MiDAS 5: Global diversity of bacteria and archaea in anaerobic digesters.

Anaerobic digestion of organic waste into methane and carbon dioxide (biogas) is carried out by complex microbial communities. Here, we use full-length 16S rRNA gene sequencing of 285 full-scale anaerobic digesters (ADs) to expand our knowledge about diversity and function of the bacteria and archaea in ADs worldwide. The sequences are processed into full-length 16S rRNA amplicon sequence variants (FL-ASVs) and are used to expand the MiDAS 4 database for bacteria and archaea in wastewater treatment systems, creating MiDAS 5. The expansion of the MiDAS database increases the coverage for bacteria and archaea in ADs worldwide, leading to improved genus- and species-level classification. Using MiDAS 5, we carry out an amplicon-based, global-scale microbial community profiling of the sampled ADs using three common sets of primers targeting different regions of the 16S rRNA gene in bacteria and/or archaea. We reveal how environmental conditions and biogeography shape the AD microbiota. We also identify core and conditionally rare or abundant taxa, encompassing 692 genera and 1013 species. These represent 84-99% and 18-61% of the accumulated read abundance, respectively, across samples depending on the amplicon primers used. Finally, we examine the global diversity of functional groups with known importance for the anaerobic digestion process.

New Perspectives on the Interactions between Adsorption and Degradation of Organic Micropollutants in Granular Activated Carbon Filters.

The removal of organic micropollutants in granular activated carbon (GAC) filters can be attributed to adsorption and biological degradation. These two processes can interact with each other or proceed independently. To illustrate the differences in their interaction, three 14C-labeled organic micropollutants with varying potentials for adsorption and biodegradation were selected to study their adsorption and biodegradation in columns with adsorbing (GAC) and non-adsorbing (sand) filter media. Using 14CO2 formation as a marker for biodegradation, we demonstrated that the biodegradation of poorly adsorbing N-nitrosodimethylamine (NDMA) was more sensitive to changes in the empty bed contact time (EBCT) compared with that of moderately adsorbing diclofenac. Further, diclofenac that had adsorbed under anoxic conditions could be degraded when molecular oxygen became available, and substantial biodegradation (≥60%) of diclofenac could be achieved with a 15 min EBCT in the GAC filter. These findings suggest that the retention of micropollutants in GAC filters, by prolonging the micropollutant residence time through adsorption, can enable longer time periods for degradations than what the hydraulic retention time would allow for. For the biologically recalcitrant compound carbamazepine, differences in breakthrough between the 14C-labeled and nonradiolabeled compounds revealed a substantial retention via successive adsorption-desorption, which could pose a potential challenge in the interpretation of GAC filter performance.

Biological degradation of organic micropollutants in GAC filters-temporal development and spatial variations.

The capacity for organic micropollutant removal in granular activated carbon (GAC) filters for wastewater treatment changes over time. These changes are in general attributed to changes in adsorption, but may in some cases also be affected by biological degradation. Knowledge on the degradation of organic micropollutants, however, is scarce. In this work, the degradation of micropollutants in several full-scale GAC and sand filters was investigated through incubation experiments over a period of three years, using 14C-labeled organic micropollutants with different susceptibilities to biological degradation (ibuprofen, diclofenac, and carbamazepine), with parallel 16S rRNA gene sequencing. The results showed that the degradation of diclofenac and ibuprofen in GAC filters increased with increasing numbers of bed volumes when free oxygen was available in the filter, while variations over filter depth were limited. Despite relatively large differences in bacterial composition between filters, a degradation of diclofenac was consistently observed for the GAC filters that had been operated with high influent oxygen concentration (DO >8 mg/L). The results of this comprehensive experimental work provide an increased understanding of the interactions between microbial composition, filter material, and oxygen availability in the biological degradation of organic micropollutants in GAC filters.

Assessing the potential of a membrane bioreactor and granular activated carbon process for wastewater reuse - A full-scale WWTP operated over one year in Scania, Sweden.

