Microbial synergies drive simultaneous biodegradation of ethoxy and alkyl chains of Nonylphenol Ethoxylate in fluidized bed reactors.

The widely-used surfactant Nonylphenol Ethoxylate (NPEO) produces endocrine-disrupting compounds during biodegradation, with these byproducts being more harmful than untreated NPEO. This study investigates the effectiveness of a Fluidized Bed Reactor (FBR) in reducing the production of 4-Nonylphenol (4-NP) during the biodegradation of NPEO. Two identical FBR filled with sand were used to assess the NPEO degradation and to enhance the microbial consortia capable of breaking down the complex byproducts, ethanol and fumarate were introduced as co-substrates. Our findings demonstrate the significant potential of the FBR, especially when coupled with fumarate, for enhancing the surfactant degradation. It outperforms the efficiency achieved with ethanol as the primary electron donor, albeit with a higher rate of byproduct production. Microbial community taxonomy and metabolic prediction revealed the high abundance of Geobacter (1.51-31.71%) and Methanobacterium (1.08-13.81%) in non-conductive sand. This may hint a new metabolic interaction and expand our understanding of Direct Interspecies Electron Transfer (DIET) in bioreactors applied to micropollutants degradation. Such an intricate relationship between facultative and anaerobes working together to simultaneously biodegrade the ethoxy and alkyl chains presents a new perspective on NPEO degradation and can potentially be extended to other micropollutants.

Bio-CH4 yield of swine manure and food waste optimized by co-substrate proportions diluted in domestic sewage and pH interactions using the response surface approach.

This research aimed at evaluating optimal conditions to obtain value-added metabolites, such as bio-CH4, by co-digesting swine manure and food waste diluted in domestic sewage. The assays were carried out in batches using the statistical methods of Rotational Central Composite Design (RCCD) and Surface Response to evaluate the ranges of food waste (1.30-9.70 gTS.L-1), pH (6.16-7.84) and granular Upflow Anaerobic Sludge Blanket sludge as inoculum (2.32-5.68 gTS.L-1), besides about 250 mL of swine manure in 500 mL Duran flasks. According to the RCCD matrix, bio-CH4 yields among 600.6 ± 60.1 and 2790.0 ± 112.0 mL CH4 gTS.L-1 were observed, besides the maximum CH4 production rate between 0.4 ± 0.5 and 49.7 ± 2.0 mL CH4 h-1 and λ between ≤0.0 and 299.3 ± 4.5 h. In the validation assay, the optimal conditions of 9.98 gTS.L-1 of food waste, pH adjusted to 8.0 and 2.20 gTS.L-1 of inoculum were considered, and the bio-CH4 yield obtained (5640.79 ± 242.98 mL CH4 gTS.L-1 or also 5201.83 ± 224.07 mL CH4 gTVS.L-1) was 11.3 times higher than in assays before optimization (499.3 ± 16.0 mL CH4 gTS.L-1) with 5 gTS.L-1 of food waste, 3 gTS.L-1 of inoculum and pH 7.0. Besides, the results observed about the energetic balance of the control and validation assays highlight the importance of process optimization, as this condition was the only one with energy supply higher than the energy required for its operation, exceeding max consumption sevenfold. Based on the most dominant microorganisms (Methanosaeta, 31.06%) and the metabolic inference of the validation assay, it could be inferred that the acetoclastic methanogenesis was the predominant pathway to CH4 production.

Application of nanoparticles to increase biological hydrogen production: the difference in metabolic pathways in batch and continuous reactors.

An alternative to improve the production of biorefinery products, such as biohydrogen (H2) and volatile fatty acids (VFA), is the combination of nanotechnology and biological processes. In order to compare the use of both processes in two different reactor configurations, batch reactors and continuous anaerobic fluidized bed reactors (AFBR) were studied under the same conditions (37°C, pH 6.8, Clostridium butyricum as an inoculum and glucose as a substrate) to evaluate the influence of zero valence iron and nickel nanoparticles (NPs) on H2 and VFA production. There was a shift in the production of acetic and butyric acids to produce mainly valeric acid when NPs were added in batch reactors. Meanwhile, in AFBR the change was from lactic acid to butyric and acetic acids with the addition of NPs. It showed that the effect of NPs on the fermentation process was different when the configuration of batch and continuous reactors was compared. The H2 yield in both reactor configurations increased with the addition of NPs. In batch reactors from 6.6 to 8.0 mmol H2 g-1 of COD and in AFBR from 4.9 to 6.2 mmol of H2 g-1 of COD. Therefore, given the simplicity and low cost of the synthesis of metallic NPs, it is a promising additive to optimize the fermentation process in different reactor configurations.

