Microalgae produced during phycoremediation of swine wastewater contains effective bacteriostatic compounds against antibiotic-resistant bacteria.

Studies on the antimicrobial effects of microalgae extracts are commonly reported using algae biomass grown in sterile synthetic mineral medium and controlled laboratory conditions. However, variations in environmental conditions and culture medium composition are known to alter microalgae biochemical structure possibly affecting the type and concentrations of bioactive compounds with antimicrobial properties. In this work, solvent extracts of the microalgae Chlorella spp. were tested for antimicrobial effects against gram-positive and multidrug resistant pathogenic bacteria Staphylococcus hyicus, Enterococcus faecalis and Streptococcus suis. Microalgae was cultivated at field scale open pond reactor using raw swine wastewater as growth substrate. Dichloromethane or methanol were used to obtain the microalgae extracts. Characterization of the extracts by ultra-high performance liquid chromatography-quadrupole mass spectrometry revealed the presence of 23 phytochemicals with recognized antimicrobial properties. Bacteriostatic activity was observed in plating assays by formation of inhibition zones ranging from 7 to 18 mm in diameter. Only dichloromethane extracts were inhibitory to all three model bacteria. The minimum inhibitory concentration assessed for dichloromethane extracts were 0.5 mg mL-1 for Staphylococcus hyicus and Enterococcus faecalis and 0.2 mg mL-1 for Streptococcus suis. Bactericidal effects were not observed using solvent-extracts at 2 or 5 mg L-1. To the best of authors knowledge, this is the first report on the antimicrobial effects of Chlorella spp. extracts against Staphylococcus hyicus and Streptococcus suis. Overall, Chlorella spp. grown on swine wastewater contains several phytochemicals that could be further explored for the treatment of infections caused by antibiotic-resistant bacteria pathogens.

Enhanced long-term attenuation of 1,4-dioxane in bioaugmented flow-through aquifer columns.

Long term natural attenuation of 1,4-dioxane (dioxane) and its enhanced biodegradation after bioaugmentation with Pseudonocardia dioxanivorans CB1190 were assessed using flow-through aquifer columns. Natural attenuation of dioxane was not observed even after 2 years of acclimation. However, dioxane removal was observed in the bioaugmented columns (34% when the influent was 200 µg/L and 92% for 5 mg/L). The thmA gene that encodes the tetrahydrofuran monooxygenase that initiates dioxane degradation by CB1190 was only detected at the inoculation port and persisted for months after inoculation, implying the resiliency of bioaugmentation and its potential to offer long-term enhanced biodegradation capabilities. However, due to extensive clumping and limited mobility of CB1190, the augmented catabolic potential may be restricted to the immediate vicinity of the inoculation port. Accordingly, bioaugmentation with CB1190 seems more appropriate for the establishment of biobarriers. Bioaugmentation efficiency was associated with the availability of oxygen. Aeration of the column influent to increase dissolved oxygen significantly improved dioxane removal (p < 0.05), suggesting that (for sites with oxygen-limiting conditions) bioaugmentation can benefit from engineered approaches for delivering additional oxygen.

Residual sugar from microalgae biomass harvested from phycoremediation of swine wastewater digestate.

The present study assessed the carbohydrate and sugar production from Chlorella spp. biomass harvested from a field scale reactor simulating phycoremediation of swine wastewater. The microalgae biomass was mainly composed by (%): carbohydrates (41 ± 0.4), proteins (50 ± 0.4), and lipids (1.3 ± 0.5). The residual sugar present in the biomass was extracted via acid hydrolysis. Among different concentrations of sulfuric acid tested (i.e., 47, 94, 188, 281 and 563 mM), significantly higher sugar content was obtained with 188 mM (0.496 g-sugar g-1 microalgae-DW). The concentration of sugar present in the microalgae did not differ significantly between the biomasses harvested by either centrifugation or coagulation-flocculation. Two commercially available strains of yeast (i.e., Saccharomyces cerevisiae and S. cerevisiae chardonnay) were tested for their capability to ferment sugar from lyophilized microalgae biomass. S. cerevisiae chardonnay showed a significantly faster consumption of sugar during the exponential growth phase. Both strains of yeast were capable of consuming most of the sugar added ≅ 8 g L-1 within 24 h. Overall, the results suggest that carbohydrate-rich microalgae biomass obtained from the phycoremediation of swine wastewaters can play an important role in green design for industries seeking alternative sources of feedstock rich in sugar.

Associating potential 1,4-dioxane biodegradation activity with groundwater geochemical parameters at four different contaminated sites.

