Advancing towards electro-bioremediation scaling-up: On-site pilot plant for successful nitrate-contaminated groundwater treatment.

The potential of nitrate electro-bioremediation has been fully demonstrated at the laboratory scale, although it has not yet been fully implemented due to the challenges associated with scaling-up bioelectrochemical reactors and their on-site operation. This study describes the initial start-up and subsequent stable operation of an electro-bioremediation pilot plant for the treatment of nitrate-contaminated groundwater on-site (Navata site, Spain). The pilot plant was operated under continuous flow mode for 3 months, producing an effluent suitable for drinking water in terms of nitrates and nitrites (<50 mg NO3- L-1; 0 mg NO2- L-1). A maximum nitrate removal rate of 0.9 ± 0.1 kg NO3- m-3 d-1 (efficiency 82 ± 18 %) was achieved at a cathodic hydraulic retention time (HRTcat) of 2.0 h with a competitive energy consumption of 4.3 ± 0.4 kWh kg-1 NO3-. Under these conditions, the techno-economic analysis estimated an operational cost of 0.40 € m-3. Simultaneously, microbiological analyses revealed structural heterogeneity in the reactor, with denitrification functionality concentrated predominantly from the centre to the upper section of the reactor. The most abundant groups were Pseudomonadaceae, Rhizobiaceae, Gallionellaceae, and Xanthomonadaceae. In conclusion, this pilot plant represents a significant advancement in implementing this technology on a larger scale, validating its effectiveness in terms of nitrate removal and cost-effectiveness. Moreover, the results validate the electro-bioremediation in a real environment and encourage further investigation of its potential as a water treatment.

Design and validation of a multiplex PCR method for the simultaneous quantification of Clostridium acetobutylicum, Clostridium carboxidivorans and Clostridium cellulovorans.

Co-cultures of clostridia with distinct physiological properties have emerged as an alternative to increase the production of butanol and other added-value compounds from biomass. The optimal performance of mixed tandem cultures may depend on the stability and fitness of each species in the consortium, making the development of specific quantification methods to separate their members crucial. In this study, we developed and tested a multiplex qPCR method targeting the 16S rRNA gene for the simultaneous quantification of Clostridium acetobutylicum, Clostridium carboxidivorans and Clostridium cellulovorans in co-cultures. Designed primer pairs and probes could specifically quantify the three Clostridium species with no cross-reactions thus allowing significant changes in their growth kinetics in the consortia to be detected and correlated with productivity. The method was used to test a suitable medium composition for simultaneous growth of the three species. We show that higher alcohol productions were obtained when combining C. carboxidivorans and C. acetobutylicum compared to individual cultures, and further improved (> 90%) in the triplet consortium. Altogether, the methodology could be applied to fermentation processes targeting butanol productions from lignocellulosic feedstocks with a higher substrate conversion efficiency.

Direct ammonium oxidation to nitrogen gas (Dirammox) in Alcaligenes strain HO-1: The electrode role.

It has been recently suggested that Alcaligenes use a previously unknown pathway to convert ammonium into dinitrogen gas (Dirammox) via hydroxylamine (NH2OH). This fact alone already implies a significant decrease in the aeration requirements for the process, but the process would still be dependent on external aeration. This work studied the potential use of a polarised electrode as an electron acceptor for ammonium oxidation using the recently described Alcaligenes strain HO-1 as a model heterotrophic nitrifier. Results indicated that Alcaligenes strain HO-1 requires aeration for metabolism, a requirement that cannot be replaced for a polarised electrode alone. However, concomitant elimination of succinate and ammonium was observed when operating a previously grown Alcaligenes strain HO-1 culture in the presence of a polarised electrode and without aeration. The usage of a polarised electrode together with aeration did not increase the succinate nor the nitrogen removal rates observed with aeration alone. However, current density generation was observed along a feeding batch test representing an electron share of 3% of the ammonium removed in the presence of aeration and 16% without aeration. Additional tests suggested that hydroxylamine oxidation to dinitrogen gas could have a relevant role in the electron discharge onto the anode. Therefore, the presence of a polarised electrode supported the metabolic functions of Alcaligenes strain HO-1 on the simultaneous oxidation of succinate and ammonium.