Metataxonomic characterization of an autochthonous and allochthonous microbial consortium involved in a two-stage anaerobic batch reactor applied to hydrogen and methane production from sugarcane bagasse.

Enzymatically pre-treated sugarcane bagasse (SCB) was used as substrate for sequential production of hydrogen (H2) and methane (CH4). In stage I, SCB was used by autochthonous bacteria, such as Enterococcus (42.4 % relative abundance) and Paraclostridium (16.8 %) for H2 production (166.8 mL H2/L), while the metabolites accumulated at the end of this stage (9140.5 mg HAc/L) were used by allochthonous inoculum for CH4 production (870.8 mL CH4/L) in stage II. In stage II, hydrogenotrophic (Methanoculleus, 49.1 %) and acetoclastic (Methanosaeta, 15.5 %) archaeal genera were identified and considered important to maintain low H2 pressure in the system. According to gene inference, the hydrolysis of the SCB fiber was performed in both stages, as potential ß-glucosidase and 1,4-ß-xylosidase encoding genes were predicted. However, the energy metabolism of microbial populations differed, as potential genes involved in CH4 metabolism were predominant in phase II (39.0 %).

Influence of cosubstrate and hydraulic retention time on the removal of drugs and hygiene products in sanitary sewage in an anaerobic Expanded Granular Sludge Bed reactor.

Diclofenac (DCF), ibuprofen (IBU), propranolol (PRO), triclosan (TCS) and linear alkylbenzene sulfonate (LAS) can be recalcitrant in Wastewater Treatment Plants (WWTP). The removal of these compounds was investigated in scale-up (69 L) Expanded Granular Sludge Bed (EGSB) reactor, fed with sanitary sewage from the São Carlos-SP (Brazil) WWTP and 200 mg L-1 of ethanol. The EGSB was operated in three phases: (I) hydraulic retention time (HRT) of 36±4 h; (II) HRT of 20±2 h and (III) HRT of 20±2 h with ethanol. Phases I and II showed no significant difference in the removal of LAS (63 ± 11-65 ± 12 %), DCF (37 ± 18-35 ± 11 %), IBU (43 ± 18-44 ± 16 %) and PRO (46 ± 25-51 ± 23 %) for 13±2-15 ± 2 mg L-1, 106 ± 32-462 ± 294 µg L-1, 166 ± 55-462 ± 213 µg L-1 and 201 ± 113-250 ± 141 µg L-1 influent, respectively. Higher TCS removal was obtained in phase I (72 ± 17 % for 127 ± 120 µg L-1 influent) when compared to phase II (51 ± 13 % for 135 ± 119 µg L-1 influent). This was due to its greater adsorption (40 %) in the initial phase. Phase III had higher removal of DCF (42 ± 10 % for 107 ± 26 µg L-1 influent), IBU (50 ± 15 % for 164 ± 47 µg L-1 influent) and TCS (85 ± 15 % for 185 ± 148 µg L-1 influent) and lower removal of LAS (35 ± 14 % for 12 ± 3 mg L-1 influent) and PRO (-142 ± 177 % for 188 ± 88 µg L-1 influent). Bacteria similar to Syntrophobacter, Smithella, Macellibacteroides, Syntrophus, Blvii28_wastewater-sludge_group and Bacteroides were identified in phase I with relative abundance of 3.1 %-4.7 %. Syntrophobacter was more abundant (15.4 %) in phase II, while in phase III, it was Smithella (12.7 %) and Caldisericum (15.1 %). Regarding the Archaea Domain, Methanosaeta was more abundant in phases I (84 %) and II (67 %), while in phase III it was Methanobacterium (86 %).

Influence of metabolic cosubstrates on methanogenic potential and degradation of triclosan and propranolol in sanitary sewage.

