Exploring resistomes and microbiomes in pilot-scale microalgae-bacteria wastewater treatment systems for use in low-resource settings.

Antibiotic resistance genes (ARGs) released into the environment are an emerging human and environmental health concern, including ARGs spread in wastewater treatment effluents. In low-to-middle income countries (LMICs), an alternate wastewater treatment option instead of conventional systems are low-energy, high-rate algal ponds (HRAP) that use microalgae-bacteria aggregates (MABA) for waste degradation. Here we studied the robustness of ARG removal in MABA-based pilot-scale outdoor systems for 140 days of continuous operation. The HRAP system successfully removed 73 to 88 % chemical oxygen demand and up to 97.4 % ammonia, with aggregate size increasing over operating time. Fourteen ARG classes were identified in the HRAP influent, MABA, and effluent using metagenomics, with the HRAP process reducing total ARG abundances by up to 5-fold from influent to effluent. Parallel qPCR analyses showed the HRAP system significantly reduced exemplar ARGs (p < 0.05), with 1.2 to 4.9, 2.7 to 6.3, 0 to 1.5, and 1.2 to 4.8 log-removals for sul1, tetQ, blaKPC, and intl1 genes, respectively. Sequencing of influent, effluent and MABAs samples showed associated microbial communities differed significantly, with influent communities by Enterobacteriales (clinically relevant ARGs carrying bacteria), which were less evident in MABA and effluent. In this sense, such bacteria might be excluded from MABA due to their good settling properties and the presence of antimicrobial peptides. Microalgae-bacteria treatment systems steadily reduced ARGs from wastewater during operation time, using sunlight as the energetic driver, making them ideal for use in LMIC wastewater treatment applications.

Monitoring of SARS-CoV-2 RNA in wastewater: A surveillance tool to foresee infection's evolution in the Mexican Caribbean.

The study aims to determine SARS-CoV-2 RNA in sewage of Cancun wastewater treatment plants, the main touristic destination of Mexico, and to estimate the infected persons during the sampling period. SARS-CoV-2 RNA traces were detected in the inlet of the five plants during almost all the sampling months. However, there is no presence of SARS-CoV-2 RNA traces in the effluent of the five WWTPs during the study period. ANOVA analysis showed differences in the concentrations of RNA traces of SARS-CoV-2 between the sample dates, but no differences were found from one WWTP to another. Estimated infected individuals by Markov chain Monte Carlo simulation are higher (between 77% and 91%) than the cases reported by the health authority. Wastewater monitoring and the estimation of infected individuals are a helpful tool, because estimation provides early warning signs on how broadly SARS-CoV-2 is circulating in the city, and led to the authorities to take measures wisely. PRACTITIONER POINTS: There is no presence of SARS-CoV-2 RNA traces in the effluent of the facilities, suggesting the effectiveness of treatment. Surveillance of viral RNA concentrations at treatment plants revealed presence in the influent of five plants Estimated infected individuals by MCMC simulation are higher than cases reported by health authority Environmental surveillance approach in wastewater influent is helpful to identify the clusters and to take informed decisions.

Loop-mediated isothermal amplification-based electrochemical sensor for detecting SARS-CoV-2 in wastewater samples.

The current pandemic COVID-19 caused by the coronavirus SARS-CoV-2, has generated different economic, social and public health problems. Moreover, wastewater-based epidemiology could be a predictor of the virus rate of spread to alert on new outbreaks. To assist in epidemiological surveillance, this work introduces a simple, low-cost and affordable electrochemical sensor to specifically detect N and ORF1ab genes of the SARS-CoV-2 genome. The proposed sensor works based on screen-printed electrodes acting as a disposable test strip, where the reverse transcription loop-mediated isothermal amplification (RT-LAMP) reaction takes place. Electrochemical detection relies upon methylene blue as a redox intercalator probe, to provide a diffusion-controlled current encoding the presence and concentration of RT-LAMP products, namely amplicons or double-stranded DNA. We test the performance of the sensor by testing real wastewater samples using end-point and time course measurements. Results show the ability of the electrochemical test strip to specifically detect and quantify RT-LAMP amplicons below to ~ 2.5 × 10-6 ng/µL exhibiting high reproducibility. In this sense, our RT-LAMP electrochemical sensor is an attractive, efficient and powerful tool for rapid and reliable wastewater-based epidemiology studies.

Continuous anaerobic oxidation of methane: Impact of semi-continuous liquid operation and nitrate load on N2O production and microbial community.