A full-scale membrane bioreactor (MBR) with ultrafiltration, followed by granular activated carbon (GAC), was examined to determine the potential of reusing treated water as a source of drinking water or for irrigation. The major part of the bacteria removal took place in the MBR, whereas the GAC removed substantial amounts of organic micropollutants. Annual variations in inflow and infiltration resulted in a concentrated influent during summer and a diluted influent in the winter. The removal of E. coli was high throughout the process (average log removal 5.8), with effluent concentrations meeting the threshold for class B water standards for irrigation (EU 2020/741) but exceeding those for drinking water in Sweden. The total bacterial concentration increased over the GAC, indicating the growth and release of bacteria; however, E. coli concentrations declined. The effluent concentrations of metals met the Swedish criteria for drinking water. The removal of organic micropollutants decreased during the initial operation of the treatment plant, but after 1 year and 3 months, corresponding to 15,000 bed volumes, the removal increased. Maturation of the biofilm in the GAC filters might have resulted in biodegradation of certain organic micropollutants, in combination with bioregeneration. Although there is no legislation in Scandinavia with regard to many organic micropollutants in drinking water and water for irrigation, the effluent concentrations were generally in the same order of magnitude as to those in Swedish source waters that are used for drinking water production.

Chemically enhanced primary treatment, microsieving, direct membrane filtration and GAC filtration of municipal wastewater: a pilot-scale study.

Chemically enhanced primary treatment (CEPT) followed by microsieving and direct membrane filtration (DMF) as ultrafiltration, was evaluated on pilot scale at a municipal wastewater treatment plant. In addition, a granular activated carbon (GAC) filter downstream of DMF was evaluated for the removal of organic micropollutants. Up to 80% of the total organic carbon (TOC) and 96% of the total phosphorus were removed by CEPT with microsieving. The additional contribution of subsequent DMF was minor, and only five days of downstream GAC filtration was possible due to fouling of the membrane. Of the 21 organic micropollutants analysed, all were removed (≥ 98%) by the GAC filter until 440 bed volumes, while CEPT with microsieving and DMF removed only a few compounds. Measurements of the oxygen uptake rate indicated that the required aeration for supplementary biological treatment downstream of CEPT with microsieving, both with and without subsequent DMF, was 20-25% of that in the influent wastewater. This study demonstrated the potential of using compact physicochemical processes to treat municipal wastewater, including the removal of organic micropollutants.

Ozonation of 14C-labeled micropollutants - mineralization of labeled moieties and adsorption of transformation products to activated carbon.

Ozonation transformation products (OTPs) are largely unknown compounds that are formed during the ozonation of micropollutants, and it is uncertain to which extent these compounds can be removed by subsequent adsorption to activated carbon. Thus, 14C-labeled micropollutants were ozonated to generate 14C-labeled OTPs, for which the adsorption of the sum of all 14C-labeled OTPs to activated carbon could be determined, based on the adsorption of the labeled carbon. Further, 14CO2 traps were used to examine the mineralization of 14C-labeled moieties during ozonation. 14CO2-formation revealed a partial mineralization of the 14C-labeled moieties in all compounds except for propyl-labeled bisphenol A and O-methyl-labeled naproxen. A similar degree of mineralization was noted for different compounds labeled at the same moiety, including the carboxylic carbon in diclofenac and ibuprofen (∼40% at 1 g O3/g DOC) and the aniline ring in sulfamethoxazole and sulfadiazine (∼30% at 1 g O3/g DOC). Aromatic ring cleavage was also confirmed for bisphenol A, sulfamethoxazole, and sulfadiazine through the formation of 14CO2. The adsorption experiments demonstrated increased adsorption of micropollutants to powdered activated carbon after ozonation, which was connected to a decreased adsorption of dissolved organic matter (DOM). Conversely, the OTPs showed a substantial and successive decline in adsorption at increased ozone doses for all compounds, likely due to decreased hydrophobicity and aromaticity of the OTPs. These findings indicate that adsorption to activated carbon alone is not a viable removal method for a wide range of ozonation transformation products.

A comparison of adsorption of organic micropollutants onto activated carbon following chemically enhanced primary treatment with microsieving, direct membrane filtration and tertiary treatment of municipal wastewater.