Triclosan (TCS) and propranolol (PRO) are emerging micropollutants that are difficult to remove in wastewater treatment plants. In this study, methanogenic potential (P) of anaerobic sludge submitted to TCS (3.6 ± 0.1 to 15.5 ± 0.1 mg L-1) and PRO (6.1 ± 0.1 to 55.9 ± 1.2 mg L-1) in sanitary sewage, was investigated in batch reactors. The use of cosubstrates (200 mg L-1 of organic matter) ethanol, methanol:ethanol and fumarate was evaluated for micropollutant degradation. Without cosubstrates, P values for 5.0 ± 0.1 mgTCS L-1, 15.5 ± 0.1 mgTCS L-1 and 55.0 ± 1.3 mgPRO L-1 were 50.53%, 98.24% and 17.66% lower in relation to Control assay (855 ± 5 µmolCH4) with sanitary sewage, without micropollutants and cosubstrates, respectively. The use of fumarate, ethanol and methanol:ethanol favored greater methane production, with P values of 2144 ± 45 µmolCH4, 2960 ± 185 µmolCH4 and 2239 ± 171 µmolCH4 for 5.1 ± 0.1 mgTCS L-1, respectively; and of 10,827 ± 185 µmolCH4, 10,946 ± 108 µmolCH4 and 10,809 ± 210 µmolCH4 for 55.0 ± 1.3 mgPRO L-1, respectively. Greater degradation of TCS (77.1 ± 0.1% for 5.1 ± 0.1 mg L-1) and PRO (24.1 ± 0.1% for 55.9 ± 1.2 mg L-1) was obtained with ethanol. However, with 28.5 ± 0.5 mg PRO L-1, greater degradation (88.4 ± 0.9%) was obtained without cosubstrates. With TCS, via sequencing of rRNA 16S gene, for Bacteria Domain, greater abundance of phylum Chloroflexi and of the genera Longilinea, Arcobacter, Mesotoga and Sulfuricurvum were identified. With PRO, the genus VadinBC27 was the most abundant. Methanosaeta was dominant in TCS with ethanol, while in PRO without cosubstrates, Methanobacterium and Methanosaeta were the most abundant. The use of metabolic cosubstrates is a favorable strategy to obtain greater methanogenic potential and degradation of TCS and PRO.

Methanogenic potential of diclofenac and ibuprofen in sanitary sewage using metabolic cosubstrates.

Diclofenac (DCF) and ibuprofen (IBU) are widely used anti-inflammatory drugs and are frequently detected in wastewater from Wastewater Treatment Plants and in aquatic environments. In this study, the methanogenic potential (P) of anaerobic sludge subjected to DCF (7.11 ± 0.02 to 44.41 ± 0.05 mg L-1) and IBU (6.11 ± 0.01 to 42.61 ± 0.05 mg L-1), in sanitary sewage, was investigated in batch reactors. Cosubstrates (200 mg L-1 of organic matter) in the form of ethanol, methanol:ethanol and fumarate were tested separately for the removal of drugs. In the DCF assays, P was 6943 ± 121 µmolCH4, 9379 ± 259 µmolCH4, 9897 ± 212 µmolCH4 and 11,530 ± 368 µmolCH4 for control, fumarate, methanol:ethanol and ethanol conditions, respectively. In the IBU assays, under the same conditions, P was 6145 ± 101 µmolCH4, 6947 ± 66 µmolCH4, 8141 ± 191 µmolCH4and 10,583 ± 512 µmolCH4, respectively. Without cosubstrates, drug removal was below 18% for 43.10 ± 0.01 mgDCF L-1 and 43.12 ± 0.03 mgIBU L-1, respectively. Higher P and removal of DCF (28.24 ± 1.10%) and IBU (18.72 ± 1.60%) with ethanol was observed for 43.20 ± 0.01 mgDCF L-1 and 43.42 ± 0.03 mgIBU L-1, respectively. This aspect was better evidenced with DCF due to its molecular structure, a condition that resulted in a higher diversity of bacterial populations. Through the 16S rRNA sequencing, bacteria genera capable of performing aromatic ring cleavage, ß-oxidation and oxidation of ethanol and fatty acids were identified. Higher relative abundance (>0.6%) was observed for Smithella, Sulfuricurvum and Synthophus for the Bacteria Domain and Methanosaeta (>79%) for the Archaea Domain. The use of ethanol favored greater mineralization of organic matter and greater methane production, which can directly assist in the metabolic pathways of microorganisms.

Influence of linear alkylbenzene sulfonate and ethanol on the degradation kinetics of domestic sewage in co-digestion with commercial laundry wastewater.

