biomass: What to expect from anaerobic fungus Orpinomyces sp.

Energy-efficient biogas reactors are often designed and operated mimicking natural microbial ecosystems such as the digestive tracts of ruminants. Anaerobic fungi play a crucial role in the degradation of lignocellulose-rich fiber thanks to their high cellulolytic activity. Fungal bioaugmentation is therefore at the heart of our understanding of enhancing anaerobic digestion (AD). The efficiency of bioaugmentation with anaerobic fungus Orpinomyces sp. was evaluated in lignocellulose-based AD configurations. Fungal bioaugmentation increased the methane yield by 15-33% during anaerobic co-digestion of cow manure and selected cereal crops/straws. Harvesting stage of the crops was a decisive parameter to influence methane production together with fungal bioaugmentation. A more efficient fermentation process in the bioaugmented digesters was distinguished by relatively-higher abundance of Synergistetes, which was mainly represented by the genus Anaerobaculum. On the contrary, the composition of the methanogenic archaea did not change, and the majority of methanogens was assigned to Methanosarcina.

Effect of bioaugmentation by cellulolytic bacteria enriched from sheep rumen on methane production from wheat straw.

The aim of this study was to determine the potential of bioaugmentation with cellulolytic rumen microbiota to enhance the anaerobic digestion of lignocellulosic feedstock. An anaerobic cellulolytic culture was enriched from sheep rumen fluid using wheat straw as substrate under mesophilic conditions. To investigate the effects of bioaugmentation on methane production from straw, the enrichment culture was added to batch reactors in proportions of 2% (Set-1) and 4% (Set-2) of the microbial cell number of the standard inoculum slurry. The methane production in the bioaugmented reactors was higher than in the control reactors. After 30 days of batch incubation, the average methane yield was 154 mLN CH4 gVS-1 in the control reactors. Addition of 2% enrichment culture did not enhance methane production, whereas in Set-2 the methane yield was increased by 27%. The bacterial communities were examined by 454 amplicon sequencing of 16S rRNA genes, while terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of mcrA genes was applied to analyze the methanogenic communities. The results highlighted that relative abundances of Ruminococcaceae and Lachnospiraceae increased during the enrichment. However, Cloacamonaceae, which were abundant in the standard inoculum, dominated the bacterial communities of all batch reactors. T-RFLP profiles revealed that Methanobacteriales were predominant in the rumen fluid, whereas the enrichment culture was dominated by Methanosarcinales. In the batch rectors, the most abundant methanogens were affiliated to Methanobacteriales and Methanomicrobiales. Our results suggest that bioaugmentation with sheep rumen enrichment cultures can enhance the performance of digesters treating lignocellulosic feedstock.

Use of PCR-DGGE based molecular methods to assessment of microbial diversity during anaerobic treatment of antibiotic combinations.

As it is currently often not know how anaerobic bioreactors, e.g. for biogas production, react if the substrate is contaminated by toxic compounds like antibiotics. This study evaluated how anaerobic sequencing batch reactors were affected by amendments of different antibiotics and stepwise increasing concentrations. The compositions of microbial community were determined in the seed sludge using 16S rRNA gene clone libraries and PCR-DGGE analyses were used for the detection of microbial community changes upon antibiotics additions. According to PCR-DGGE results, the syntrophic interaction of acetogens and methanogens is critical to the performance of the reactors. Failure to maintain the stability of these microorganisms resulted in a decrease in the performance and stability of the anaerobic reactors. Assessment of DGGE data is also useful for suggesting the potential to control ultimate microbial community structure, especially derived from Gram-negative bacteria, through bioaugmentation to successful for antibiotic biodegradation.

Combined effect of erythromycin, tetracycline and sulfamethoxazole on performance of anaerobic sequencing batch reactors.

The combined effects of erythromycin-tetracycline-sulfamethoxazole (ETS) and sulfamethoxazole-tetracycline (ST) antibiotics on the performance of anaerobic sequencing batch reactors were studied. A control reactor was fed with wastewater that was free of antibiotics, while two additional reactors were fed with ETS and ST. The way in which the ETS and ST mixtures impact COD removal, VFA production, antibiotic degradation, biogas production and composition were investigated. The effects of the ETS mixtures were different from the ST mixtures, erythromycin can have an antagonistic effect on sulfamethoxazole and tetracycline. The anaerobic pre-treatment of these antibiotics can represent a suitable alternative to the use of chemical treatments for concentrations at 10 mg/L of S and 1 mg/L of T; 2 mg/L of E, 2 mg/L of T and 20 mg/L of S for the ST and ETS reactors respectively, which corresponds to min 70% COD removal efficiency.