This work proves the feasibility of employing regular secondary activated sludge for the enrichment of a microbial community able to perform the anaerobic oxidation of methane coupled to nitrate reduction (N-AOM). After 96 days of activated sludge enrichment, a clear N-AOM activity was observed in the resulting microbial community. The methane removal potential of the enriched N-AOM culture was then studied in a stirred tank reactor (STR) operated in continuous mode for methane supply and semi-continuous mode for the liquid phase. The effect of applying nitrate loads of ∼22, 44, 66, and 88 g NO3- m-3 h-1 on (i) STR methane and nitrate removal performance, (ii) N2O emission, and (iii) microbial composition was investigated. Methane elimination capacities from 21 ± 13.3 to 55 ± 12 g CH4 m-3 h-1 were recorded, coupled to nitrate removal rates ranging from 6 ± 3.2 to 43 ± 14.9 g NO3- m-3 h-1. N2O production was not detected under the three nitrate loading rates applied for the assessment of potential N2O emission in the continuous N-AOM process (i.e. ∼22-66 g NO-3 m-3 h-1). The lack of N2O emissions during the process was attributed to the N2O reducing capacity of the bacterial taxa identified and the rigorous control of dissolved O2 and pH implemented (dissolved O2 values ≤ 0.07 g m-3 and pH of 7.6 ± 0.4). Microbial characterization showed that the N-AOM process was performed in absence of putative N-AOM archaea and bacteria (ANME-2d, M. oxyfera). Instead, microbial activity was driven by methane-oxidizing bacteria and denitrifying bacteria (Bacteroidetes, α-, and γ-proteobacteria).

Thermophilic biogas production from microalgae-bacteria aggregates: biogas yield, community variation and energy balance.

Biogas production through anaerobic mesophilic digestion is the most straightforward biofuel production route integrated into microalgae-bacteria wastewater treatment plants. Improvement of this biofuel route without adding pretreatment units is possible through the temperature increase. This paper presents a comprehensive evaluation of the transitory effect of different temperatures (35 °C and 55 °C) and hydraulic retention times (HRT) of 15 and 30 d on the long-term methane production using non-pretreated microalgae-bacteria aggregates as a feedstock. The thermophilic transition from mesophilic inoculum adapted to microalgae-bacteria aggregate increased 1.7-fold the methane production (0.41 m3CH4 kgVS-1) at HRT of 30 d. A substantial decrease in the microbial community's diversity present in the anaerobic reactor was observed when thermophilic conditions were applied, explaining the long adaptation period needed. The increase of the operative temperature condition promotes changes in the dominance pathway of methanogenesis from hydrogenotrophic to acetolactic. The energy balance assessment showed a positive net energy ratio when the digester was operated at an HRT of 30 d. A maximum net energy ratio of 1.5 was achieved at mesophilic temperature. This study demonstrated, based on experimental data, that microalgal digestion with an HRT of 30 d favors energy self-sustainability in microalgal wastewater treatment plants.

A review on the factors influencing biohydrogen production from lactate: The key to unlocking enhanced dark fermentative processes.

Dark fermentation (DF) is one of the most promising biological methods to produce bio-hydrogen and other value added bio-products from carbohydrate-rich wastes and wastewater. However, process instability and low hydrogen production yields and rates have been highlighted as the major bottlenecks preventing further development. Numerous studies have associated such concerns with the inhibitory activity of lactate-producing bacteria (LAB) against hydrogen producers. However, an increasing number of studies have also shown lactate-based metabolic pathways as the prevailing platform for hydrogen production. This opens a vast potential to develop new strategies to deal with the "Achilles heel" of DF - LAB overgrowth - while untapping high-performance DF. This review discusses the key factors influencing the lactate-driven hydrogen production, paying particular attention to substrate composition, the operating conditions, as well as the microbiota involved in the process and its potential functionality and related biochemical routes. The current limitations and future perspectives in the field are also presented.

Innovative Control of Biofilms on Stainless Steel Surfaces Using Electrolyzed Water in the Dairy Industry.

Biofilms on food-contact surfaces can lead to recurrent contamination. This work aimed to study the biofilm formation process on stainless steel plates used in the dairy industry: 304 surface finish 2B and electropolished; and the effect of a cleaning and disinfection process using alkaline (AEW) and neutral (NEW) electrolyzed water. Milk fouling during heat processing can lead to type A or B deposits, which were analyzed for composition, surface energy, thickness, and roughness, while the role of raw milk microbiota on biofilm development was investigated. Bacteria, yeasts, and lactic acid bacteria were detected using EUB-338, PF2, and Str-493 probes, respectively, whereas Lis-637 probe detected Listeria sp. The genetic complexity and diversity of biofilms varied according to biofilm maturation day, as evaluated by 16S rRNA gene sequence, denaturing gradient gel electrophoresis, and fluorescence in situ hybridization microscopy. From analysis of the experimental designs, a cleaning stage of 50 mg/L NaOH of AEW at 30 °C for 10 min, followed by disinfection using 50 mg/L total available chlorine of NEW at 20 °C for 5 min is a sustainable alternative process to prevent biofilm formation. Fluorescence microscopy was used to visualize the effectiveness of this process.