The influence of ethanol on the degradation kinetics of linear alkyl benzene sulfonate (LAS) and organic matter was investigated using batch experiments with different initial LAS concentrations (8.3 mg L-1 to 66.9 mg L-1) and biomass immobilized on sand. Data were fitted with a substrate inhibition model. Concentrations of 2.4 mg LAS L-1 and 18.9 mg LAS L-1 (without and with ethanol) provided the maximum LAS utilization rate by the biomass (Sbm). For LAS degradation, ethanol addition favored a lower decrease in the specific substrate utilization rate (robs), even at the LAS concentration usually reported as inhibitory (> 14.4 mg L-1). For organic matter degradation, robs was higher with ethanol. Higher biomass differentiation was observed at higher LAS concentrations. With ethanol, microbial selection occurred at LAS concentrations near Sbm. At higher LAS concentrations, the dominance and diversity values did not change significantly with ethanol, whereas without ethanol, their behaviors were irregular.

Microbial diversity of a full-scale UASB reactor applied to poultry slaughterhouse wastewater treatment: integration of 16S rRNA gene amplicon and shotgun metagenomic sequencing.

The 16S rRNA gene amplicon and whole-genome shotgun metagenomic (WGSM) sequencing approaches were used to investigate wide-spectrum profiles of microbial composition and metabolic diversity from a full-scale UASB reactor applied to poultry slaughterhouse wastewater treatment. The data were generated by using MiSeq 2 × 250 bp and HiSeq 2 × 150 bp Illumina sequencing platforms for 16S amplicon and WGSM sequencing, respectively. Each approach revealed a distinct microbial community profile, with Pseudomonas and Psychrobacter as predominant genus for the WGSM dataset and Clostridium and Methanosaeta for the 16S rRNA gene amplicon dataset. The virome characterization revealed the presence of two viral families with Bacteria and Archaea as host, Myoviridae, and Siphoviridae. A wide functional diversity was found with predominance of genes involved in the metabolism of acetone, butanol, and ethanol synthesis; and one-carbon metabolism (e.g., methanogenesis). Genes related to the acetotrophic methanogenesis pathways were more abundant than methylotrophic and hydrogenotrophic, corroborating the taxonomic results that showed the prevalence of the acetotrophic genus Methanosaeta. Moreover, the dataset indicated a variety of metabolic genes involved in sulfur, nitrogen, iron, and phosphorus cycles, with many genera able to act in all cycles. BLAST analysis against Antibiotic Resistance Genes Database (ARDB) revealed that microbial community contained 43 different types of antibiotic resistance genes, some of them were associated with growth chicken promotion (e.g., bacitracin, tetracycline, and polymyxin).

Simultaneous determination of anionic and nonionic surfactants in commercial laundry wastewater and anaerobic fluidized bed reactor effluent by online column-switching liquid chromatography/tandem mass spectrometry.

This study presents a new method developed for the simultaneous determination of anionic surfactant (linear alkylbenzene sulfonate - LAS, 4 homologs) and nonionic surfactant (linear alcohol ethoxylate - LAE) in commercial laundry wastewater. The surfactants were identified and quantified using online column-switching solid-phase extraction (SPE) coupled with liquid chromatography/tandem mass spectrometry (LC-MS/MS). Ten and three transitions (m/z) were identified for LAS and LAE, respectively. The detection and quantification limits were 75 and 200µg/L for LAS, respectively, and 75µg/L for LAE. This method was applied to the determination of the surfactants in the influent and effluent of an anaerobic fluidized bed reactor that was used for the treatment of commercial laundry wastewater. After 480days of operation with a hydraulic retention time (HRT) of 18h, the removal of 45.9±5.6% LAS and 99.2±4.3% LAE from an influent with surfactant concentrations of 26.1±12.9mg/L and 23.8±6.8mg/L, respectively, was obtained. Under these conditions, the breakage of longer-chain LAS homologs with the release of carbon units was observed with an increase in the number of shorter homolog chains. This SPE online sample treatment method is simple, fast and effective for the analysis of both surfactants. This technique is pioneering in its simultaneous measurement of two surfactant categories in anaerobic fluidized bed reactors.

Kinetics of methane production and biodegradation of linear alkylbenzene sulfonate from laundry wastewater.