Let's chat: Communication between electroactive microorganisms.

Electroactive microorganisms can exchange electrons with other cells or conductive interfaces in their extracellular environment. This property opens the way to a broad range of practical biotechnological applications, from manufacturing sustainable chemicals via electrosynthesis, to bioenergy, bioelectronics or improved, low-energy demanding wastewater treatments. Besides, electroactive microorganisms play key roles in environmental bioremediation, significantly impacting process efficiencies. This review highlights our present knowledge on microbial interactions promoting the communication between electroactive microorganisms in a biofilm on an electrode in bioelectrochemical systems (BES). Furthermore, the immediate knowledge gaps that must be closed to develop novel technologies will also be acknowledged.

The core microbiome is responsible for volatile silicon and organic compounds degradation during anoxic lab scale biotrickling filter performance.

Volatile silicon compounds present in the biogas of anaerobic digesters can cause severe problems in the energy recovery systems, inducing costly damages. Herein, the microbial community of a lab-scale biotrickling filter (BTF) was studied while testing its biodegradation capacity on octamethylcyclotetrasiloxane (D4) and decamethylcyclopentasiloxane (D5), in the presence of toluene, limonene and hexane. The reactor performance was tested at different empty bed residence times (EBRT) and packing materials. Community structure was analysed by bar-coded amplicon sequencing of the 16S rRNA gene. Microbial diversity and richness were higher in the inoculum and progressively decreased during BTF operation (Simpson's diversity index changing from 0.98-0.90 and Richness from 900 to 200 OTUs). Minimum diversity was found when reactor was operated at relatively low EBRT (7.3 min) using a multicomponent feed. The core community was composed of 36 OTUs (accounting for 55% of total sequences). Packing material played a key role in the community structure. Betaproteobacteriales were dominant in the presence of lava rock and were partially substituted by Corynebacteriales and Rhizobiales when activated carbon was added to the BTF. Despite these changes, a stable and resilient core microbiome was selected defining a set of potentially degrading bacteria for siloxane bioremoval as a complementary alternative to non-regenerative adsorption onto activated carbon.

Electro-bioremediation of nitrate and arsenite polluted groundwater.

The coexistence of different pollutants in groundwater is a common threat. Sustainable and resilient technologies are required for their treatment. The present study aims to evaluate microbial electrochemical technologies (METs) for treating groundwater contaminated with nitrate (NO3-) while containing arsenic (in form of arsenite (As(III)) as a co-contaminant. The treatment was based on the combination of nitrate reduction to dinitrogen gas and arsenite oxidation to arsenate (exhibiting less toxicity, solubility, and mobility), which can be removed more easily in further post-treatment. We operated a bioelectrochemical reactor at continuous-flow mode with synthetic contaminated groundwater (33 mg N-NO3- L-1 and 5 mg As(III) L-1) identifying the key operational conditions. Different hydraulic retention times (HRT) were evaluated, reaching a maximum nitrate reduction rate of 519 g N-NO3- m3Net Cathodic Compartment d-1 at HRT of 2.3 h with a cathodic coulombic efficiency of around 100 %. Simultaneously, arsenic oxidation was complete at all HRT tested down to 1.6 h reaching an oxidation rate of up to 90 g As(III) m-3Net Reactor Volume d -1. Electrochemical and microbiological characterization of single granules suggested that arsenite at 5 mg L-1 did not have an inhibitory effect on a denitrifying biocathode mainly represented by Sideroxydans sp. Although the coexistence of abiotic and biotic arsenic oxidation pathways was shown to be likely, microbial arsenite oxidation linked to denitrification by Achromobacter sp. was the most probable pathway. This research paves the ground towards a real application for treating groundwater with widespread pollutants.

Bacteria coated cathodes as an in-situ hydrogen evolving platform for microbial electrosynthesis.