The adsorption of organic micropollutants onto powdered activated carbon (PAC) was investigated in laboratory scale based on samples from four wastewater process streams (matrices); three from a pilot-scale plant with different degrees of physicochemical treatment of municipal wastewater and one from a full-scale activated sludge plant with post-precipitation. The pilot-scale treatment consisted of chemically enhanced primary treatment with microsieving followed by direct membrane filtration as microfiltration or ultrafiltration. The results showed highest adsorption of micropollutants in the tertiary (biologically and chemically) treated wastewater and lowest adsorption in the microsieve filtrate. Adsorption of micropollutants in the direct membrane microfiltration (200 nm) permeate was generally similar to that in the direct membrane ultrafiltration (3 nm) permeate. The higher adsorption of micropollutants in the tertiary treated wastewater could be related to a lower concentration of dissolved organic carbon (DOC) and lower affinity of DOC for PAC at low dosage (<15 mg PAC/L) in this matrix. At a PAC dose of 10 mg/L, sulfamethoxazole was removed by 33% in the tertiary treated wastewater and 7% in the direct membrane microfiltration permeate. In addition to the PAC experiments, a pilot scale sand filter and a proceeding GAC filter was operated on tertiary treated wastewater from the full-scale treatment plant. Similar removal trends in the PAC and GAC experiments were observed when studying a weighted average micropollutant removal in the GAC filter and a similar dose of activated carbon for both PAC and GAC. Positively charged micropollutants were removed to a higher extent than negatively charged ones by both PAC and GAC.

Tracking 14C-Labeled Organic Micropollutants to Differentiate between Adsorption and Degradation in GAC and Biofilm Processes.

Granular activated carbon (GAC) filters can be used to reduce emissions of organic micropollutants via municipal wastewater, but it is still uncertain to which extent biological degradation contributes to their removal in GAC filters. 14C-labeled organic micropollutants were therefore used to distinguish degradation from adsorption in a GAC-filter media with associated biofilm. The rates and extents of biological degradation and adsorption were investigated and compared with other biofilm systems, including a moving bed biofilm reactor (MBBR) and a sand filter, by monitoring 14C activities in the liquid and gas phases. The microbial cleavage of ibuprofen, naproxen, diclofenac, and mecoprop was confirmed for all biofilms, based on the formation of 14CO2, whereas the degradation of 14C-labeled moieties of sulfamethoxazole and carbamazepine was undetected. Higher degradation rates for diclofenac were observed for the GAC-filter media than for the other biofilms. Degradation of previously adsorbed diclofenac onto GAC could be confirmed by the anaerobic adsorption and subsequent aerobic degradation by the GAC-bound biofilm. This study demonstrates the potential use of 14C-labeled micropollutants to study interactions and determine the relative contributions of adsorption and degradation in GAC-based treatment systems.

Integrating dissolved and particulate matter into a prediction tool for ozonation of organic micropollutants in wastewater.

Ozonation is an established technique used to reduce the discharge of organic micropollutants into the aquatic environment, but the possibility of predicting the ozone demand for different wastewater matrices is still limited, especially in the presence of suspended solids (SS). A new tool for the prediction of the removal of organic micropollutants with ozone, based on dissolved and particulate matter in activated sludge effluents, was therefore developed. The removal of 25 organic micropollutants was determined on laboratory scale in the presence and absence of suspended solids. The linear trajectories of the dose-response curves enabled the determination of a new set of removal constants, based on dissolved chemical oxygen demand (COD) and SS. The presence of SS had a more negative effect on the removal of slow-reacting micropollutants (removal constant <3.5 mg CODCr,diss·mg O3-1) with ozone than on the fast-reacting micropollutants (removal constant >3.5 mg CODCr,diss·mg O3-1). However, the decreased removal of the organic micropollutants was generally small, <10%, at typical SS concentrations, <25 mg SS·L-1. Integration of the new removal constants based on COD and SS enabled the removal in an ozone pilot plant to be modelled with an average deviation of <10% for several organic micropollutants. The use of the frequently measured parameters, COD and SS, as input parameters could facilitate the future use of the tool to predict the removal of micropollutants during ozonation.

Characterisation of microbial communities for improved management of anaerobic digestion of food waste.