Acute effects of various antibiotic combinations on acetoclastic methanogenic activity.

Pharmaceutical production industries are one of the main sources of antibiotics, and they release considerable amounts of antibiotics to ecosystem. Antibiotics usually present as mixtures in treatment plants and have negative effect on biological processes. In this study, batch acute tests were performed to assess the inhibitory impacts of selected antibiotic combinations of sulfamethoxazole and tetracycline (ST), erythromycin and sulfamethoxazole (ES), and erythromycin and tetracycline (ET) on acetoclastic methanogenic activity. Each antibiotic was equally applied, making the total concentrations in the mixtures 0 (control), 2, 20, 50, 100, 250, and 500 mg/L. Results showed decline characteristic on methane production with increasing antibiotic concentrations. EC50 values were calculated as 275 mg/L for ES, 219 mg/L for ST, and 130 mg/L for ET. Mixture inhibition of ST and ET combinations were accurately predicted using the concept of independent action, while ES combination resulted in almost the same inhibition with that of single antibiotic response. Inhibition on acetate utilization followed similar trend with methane production inhibition.

Inhibitory effects of antibiotic combinations on syntrophic bacteria, homoacetogens and methanogens.

Antibiotics have the potential to adversely affect the microbial community that is present in biological wastewater treatment processes. The antibiotics that exist in waste streams directly inhibit substrate degradation and also have an influence on the composition of the microbial community. The aim of this study was to evaluate the short-term inhibition impact that various antibiotic combinations had on the syntrophic bacteria, homoacetogenic and methanogenic activities of a microbial community that had been fed with propionate and butyrate as the sole carbon source and VFA mixture (acetate, propionate and butyrate). Acute tests were constructed using on a two way-factorial design, where one factor was the composition of antibiotic mixture and another was the concentration of antibiotics added. In addition, the inhibitory effect of antibiotics was evaluated by monitoring biogas production and the accumulation of individual volatile fatty acids. Specific methanogenic activity batch tests showed a significant (p<0.05) decrease in the maximum methane production rate in the presence of 1 mg L(-1) of antibiotics for the substrate in a VFA mixture and propionate; 1 mg L(-1) of ETS, 25 mg L(-1) of ET, 10 mg L(-1) of ST and ES combination for substrates butyrate. The addition of antibiotics to the batch tests affected the utilization of acetate, propionate and butyrate. This study indicated that antibiotic mixtures have an effect on homoacetogenic bacteria and methanogens, which may exert inhibitory effects on propionate and butyrate-oxidizing syntrophic bacteria, resulting in unfavorable effects on methanogenesis.

Bioenergy production from diluted poultry manure and microbial consortium inside Anaerobic Sludge Bed Reactor at sub-mesophilic conditions.

In this study, anaerobic treatability of diluted chicken manure (with an influent feed ratio of 1 kg of fresh chicken manure to 6 L of tap water) was investigated in a lab-scale anaerobic sludge bed (ASB) reactor inoculated with granular seed sludge. The ASB reactor was operated at ambient temperature (17-25°C) in order to avoid the need of external heating up to higher operating temperatures (e.g., up to 35°C for mesophilic digestion). Since heat requirement for raising the temperature of incoming feed for digestion is eliminated, energy recovery from anaerobic treatment of chicken manure could be realized with less operating costs. Average biogas production rates were calculated ca. 210 and 242 L per kg of organic matter removed from the ASB reactor at average hydraulic retention times (HRTs) of 13 and 8.6 days, respectively. Moreover, average chemical oxygen demand (COD) removal of ca. 89% was observed with suspended solids removal more than 97% from the effluent of the ASB reactor. Influent ammonia, on the other hand, did not indicate any free ammonia inhibition due to dilution of the raw manure while pH and alkalinity results showed stability during the study. Microbial quantification results indicated that as the number of bacterial community decreased, the amount of Archaea increased through the effective digestion volume of the ASB reactor. Moreover, the number of methanogens displayed an uptrend like archaeal community and a strong correlation (-0.645) was found between methanogenic community and volatile fatty acid (VFA) concentration especially acetate.