1,4-Dioxane (dioxane) is a groundwater contaminant of emerging concern for which bioremediation may become a practical remediation strategy. Therefore, it is important to advance our heuristic understanding of geochemical parameters that are most influential on the potential success of intrinsic bioremediation of dioxane-impacted sites. Here, Pearson's and Spearman's correlation and linear regression analyses were conducted to discern associations between 1,4-dioxane biodegradation activity measured in aerobic microcosms and groundwater geochemical parameters at four different contaminated sites. Dissolved oxygen, which is known to limit dioxane biodegradation, was excluded as a limiting factor in this analysis. Biodegradation activity was positively associated with dioxane concentrations (p < 0.01; R < 0.70) as well as the number of catabolic thmA gene copies (p < 0.01; R = 0.80) encoding dioxane monooxygenase. Thus, whereas environmental factors such as pH, temperature, and nutrients may influence dioxane biodegradation, these parameters did not exert as strong of an influence on potential biodegradation activity as the in situ concentration of substrate dioxane at the time of sampling. This analysis infers that aerobic sites with higher dioxane concentrations are more likely to select and sustain a thriving population of dioxane degraders, while sites with relatively low dioxane concentrations would be more difficult to attenuate naturally and may require alternative remediation strategies.

Enrichment and acclimation of an anaerobic mesophilic microorganism's inoculum for standardization of BMP assays.

Appropriate enrichment of anaerobic microorganism's consortium is crucial for accurate biochemical methane potential (BMP) assays. An alternative method to produce and maintain a mesophilic methanogenic inoculum was demonstrated. Three sources of inoculum were mixed and acclimated for 857days in order to reach steady conditions (pH=7.90±0.46; VS/TS>50%; VFA/alkalinity=0.16±0.04gAcetic Acid/ [Formula: see text] ). Biogas yield >80% was obtained after 70days of inoculum acclimation in comparison to standard cellulose (>600mLN/gVS). Methanogen community analysis based on 16S rDNA of the inoculum revealed Archaea concentration of 3×10(12) gene copies/g (Methanobacteriales 8×10(10); Methanomicrobiales 8×10(10); and Methanosarcinales 4×10(11) gene copies/g). The proposed method for development and maintenance of microorganism enrichment inoculum demonstrates consistent BMP data which is a requirement for dependable prediction of biogas production at field scale operations.

Methane production from a field-scale biofilter designed for desulfurization of biogas stream.

The development of a simple and low maintenance field-scale biotrickling filter (BTF) for desulfurization of swine wastewater-derived biogas stream that was also capable of increasing biomethane concentrations was investigated. BTF was continuously fed with wastewater effluent from an air sparged nitrification-denitrification bioreactor installed downgradient from an UASB-type digester. BTF maximum removal efficiency (RE) of 99.8% was achieved with a maximum elimination capacity (EC) of 1,509 g H2S m(-3) h(-1). Average EC obtained with inlet biogas flow rates of 0.024, 0.036 and 0.048 m(3) h(-1) was 718, 1,013 and 438 g H2S m(-3) h(-1), respectively. SO4(-2) and S(0) were the major metabolites produced from biological conversion of H2S. Additionally to the satisfactory biodesulfurization capacity, an average increase in methane concentration of ≅ 3.8 ± 1.68 g m(-3) was measured in the filtered gas stream throughout 200 days of BTF operation. RT-PCR analyses of archaea communities in the biofilm confirmed dominance of hydrogenotrophic methanogens thus corroborating with the observed strong correlation between CO2 removal and CH4 production. Among the three major archaea orders investigated (i.e., Methanosarcinales, Methanobacteriales, and Methanomicrobiales), Methanobacteriales were encountered at highest concentrations (1.9 × 10(11) gene copies mL(-1)). The proposed BTF was robust efficiently removing H2S from biogas stream while concomitantly enhancing the concentration of valuable methane as source of renewable fuel.

Effects of Nitrogen and Phosphorus on Biochemical Composition of Microalgae Polyculture Harvested from Phycoremediation of Piggery Wastewater Digestate.

The effects of nitrogen (N) and/or phosphorus (P) starvation on the biochemical composition of native microalgae Chlorella spp. polyculture obtained from the phycoremediation of swine wastewaters were investigated. Microalgae-specific growth rate of 1.2 day(-1) was achieved (30.3 mg L(-1) day(-1)). PO4 (-2) and NH3 were completely removed from swine digestate effluent after 3 and 11 days, respectively. Microalgae harvested immediately after nutrient removal showed high protein (56-59 %) and carbohydrate (25-34 %) but low lipid (1.8-3 %) contents. Depletion of N or P alone stimulated carbohydrate production at the expenses of proteins. Significant lipid accumulation from 3 % ± 0.5 to 16.3 % ± 0.8 was reached only after 25 days following N and P starvation as demonstrated by Nile red-stained cells. Regarding to the effects of harvesting methods on cellular biochemical composition, circumstantial evidences indicate that coagulation-flocculation with tannin may lead to lower protein and lipid amounts but increased carbohydrate content (p < 0.01) as compared to centrifugation.