Surveillance of SARS-CoV-2 in sewage and wastewater treatment plants in Mexico.

The SARS-CoV-2 virus causing COVID-19 is spread in sewage by the stool of infected individuals, and viral material in sewage can be quantified using molecular tools. This study aimed to monitor the presence of SARS-CoV-2 RNA in sewage in Mexico based on RdRP, S, and N gene analysis. The influent, effluent, and activated sludge from two domestic wastewater treatment plants (WWTP) were evaluated from the early stage of the epidemic to July 2020. Additionally, sampling points in sewer systems were examined, comparing two different RNA-concentration methods: centrifugal ultrafiltration and adsorption-based methods. The adsorption method resulted in RNA titration that was two orders of magnitude higher than with ultrafiltration (up to 3.38 log10 copies RdRP gene/mL of sewage). The surveillance of SARS-CoV-2 RNA in the influent of two WWTP correlated with the cumulative COVID-19 cases in Queretaro city. The higher RNA level in secondary sludge compared to influent suggests that viral RNA becomes concentrated in activated sludge. This result supports SARS-CoV-2 RNA removal in WWTP, where all effluent samples were negative for virus quantification. This work proves that wastewater-based epidemiology is a very valuable tool in developing countries where diagnostic tests for COVID-19 are limited.

Approaches applied to detect SARS-CoV-2 in wastewater and perspectives post-COVID-19.

Currently, SARS-CoV-2 has been detected in the influent of wastewater treatment plants (WWTP), pumping stations, manholes, sewer networks and sludge of WWTP and facilities of countries as France, Spain, Italy, Netherlands, United States, Australia, Ecuador, Brazil and Japan. Although this virus has been detected in the wastewater streams, there is no robust method for its detection and quantification in wastewater. This review compiled and analyzed the virus concentration approaches applied to detect the SARS-CoV-2, besides to provide insights about the methodology for viral concentration, limit of detection, occurrence, persistence, and perspectives post-COVID-19 related with the implications of the virus presence in wastewater. The SARS-COV-2 detection in wastewater has been related to virus concentration methods, which present different recovery rates of the virus. The most used viral concentration methods have been the polyethylene glycol (PEG) for precipitation of viral material and the ultrafiltration at molecular weight level. After viral concentration, the detection and quantification of SARS-COV-2 in wastewater are mainly via quantitative reverse transcription polymerase chain reaction (RT-qPCR), which is the clinical assay adapted for environmental purposes. Although in some experiments the positive control during RT-qPCR is running a surrogated virus (e.g., Mengovirus or Dengue virus), RT-qPCR or reverse transcription droplet digital PCR (RT-ddPCR) targeting the gene encoding nucleocapsid (N1, N2 and N3) of SARS-COV-2 are highly recommended to calculate the limit of detection in wastewater samples. Current results suggest that a rigorous methodology to elucidate the positive cases in a region from genomic copies in wastewater is needed.

Standardized protocol for determination of biohydrogen potential.

Biohydrogen production potential (BHP) depends on several factors like inoculum source, substrate, pH, among many others. Batch assays are the most common strategy to evaluate such parameters, where the comparison is a challenging task due to the different procedures used. The present method introduces the first internationally validated protocol, evaluated by 8 independent laboratories from 5 different countries, to assess the biohydrogen potential. As quality criteria, a coefficient of variation of the cumulative hydrogen production (H max) was defined to be <15 %. Two options to run BHP batch tests were proposed; a manual protocol with periodic measurements of biogas production, needing conventional laboratory materials and analytical equipment for biogas characterization; and an automatic protocol, which is run in a device developed for online measurements of low biogas production. The detailed procedures for both protocol options are presented, as well as data validating them. The validation showed acceptable repeatability and reproducibility, measured as intra- and inter-laboratory coefficient of variation, which can be reduced up to 9 %.

Influence of Added Nutrients and Substrate Concentration in Biohydrogen Production from Winery Wastewaters Coupled to Methane Production.