Anaerobic digestion of food waste is an attractive and increasingly popular technology within waste management and energy recovery. A better understanding of the microbiology associated with anaerobic digestion of food waste will provide new insight into the operational conditions required for optimizing this process, as well as its potential for utilisation in co-digestion systems. Eighteen full-scale reactors processing varying proportions of food waste under diverse operational configurations were subjected to microbial community analysis by amplicon sequencing of the 16S rRNA and mcrA genes to capture the bacterial and methanogenic populations. Statistical correlations between microbial populations, plant design and operating conditions revealed that the microbial communities were shaped by operational parameters such as the primary substrate type and operational temperature, while the methanogenic communities showed a more reactor specific distribution. The distribution of microbes based on the waste processed in the surveyed digesters was explored, as well as the presence of specialist populations such as syntrophs and methanogens. Food waste digester communities were not associated with a strong microbial fingerprint compared to other waste types (wastewater and manure) but contained greater abundance and unique syntrophic acetate oxidising populations, suggesting that co-digestion with food waste may improve the functional diversity of anaerobic digesters.

Impact of activated sludge ozonation on filamentous bacteria viability and possible added benefits.

Ozone was applied to return activated sludge in full-scale to study how ozone impacts filamentous bacteria viability (Live/Dead®). Additionally, the ozonated sludges were subjected to anaerobic digestion trials and analysis of micropollutants (MPs). Ozone treatment (3-4.8 g O3/kg total suspended solids) improved the settling properties of the sludge by lowering the diluted sludge volume with 7-35%. Ozone inactivated filamentous bacteria outside the floc structures and the fraction of inactivated filaments increased with an increasing ozone dose. It was observed that ozone treatment may act selectively towards different types of filaments. With respect to the two dominating morphotypes present, Type 0041 filamentous bacteria were found to be more resistant to ozone attack than Microthrix parvicella. Thus, higher ozone doses may be required to mitigate sludge bulking caused by Type 0041 filaments. No effects could be discerned by ozone addition on neither the methane production of the sludge nor on the concentrations of MPs analysed for this study. The lack of effect on both methane production and micropollutant removal was deemed to be caused by insufficient ozone doses.

Evaluation of direct membrane filtration and direct forward osmosis as concepts for compact and energy-positive municipal wastewater treatment.

Municipal wastewater treatment commonly involves mechanical, biological and chemical treatment steps to protect humans and the environment from adverse effects. Membrane technology has gained increasing attention as an alternative to conventional wastewater treatment due to increased urbanization. Among the available membrane technologies, microfiltration (MF) and forward osmosis (FO) have been selected for this study due to their specific characteristics, such as compactness and efficient removal of particles. In this study, two treatment concepts were evaluated with regard to their specific electricity, energy and area demands. Both concepts would fulfil the Swedish discharge demands for small- and medium-sized wastewater treatment plants at full scale: (1) direct MF and (2) direct FO with seawater as the draw solution. The framework of this study is based on a combination of data obtained from bench- and pilot-scale experiments applying direct MF and FO, respectively. Additionally, available complementary data from a Swedish full-scale wastewater treatment plant and the literature were used to evaluate the concepts in depth. The results of this study indicate that both concepts are net positive with respect to electricity and energy, as more biogas can be produced compared to that using conventional wastewater treatment. Furthermore, the specific area demand is significantly reduced. This study demonstrates that municipal wastewater could be treated in a more energy- and area-efficient manner with techniques that are already commercially available and with future membrane technology.

Detection of acidification limit in anaerobic membrane bioreactors at ambient temperature.

High-volume, low-strength industrial wastewaters constitute a large potential for biogas production, which could be realized by membrane bioreactors operating at the ambient temperature of the wastewater. However, the start-up of low-temperature anaerobic processes using unadapted inoculum can be sensitive to overloading, which results in acidification. This study assessed if a novel acidification limit test can be used to identify stable organic loading rates as well as process over-loading. The test is based on easy-to-apply batch experiments for determination of the hydrolysis rate constant and the specific methanogenic activity of the acetotrophic and hydrogenotrophic pathways. For evaluation, two anaerobic membrane bioreactors, treating synthetic dairy wastewater at an ambient temperature of 24 °C, were used with a slow or a rapid start-up regime, respectively. Tests for hydrolysis rate and methanogenic activity were performed throughout the experiment and were used to calculate acidification limits for each system throughout the start-up. The acidification limit test was able to successfully identify both stable operation of one reactor and process failure of the other reactor as the organic loading rate increased. The reactor failure was caused by over-loading the acetotrophic pathway and coincided with microbial changes observed in real-time PCR and moving window analysis. Overall, the acidification limit tests seem promising as an easy applicable method for estimating what organic loading rate can be utilized, without risking acidification of anaerobic systems.