Enhancement of nutrient removal from swine wastewater digestate coupled to biogas purification by microalgae Scenedesmus spp.

This work investigated the effects of swine wastewater-derived biogas on microalgae biomass production and nutrient removal rates from piggery wastewater concomitantly with biogas filtration. Photobioreactors with dominant Scenedesmus spp. were prepared using non-sterile digestate and exposed to different photoperiods. In the presence of biogas and autotrophic conditions microalgae yield of 1.1±0.2 g L(-1) (growth rate of 141.8±3.5 mg L(-1) d(-1)) was obtained leading to faster N-NH3 and P-PO4(3-) assimilation rate of 21.2±1.2 and 3.5±2.5 mg L(-1) d(-1), respectively. H2S up to 3000 ppmv was not inhibitory and completely removed. Maximum CO2 assimilation of 219±4.8 mg L(-1) d(-1) was achieved. Biological consumption of CH4 up to 18% v/v was verified. O2 up to 22% v/v was controlled by adding acetate to exacerbate oxygen demand by microorganisms. Microalgae-based wastewater treatment coupled to biogas purification accelerates nutrient removal concomitantly producing valuable biomass and biomethane.

Assessment of bacterial and archaeal community structure in Swine wastewater treatment processes.

Microbial communities from two field-scale swine wastewater treatment plants (WWTPs) were assessed by pyrosequencing analyses of bacterial and archaeal 16S ribosomal DNA (rDNA) fragments. Effluent samples from secondary (anaerobic covered lagoons and upflow anaerobic sludge blanket [UASB]) and tertiary treatment systems (open-pond natural attenuation lagoon and air-sparged nitrification-denitrification tank followed by alkaline phosphorus precipitation process) were analyzed. A total of 56,807 and 48,859 high-quality reads were obtained from bacterial and archaeal libraries, respectively. Dominant bacterial communities were associated with the phylum Firmicutes, Bacteroidetes, Proteobacteria, or Actinobacteria. Bacteria and archaea diversity were highest in UASB effluent sample. Escherichia, Lactobacillus, Bacteroides, and/or Prevotella were used as indicators of putative pathogen reduction throughout the WWTPs. Satisfactory pathogen reduction was observed after the open-pond natural attenuation lagoon but not after the air-sparged nitrification/denitrification followed by alkaline phosphorus precipitation treatment processes. Among the archaeal communities, 80% of the reads was related to hydrogeno-trophic methanogens Methanospirillum. Enrichment of hydrogenotrophic methanogens detected in effluent samples from the anaerobic covered lagoons and UASB suggested that CO2 reduction with H2 was the dominant methanogenic pathway in these systems. Overall, the results served to improve our current understanding of major microbial communities' changes downgradient from the pen and throughout swine WWTP as a result of different treatment processes.

Effects of nitrate injection on microbial enhanced oil recovery and oilfield reservoir souring.

Column experiments were utilized to investigate the effects of nitrate injection on sulfate-reducing bacteria (SRB) inhibition and microbial enhanced oil recovery (MEOR). An indigenous microbial consortium collected from the produced water of a Brazilian offshore field was used as inoculum. The presence of 150 mg/L volatile fatty acids (VFA´s) in the injection water contributed to a high biological electron acceptors demand and the establishment of anaerobic sulfate-reducing conditions. Continuous injection of nitrate (up to 25 mg/L) for 90 days did not inhibit souring. Contrariwise, in nitrogen-limiting conditions, the addition of nitrate stimulated the proliferation of δ-Proteobacteria (including SRB) and the associated sulfide concentration. Denitrification-specific nirK or nirS genes were not detected. A sharp decrease in water interfacial tension (from 20.8 to 14.5 mN/m) observed concomitantly with nitrate consumption and increased oil recovery (4.3 % v/v) demonstrated the benefits of nitrate injection on MEOR. Overall, the results support the notion that the addition of nitrate, at this particular oil reservoir, can benefit MEOR by stimulating the proliferation of fortuitous biosurfactant-producing bacteria. Higher nitrate concentrations exceeding the stoichiometric volatile fatty acid (VFA) biodegradation demands and/or the use of alternative biogenic souring control strategies may be necessary to warrant effective SRB inhibition down gradient from the injection wells.

Assessment of microbial communities associated with fermentative-methanogenic biodegradation of aromatic hydrocarbons in groundwater contaminated with a biodiesel blend (B20).