Winery wastewaters are acidic effluents with high content of organic matter and nutrients. Different initial values of chemical oxygen demand (COD), ranging from 4 to 50 g L-1, were tested in batch assays to evaluate the fermentative hydrogen production followed by a methane production step. The influence of adding a typical nutrient solution for hydrogen production was investigated. Nutrients include N-NH4, Mg, Fe, Co, Mn, I, Ni, and Zn. The best hydrogen production potential was obtained at a COD of 50 g L-1 without nutrient addition. This condition produced 528 mL H2 L-1. At a COD ≥ 35 g L-1, tests with only WW had a hydrogen potential 1.6 to 1.9 times higher than did tests where nutrients were added. The use of added nutrients reduced the hydrogen production by producing additional reduced acids, such as propionate and valerate. In a second stage, biomethane potential was evaluated using the effluent of a selected condition from hydrogen production tests. The methane production reached values of 207 ± 2.2 mL CH4 g-1 COD at 10 g COD L-1. The COD affected the specific methane production. The results of this study demonstrated the potential of winery effluents as a substrate for sequential hydrogen and methane production to increase the energy recovery from this effluent, with a maximum energetic yield and productivity of 7.15 kJ gCOD-1 and 11.51 kJ d-1.

From mesophilic to thermophilic conditions: one-step temperature increase improves the methane production of a granular sludge treating agroindustrial effluents.

OBJECTIVES: To assess the effect of one-step temperature increase, from 35 to 55 °C, on the methane production of a mesophilic granular sludge (MGS) treating wine vinasses and the effluent of a hydrogenogenic upflow anaerobic sludge blanket (UASB) reactor. RESULTS: One-step temperature increase from mesophilic to thermophilic conditions improved methane production regardless of the substrate tested. The biomethane potentials obtained under thermophilic conditions were 1.8-2.9 times higher than those obtained under mesophilic conditions. The MGS also performed better than an acclimated thermophilic digestate, producing 2.2-2.5 times more methane than the digestate under thermophilic conditions. Increasing the temperature from 35 to 55 °C also improved the methane production rate of the MGS (up to 9.4 times faster) and reduced the lag time (up to 1.9 times). Although the temperature increase mediated a decrease in the size of the sludge granules, no negative effects on the performance of the MGS was observed under thermophilic conditions. CONCLUSIONS: More methane is obtained from real agroindustrial effluents at thermophilic conditions than under mesophilic conditions. One-step temperature increase (instead of progressive sequential increases) can be used to implement the thermophilic anaerobic digestion processes with MGS.

Hydrolysis of microalgal biomass using ruminal microorganisms as a pretreatment to increase methane recovery.

The use of ruminal fluid as a source of hydrolytic microorganisms for the pretreatment of a native consortium of microalgae (essentially Senedesmus) was investigated. The hydrolytic enzyme activity of the ruminal culture was first enriched in a bioreactor. Then, using the enriched culture, the effect of the microalgae to the ruminal fluid ratio (S/X) on the hydrolysis and subsequent production of methane was investigated. An S/X ratio of 0.5 showed the best hydrolysis efficiency (29%) reaching in a second stage process a methane yield of 193mL CH4g COD-1. The processing time (pretreatment plus methanization) was only 7days. The predominant ruminal hydrolytic bacteria selected in the enrichment were principally Clostridium, Proteocatella and Pseudomonas.

Biohydrogen and methane production via a two-step process using an acid pretreated native microalgae consortium.

A native microalgae consortium treated under thermal-acidic hydrolysis was used to produce hydrogen and methane in a two-step sequential process. Different acid concentrations were tested, generating hydrogen and methane yields of up to 45mLH2gVS-1 and 432mLCH4gVS-1, respectively. The hydrogen production step solubilized the particulate COD (chemical oxygen demand) up to 30%, creating considerable amounts of volatile fatty acids (up to 10gCODL-1). It was observed that lower acid concentration presented higher hydrogen and methane production potential. The results revealed that thermal acid hydrolysis of a native microalgae consortium is a simple but effective strategy for producing hydrogen and methane in the sequential process. In addition to COD removal (50-70%), this method resulted in an energy recovery of up to 15.9kJ per g of volatile solids of microalgae biomass, one of the highest reported.

Microbial communities from 20 different hydrogen-producing reactors studied by 454 pyrosequencing.

To provide new insight into the dark fermentation process, a multi-lateral study was performed to study the microbiology of 20 different lab-scale bioreactors operated in four different countries (Brazil, Chile, Mexico, and Uruguay). Samples (29) were collected from bioreactors with different configurations, operation conditions, and performances. The microbial communities were analyzed using 16S rRNA genes 454 pyrosequencing. The results showed notably uneven communities with a high predominance of a particular genus. The phylum Firmicutes predominated in most of the samples, but the phyla Thermotogae or Proteobacteria dominated in a few samples. Genera from three physiological groups were detected: high-yield hydrogen producers (Clostridium, Kosmotoga, Enterobacter), fermenters with low-hydrogen yield (mostly from Veillonelaceae), and competitors (Lactobacillus). Inocula, reactor configurations, and substrates influence the microbial communities. This is the first joint effort that evaluates hydrogen-producing reactors and operational conditions from different countries and contributes to understand the dark fermentation process.