Modeling of Anaerobic Digestion with a Focus on Estimation of Hydrolysis Constants at 35, 55, and 60 °C.

Hydrolysis constants of mixed sludge at 35, 55, and 60 °C were found to be 0.32, 0.44, and 0.50 1/d, respectively, in pilot-scale, semicontinuously operated anaerobic digesters. The hydrolysis constants and estimated chemical oxygen demand fractions in the feed were introduced to a mathematical model for anaerobic digestion published by Siegrist et al. (2002), which is similar to Anaerobic Digestion Model No. 1. First-order and Monod-type kinetics were tested for estimation of hydrolysis constants. The applied kinetics were found to affect the outcome of the regression study. Moreover, the free ammonia inhibition model was excluded for both propionate oxidation and acetate conversion, thanks to the apparent acclimatized biomass. No substantial accumulation of volatile fatty acids was observed in the reactors at 35, 55, and 60 °C, corresponding to free ammonia nitrogen concentrations of about 20, 110, and 130 g N/m³, respectively.

Functional movement screen normative values in a young, active population.

BACKGROUND: The Functional Movement Screen(TM) (FMS(TM)) is a screening instrument which evaluates selective fundamental movement patterns to determine potential injury risk. However, despite its global use, there are currently no normative values available for the FMS(TM). OBJECTIVES: To establish normative values for the FMS(TM) in a population of active, healthy individuals. Secondary aims were to investigate whether performance differed between males and females, between those with and without a previous history of injury, and to establish real-time inter-rater reliability of the FMS(TM). METHODS: Two hundred and nine (108 females and 101 males) physically active individuals, aged between 18 and 40 years, with no recent (<6 weeks) history of musculoskeletal injury were recruited. All participants performed the FMS(TM) and were scored using the previously established standardized FMS(TM) criteria. A representative sub-group participant sample (28%) determined inter rater reliability. RESULTS: The mean composite FMS(TM) score was 15.7 with a 95% confidence interval between 15.4 and 15.9 out of a possible total of 21. There was no statistically significant difference in scores between females and males (t(207) = .979, p = .329), or those who reported a previous injury and those who did not (t(207) = .688, p= .492). Inter-rater reliability (ICC(3,1)) for the composite FMS(TM) score was .971, demonstrating excellent reliability. Inter-rater reliability (Kappa) for individual test components of the FMS(TM) demonstrated substantial to excellent agreement (0.70 - 1.0). DISCUSSION AND CONCLUSION: This cross-sectional study provides FMS(TM) reference values for young, active individuals, which will assist in the interpretation of individual scores when screening athletes for musculoskeletal injury and performance factors.

Anaerobic digestion potential of urban organic waste: a case study in Malmö.

A study of existing organic waste types in Malmö, Sweden was performed. The purpose was to gather information about organic waste types in the city to be able to estimate the potential for anaerobic treatment in existing digesters at the wastewater treatment plan (WWTP). The urban organic waste types that could have a significant potential for anaerobic digestion amount to about 50 000 tonnes year(-1) (sludge excluded). Some of the waste types were further evaluated by methane potential tests and continuous pilot-scale digestion. Single-substrate digestion and co-digestion of pre-treated, source-sorted organic fraction of municipal solid waste, wastewater sludge, sludge from grease traps and fruit and vegetable waste were carried out. The experiments showed that codigestion of grease sludge and WWTP sludge was a better way of making use of the methane potential in the grease trap sludge than single-substrate digestion. Another way of increasing the methane production in sludge digesters is to add source-sorted organic fraction of municipal solid waste (SSOFMSW). Adding SSOFMSW (20% of the total volatile solids) gave a 10-15% higher yield than could be expected by comparison with separate digestion of sludge respective SSOFMSW. Co-digestion of sludge and organic waste is beneficial not just for increasing gas production but also for stabilizing the digestion process. This was seen when co-digesting fruit and vegetable waste and sludge. When co-digested with sludge, this waste gave a better result than the separate digestion of fruit and vegetable waste. Considering single-substrate digestion, SSOFMSW is the only waste in the study which makes up a sufficient quantity to be suitable as the base substrate in a full-scale digester that is separated from the sludge digestion. The two types of SSOFMSW tested in the pilot-scale digestion were operated successfully at mesophilic temperature. By adding SSOFMSW, grease trap sludge and fruit and vegetables waste to sludge digesters at the wastewater treatment plant, the yearly energy production from methane could be expected to increase from 24 to 43 GWh.