Hydrogen is a key intermediate element in microbial electrosynthesis as a mediator of the reduction of carbon dioxide (CO2) into added value compounds. In the present work we aimed at studying the biological production of hydrogen in biocathodes operated at - 1.0 V vs. Ag/AgCl, using a highly comparable technology and CO2 as carbon feedstock. Ten bacterial strains were chosen from genera Rhodobacter, Rhodopseudomonas, Rhodocyclus, Desulfovibrio and Sporomusa, all described as hydrogen producing candidates. Monospecific biofilms were formed on carbon cloth cathodes and hydrogen evolution was constantly monitored using a microsensor. Eight over ten bacteria strains showed electroactivity and H2 production rates increased significantly (two to eightfold) compared to abiotic conditions for two of them (Desulfovibrio paquesii and Desulfovibrio desulfuricans). D. paquesii DSM 16681 exhibited the highest production rate (45.6 ± 18.8 µM min-1) compared to abiotic conditions (5.5 ± 0.6 µM min-1), although specific production rates (per 16S rRNA copy) were similar to those obtained for other strains. This study demonstrated that many microorganisms are suspected to participate in net hydrogen production but inherent differences among strains do occur, which are relevant for future developments of resilient biofilm coated cathodes as a stable hydrogen production platform in microbial electrosynthesis.

Limited effect of radial oxygen loss on ammonia oxidizers in Typha angustifolia root hairs.

The benefits of plant-microbe interactions have been exploited extensively for nutrient removal. Radial oxygen loss in aquatic macrophytes potentially promotes nitrification and accelerates nitrogen removal through coupled nitrification-denitrification process. Nitrification is likely the limiting activity for an effective nitrogen removal in wetlands. In this work, we have quantified the effect of radial oxygen losses in Typha angustifolia plants in environments of contrasting salinities, including a temporary lagoon, a constructed wetland, and a river estuary. In all sites, radial oxygen diffusion occurred mainly at a narrow band, from 1 to 5 cm from the root tip, and were almost absent at the tip and basal sections of the root (> 5 cm). Root sections with active oxygen diffusion tended to show higher bacterial and archaeal densities in the rhizoplane according to 16S rRNA gene abundance data, except at higher salinities. Archaeal amoA /bacterial amoA gene ratios were highly variable among sites. Archaeal nitrifiers were only favoured over bacteria on the root surface of Typha collected from the constructed wetland. Collectively, radial oxygen loss had little effect on the nitrifying microbial community at the smaller scale (differences according to root-section), and observed differences were more likely related to prevailing physicochemical conditions of the studied environments or to long-term effects of the root microenvironment (root vs sediment comparisons).

Changes in the Potential Activity of Nitrite Reducers and the Microbial Community Structure After Sediment Dredging and Plant Removal in the Empuriabrava FWS-CW.

In constructed wetlands (CW), denitrification usually accounts for > 60% of nitrogen removal and is supposedly affected by wetland management practices, such as dredging (and plant removal). These practices cause an impact in sediment properties and microbial communities living therein. We have quantified the effects of a sediment dredging event on dissimilatory nitrite reduction by analysing the structure and activities of the microbial community before and after the event. Potential rates for nitrate reduction to ammonia and denitrification were in accordance with changes in the physicochemical conditions. Denitrification was the predominant pathway for nitrite removal (> 60%) and eventually led to the complete removal of nitrate. On the contrary, dissimilatory nitrite reduction to ammonia (DNRA) increased from 5 to 18% after the dredging event. Both actual activities and abundances of 16S rRNA, nirK and nirS significantly decreased after sediment dredging. However, genetic potential for denitrification (qnirS + qnirK/q16S rRNA) remained unchanged. Analyses of the 16S rRNA gene sequences revealed the importance of vegetation in shaping microbial community structures, selecting specific phylotypes potentially contributing to the nitrogen cycle. Overall, we confirmed that sediment dredging and vegetation removal exerted a measurable effect on the microbial community, but not on potential nitrite + nitrate removal rates. According to redundancy analysis, nitrate concentration and pH were the main variables affecting sediment microbial communities in the Empuriabrava CWs. Our results highlight a high recovery of the functionality of an ecosystem service after a severe intervention and point to metabolic redundancy of denitrifiers. We are confident these results will be taken into account in future management strategies in CWs.

Potential use of Methylibium sp. as a biodegradation tool in organosilicon and volatile compounds removal for biogas upgrading.