This study evaluates the kinetics of methane production and degradation of standard linear alkylbenzene sulfonate (LAS) (50 ± 3.5 mg/L) and LAS from laundry wastewater (85 ± 2.1 mg/L) in anaerobic batch reactors at 30°C with different sources of inoculum. The inocula were obtained by auto-fermentation (AFM) and UASB reactors from wastewater treatment of poultry slaughterhouse (SGH), swine production (SWT) and wastewater treatment thermophilic of sugarcane industry (THR). The study was divided into three phases: synthetic substrate (Phase I), standard LAS (Phase II) and LAS from laundry wastewater (Phase III). For SGH, the highest values for cumulative methane productions (1,844.8 ± 149 µmol-Phase II), methane production rate (70.8 ± 88 µmol/h-Phase II and 4.01 ± 07 µmol/h-Phase III) were observed. The use of thermophilic biomass (THR) incubated at 30°C was not favorable for methane production and LAS biodegradation, but the highest kinetic coefficient degradation (k1app) was obtained for LAS (0.33 ± 0.3 h) compared with mesophilic biomass (SGH and SWT) (0.13 ± 0.02 h). Therefore, both LAS sources influenced the kinetics of methane production and organic matter degradation. For SGH, inoculum obtained the highest LAS degradation. In the SGH inoculum sequenced by MiSeq-Illumina was identified genera (VadinCA02, Candidatus Cloacamonas, VadinHB04, PD-UASB-13) related to degrade toxic compounds. Therefore, it recommended the reactor mesophilic inoculum UASB (SGH) for the LAS degradation.

Biodegradation of linear alkylbenzene sulfonate in commercial laundry wastewater by an anaerobic fluidized bed reactor.

The biodegradation of linear alkylbenzene sulfonate (LAS) from commercial laundry wastewater was evaluated in an anaerobic fluidized bed reactor (FBR) fed with synthetic substrate (598 mg L(-1) to 723 mg L(-1) of organic matter) supplemented with 9.5±3.1 mg L(-1) to 27.9±9.6 mg L(-1) of LAS. The average chemical oxygen demand (COD) removal efficiency was 89% and the biodegradation of LAS was 57% during the 489 days of anaerobic FBR. Higher levels of volatile fatty acids (VFA) were observed in the effluent at the stage with the best LAS removal performance. Increasing the surfactant concentration did not increase the VFA production in the effluent. The predominant VFAs after the addition of LAS were as follows: isovaleric acid and valeric acid, followed by propionic acid, caproic acid and formic acid. The similarities of 64% and 45% to Archaea and Bacteria domains were observed in the samples taken in the operating period of anaerobic FBR fed with 23.6±10 mg L(-1) and 27.9±10 mg L(-1) of LAS. During the operation stages in the reactor, Gemmatimonas, Desulfobulbus and Zoogloea were determined as the most abundant genera related to surfactant degradation using 454-Pyrosequencing.

Application of molecular techniques to evaluate the methanogenic archaea and anaerobic bacteria in the presence of oxygen with different COD:sulfate ratios in a UASB reactor.

In this paper, the microbial characteristics of the granular sludge in the presence of oxygen (3.0+/-0.7 mg O2 l(-1)) were analyzed using molecular biology techniques. The granules were provided by an upflow anaerobic sludge blanket (UASB) operated over 469 days and fed with synthetic substrate. Ethanol and sulfate were added to obtain different COD/SO4(2-) ratios (3.0, 2.0, and 1.6). The results of fluorescent in situ hybridization (FISH) analyses showed that archaeal cells, detected by the ARC915 probe, accounted for 77%, 84%, and 75% in the COD/SO(4)(2-) ratios (3.0, 2.0, and 1.6, respectively). Methanosaeta sp. was the predominant acetoclastic archaea observed by optical microscopy and FISH analyses, and confirmed by sequencing of the excised bands of the DGGE gel with a similarity of 96%. The sulfate-reducing bacterium Desulfovibrio vulgaris subsp. vulgaris (similarity of 99%) was verified by sequencing of the DGGE band. Others identified microorganism were similar to Shewanella sp. and Desulfitobacterium hafniense, with similarities of 95% and 99%, respectively. These results confirmed that the presence of oxygen did not severely affect the metabolism of microorganisms that are commonly considered strictly anaerobic. We obtained mean efficiencies of organic matter conversion and sulfate reducing higher than 74%.