Effects of pre-treatment technologies on quantity and quality of source-sorted municipal organic waste for biogas recovery.

Source-sorted municipal organic waste collected from different dwelling types in five Danish cities and pre-treated at three different plants was sampled and characterized several times during one year to investigate the origin of any differences in composition of the pre-treated waste introduced by city, pre-treatment technology, dwelling type or annual season. The investigated pre-treatment technologies were screw press, disc screen and shredder+magnet. The average quantity of pre-treated organic waste (biomass) produced from the incoming waste varied between the investigated pre-treatment technologies: 59%, 66% and 98% wet weight, respectively (41%, 34% and 2% reject, respectively). The pre-treatment technologies showed differences with respect to distribution of the chemical components in the waste between the biomass and the rejected material (reject), especially for dry matter, ash, collection bag material (plastic or paper) and easily degradable organic matter. Furthermore, the particle size of the biomass was related to the pre-treatment technology. The content of plastic in the biomass depended both on the actual collection bag material used in the system and the pre-treatment technology. The sampled reject consisted mostly of organic matter. For cities using plastic bags for the source-separated organic waste, the expected content of plastic in the reject was up to 10% wet weight (in some cases up to 20%). Batch tests for methane potential of the biomass samples showed only minor variations caused by the factors city, pre-treatment technology, dwelling type and season when based on the VS content of the waste (overall average 459STPm(3)/tVS). The amount of methane generated from 1t of collected waste was therefore mainly determined by the efficiency of the chosen pre-treatment technology described by the mass distribution of the incoming waste between biomass and reject.

Composition of source-sorted municipal organic waste collected in Danish cities.

Source-sorted municipal organic waste from different dwelling types in five Danish cities was sampled during one year. The samples were from permanent, full-scale systems or temporary, experimental systems for collection of source-sorted municipal organic waste. Pre-treatment of the organic waste prior to biological treatment was used in all cities to remove foreign objects and provide size reduction. All sampling was performed after pre-treatment in order to obtain more homogeneous and representative samples. The sampling included both the pre-treated waste and the reject from the pre-treatment allowing for estimation of the composition of the original waste. A total of 40 waste samples were chemically characterised with respect to 15 parameters. The waste generally consisted of around 88% VS of which an average of 80% was easily degradable. The average content of N, P and K in the dry matter of the organic waste was 2.5%, 0.4% and 0.9%, respectively. A general analysis of variance was applied to show the influence of the collection system, dwelling type and annual season on the waste composition. The content of plastic and crude fibres in the waste differed the most among the samples, probably due to use of different bag types (plastic and paper) in the different collection systems. Variations in the ash content and the calorific value might be explained by differences in the sorting instructions (whether soil and cat litter are allowed in the organic fraction). Significant seasonal variations were seen for ash, S and Cl. Dwelling type showed no statistically significant influence on any waste components. A test for uniform distribution of the p-values from the analysis of variance (Kolmogorov-Smirnov test) showed that the overall composition of the collected waste was strongly affected by the collection system (city) and season, while dwelling type had no significant influence.

Methane yield in source-sorted organic fraction of municipal solid waste.

Treating the source-separated organic fraction of municipal solid waste (SS-OFMSW) by anaerobic digestion is considered by many municipalities in Europe as an environmentally friendly means of treating organic waste and simultaneously producing methane gas. Methane yield can be used as a parameter for evaluation of the many different systems that exist for sorting and pre-treating waste. Methane yield from the thermophilic pilot scale digestion of 17 types of domestically SS-OFMSW originating from seven full-scale sorting systems was found. The samples were collected during 1 year using worked-out procedures tested statistically to ensure representative samples. Each waste type was identified by its origin and by pre-sorting, collection and pre-treatment methods. In addition to the pilot scale digestion, all samples were examined by chemical analyses and methane potential measurements. A VS-degradation rate of around 80% and a methane yield of 300-400Nm(3) CH(4)/ton VS(in) were achieved with a retention time of 15 days, corresponding to approximately 70% of the methane potential. The different waste samples gave minor variation in chemical composition and thus also in methane yield and methane potential. This indicates that sorting and collection systems in the present study do not significantly affect the amount of methane produced per VS treated.