Organosilicon compounds are the most undesirable compounds for the energy recovery of biogas. These compounds are still resistant to biodegradation when biotechnologies are considered for biogas purification. Herein we isolated 52 bacterial species from anaerobic batch enrichment cultures (BEC) saturated with D4 and from an anaerobic lab-scale biotrickling filter (BTF) fed with a gas flow containing D4 as unique carbon source. Among those Methylibium sp. and Pseudomonas aeruginosa showed the highest capacity to remove D4 (53.04% ±â€¯0.03 and 24.42% ±â€¯0.02, respectively). Contrarily, co-culture evaluation treatment for the biodegradation of siloxanes together with volatile organic compounds removed a lower concentration of D4 compared to toluene and limonene, which were completely removed. Remarkably, the siloxane D5 proved to be more biodegradable than D4. Substrates removal values achieved by Methylibium sp. suggested that this bacterial isolate could be used in biological removal technologies of siloxanes.

[NiFe]-hydrogenases are constitutively expressed in an enriched Methanobacterium sp. population during electromethanogenesis.

Electromethanogenesis is the bioreduction of carbon dioxide (CO2) to methane (CH4) utilizing an electrode as electron donor. Some studies have reported the active participation of Methanobacterium sp. in electron capturing, although no conclusive results are available. In this study, we aimed at determining short-time changes in the expression levels of [NiFe]-hydrogenases (Eha, Ehb and Mvh), heterodisulfide reductase (Hdr), coenzyme F420-reducing [NiFe]-hydrogenase (Frh), and hydrogenase maturation protein (HypD), according to the electron flow in independently connected carbon cloth cathodes poised at- 800 mV vs. standard hydrogen electrode (SHE). Amplicon massive sequencing of cathode biofilm confirmed the presence of an enriched Methanobacterium sp. population (>70% of sequence reads), which remained in an active state (78% of cDNA reads), tagging this archaeon as the main methane producer in the system. Quantitative RT-PCR determinations of ehaB, ehbL, mvhA, hdrA, frhA, and hypD genes resulted in only slight (up to 1.5 fold) changes for four out of six genes analyzed when cells were exposed to open (disconnected) or closed (connected) electric circuit events. The presented results suggested that suspected mechanisms for electron capturing were not regulated at the transcriptional level in Methanobacterium sp. for short time exposures of the cells to connected-disconnected circuits. Additional tests are needed in order to confirm proteins that participate in electron capturing in Methanobacterium sp.

Specific detection of "Clostridium autoethanogenum", Clostridium ljungdahlii and Clostridium carboxidivorans in complex bioreactor samples.

The high genetic similarity between some carboxydotrophic bacteria does not allow for the use of common sequencing techniques targeting the 16S rRNA gene for species identification. 16S rRNA sequencing fails to discriminate among Clostridium ljungdahlii and 'Clostridium autoethanogenum', despite this two species exhibit significant differences in CO2 assimilation and alcohol production. In this work we designed PCR primers targeting for the DNA gyrase subunit A (gyrA) and a putative formate/nitrite transporter (fnt) to specifically detect the presence of 'C. autoethanogenum', C. ljungdahlii or Clostridium carboxidivorans. We could confirm the simultaneous presence of C. ljungdahlii and 'C. autoethanogenum' in different bioreactors, and a preference of the latter for high CO2 content.

Effect of ethanol and butanol on autotrophic growth of model homoacetogens.

Research efforts aimed at increasing ethanol and butanol productivity from syngas are currently gaining attention. For most model carboxydotrophic bacteria, production rates, yields and maximum product titres have been studied in detail, but little is known on alcohol toxicity in these bacteria. The aim of this work was to investigate the inhibitory effects of ethanol and butanol on the growth of Clostridium ljungdahlii PETC, C. carboxidivorans P7, and 'Butyribacterium methylotrophicum DSM3468'. Experiments to determine inhibitory effects due to product accumulation were carried out using a synthetic mixture of CO:CO2:H2 as a substrate. These conditions were chosen to mimic gaseous effluents of biomass and waste gasification plants. Inhibition effects were recorded as changes in growth parameters. No significant inhibition was observed for ethanol at concentrations below 15 g/L. The three species exhibited higher sensitivity to butanol. Half inhibition constants for butanol could be estimated for P7 (IC50 = 4.12 g/L), DSM3468 (IC50 = 1.79 g/L), and PETC (IC50 = 9.75 g/L). In conclusion, at least for the tested strains, alcohol toxicity is not an immediate handicap for increasing alcohol production of the tested homoacetogenic strains.