A controlled field experiment was conducted to assess the potential for fermentative-methanogenic biostimulation (by ammonium-acetate injection) to enhance biodegradation of benzene, toluene, ethylbenzene and xylenes (BTEX) as well as polycyclic aromatic hydrocarbons (PAHs) in groundwater contaminated with biodiesel B20 (20:80 v/v soybean biodiesel and diesel). Changes in microbial community structure were assessed by pyrosequencing 16S rRNA analyses. BTEX and PAH removal began 0.7 year following the release, concomitantly with the increase in the relative abundance of Desulfitobacterium and Geobacter spp. (from 5 to 52.7 % and 15.8 to 37.3 % of total Bacteria 16S rRNA, respectively), which are known to anaerobically degrade hydrocarbons. The accumulation of anaerobic metabolites acetate and hydrogen that could hinder the thermodynamic feasibility of BTEX and PAH biotransformations under fermentative/methanogenic conditions was apparently alleviated by the growing predominance of Methanosarcina. This suggests the importance of microbial population shifts that enrich microorganisms capable of interacting syntrophically to enhance the feasibility of fermentative-methanogenic bioremediation of biodiesel blend releases.

Biostimulation of anaerobic BTEX biodegradation under fermentative methanogenic conditions at source-zone groundwater contaminated with a biodiesel blend (B20).

Field experiments were conducted to assess the potential for anaerobic biostimulation to enhance BTEX biodegradation under fermentative methanogenic conditions in groundwater impacted by a biodiesel blend (B20, consisting of 20 % v/v biodiesel and 80 % v/v diesel). B20 (100 L) was released at each of two plots through an area of 1 m(2) that was excavated down to the water table, 1.6 m below ground surface. One release was biostimulated with ammonium acetate, which was added weekly through injection wells near the source zone over 15 months. The other release was not biostimulated and served as a baseline control simulating natural attenuation. Ammonium acetate addition stimulated the development of strongly anaerobic conditions, as indicated by near-saturation methane concentrations. BTEX removal began within 8 months in the biostimulated source zone, but not in the natural attenuation control, where BTEX concentrations were still increasing (due to source dissolution) 2 years after the release. Phylogenetic analysis using quantitative PCR indicated an increase in concentration and relative abundance of Archaea (Crenarchaeota and Euryarchaeota), Geobacteraceae (Geobacter and Pelobacter spp.) and sulfate-reducing bacteria (Desulfovibrio, Desulfomicrobium, Desulfuromusa, and Desulfuromonas) in the biostimulated plot relative to the control. Apparently, biostimulation fortuitously enhanced the growth of putative anaerobic BTEX degraders and associated commensal microorganisms that consume acetate and H2, and enhance the thermodynamic feasibility of BTEX fermentation. This is the first field study to suggest that anaerobic-methanogenic biostimulation could enhance source zone bioremediation of groundwater aquifers impacted by biodiesel blends.

In situ source zone sediment mixing coupled to groundwater biostimulation to enhance phenol natural attenuation.

Phenol is an industrially key compound that has a wide range of applications and also one of the most commonly found toxic pollutants in wastewaters and groundwater. This paper demonstrates the applicability of in situ remediation at a deactivated industrial site using source zone excavation and sediment mixing associated with nutrients delivery into groundwater. Sediment excavation and mixing displaced the entrapped source zone enhancing mass transfer into groundwater and contaminant bioavailability. A nutrient solution prepared with nitrate, phosphate, sodium hydroxide and hydrogen peroxide was continuously delivered into groundwater to stimulate biodegradation and restrict plume migration. The observed correlation between phenol-dependent Enterobacteriaceae concentrations throughout the remediation time frame supported circumstantial evidence of biodegradation. Phenol concentration in groundwater (up to 1,300 mg/L) was reduced >99% after 5 months following remediation and remained under the established site specific target level (4 mg/L). Nitrate and phosphate concentrations returned to background concentrations levels at the end of the remediation. Overall, the proposed in situ remediation scheme was effective to remediate this particular aquifer contaminated with phenol for over 20 years.

Indole-based assay to assess the effect of ethanol on Pseudomonas putida F1 dioxygenase activity.

Toluene dioxygenase (TDO) is ubiquitous in nature and has a broad substrate range, including benzene, toluene, ethylbenzene and xylenes (BTEX). Pseudomonas putida F1 (PpF1) induced on toluene is known to produce indigo from indole through the activity of TDO. In this work, a spectrophotometric assay previously developed to measure indole to indigo production rates was modified to characterize the effects of various ethanol concentrations on toluene aerobic biodegradation activity and assess catabolite repression of TDO. Indigo production rate by cells induced on toluene alone was 0.0012 +/- 0.0006 OD(610) min(-1). The presence of ethanol did not fully repress TDO activity when toluene was also available as a carbon source. However, indigo production rates by PpF1 grown on ethanol:toluene mixtures (3:1 w/w) decreased by approximately 50%. Overall, the proposed spectrophotometric assay is a simple approach to quantify TDO activity, and demonstrates how the presence of ethanol in groundwater contaminated with reformulated gasoline is likely to interfere with naturally occurring microorganisms from fully expressing their aerobic catabolic potential towards hydrocarbons bioremediation.