Isotope and microbiome data provide complementary information to identify natural nitrate attenuation processes in groundwater.

Natural attenuation processes alleviate the impact of fertilization practices on groundwater resources. Therefore, identifying the occurrence of denitrification has become a requirement for water quality management. Several approaches are useful for this purpose, such as isotopic and microbiological methods, each of them providing distinct but complementary information about denitrification reactions, attenuation rates and their occurrence in the aquifer. In this paper, we investigate the contribution of both approaches to describe denitrification in a consolidated rock aquifer (limestone and marls), with a porosity related to fracture networks located in the northeastern sector of the Osona basin (NE Spain). Isotopic methods indicated the origin of nitrate (fertilization using manure) and that denitrification occurred, reaching a reduction of near 25% of the nitrate mass in groundwater. The studied area could be divided in two zones with distinct agricultural pressures and, consequently, nitrate concentrations in groundwater. Denitrification occurred in both zones and at different levels, indicating that attenuation processes took place all along the whole hydrogeological unit, and that the observed levels could be attributed to a larger flow path or, in a minor extent, to mixing processes that mask the actual denitrification rates. Microbiological data showed a correlation between denitrifier genes and the isotopic composition. However, the groundwater microbiome and the distribution of denitrifying bacteria did not reveal a major influence on the denitrification level observed by isotopic methods. This focuses the interest of microbiological analysis to identify functional genes within the bacteria present in the aquifer. Results indicated that isotopic methods provide information of the overall denitrification ability of the hydrogeological unit, and that genomic data represent the processes actually acting nearby the well. A combination of both approaches is advised to support induced in situ attenuation actions in polluted sites.

Microbial electrosynthesis of butyrate from carbon dioxide: Production and extraction.

To date acetate is the main product of microbial electrosynthesis (MES) from carbon dioxide (CO2). In this work a tubular bioelectrochemical system was used to carry out MES and enhance butyrate production over the other organic products. Batch tests were performed at a fixed cathode potential of -0.8V vs SHE. The reproducibility of the results according to previous experiments was validated in a preliminary test. According to the literature butyrate production could take place by chain elongation reactions at low pH and high hydrogen partial pressure (pH2). During the experiment, CO2 supply was limited to build up pH2 and trigger the production of compounds with a higher degree of reduction. In test 1 butyrate became the predominant end-product, with a concentration of 59.7mMC versus 20.3mMC of acetate, but limitation on CO2 supply resulted in low product titers. CO2 limitation was relaxed in test 2 to increase the bioelectrochemical activity but increase pH2 and promote the production of butyrate, what resulted in the production of 87.5mMC of butyrate and 34.7mMC of acetate. The consumption of ethanol, and the presence of other products in the biocathode (i.e. caproate) suggested that butyrate production took place through chain elongation reactions, likely driven by Megasphaera sueciensis (>39% relative abundance). Extraction and concentration of butyrate was performed by liquid membrane extraction. A concentration phase with 252.4mMC of butyrate was obtained, increasing also butyrate/acetate ratio to 16.4. The results are promising for further research on expanding the product portfolio of MES.

Tracking bio-hydrogen-mediated production of commodity chemicals from carbon dioxide and renewable electricity.

This study reveals that reduction of carbon dioxide (CO2) to commodity chemicals can be functionally compartmentalized in bioelectrochemical systems. In the present example, a syntrophic consortium composed by H2-producers (Rhodobacter sp.) in the biofilm is combined with carboxidotrophic Clostridium species, mainly found in the bulk liquid. The performance of these H2-mediated electricity-driven systems could be tracked by the activity of a biological H2 sensory protein identified at cathode potentials between -0.2V and -0.3V vs SHE. This seems to point out that such signal is not strain specific, but could be detected in any organism containing hydrogenases. Thus, the findings of this work open the door to the development of a biosensor application or soft sensors for monitoring such systems.

External Resistances Applied to MFC Affect Core Microbiome and Swine Manure Treatment Efficiencies.

Microbial fuel cells (MFCs) can be designed to combine water treatment with concomitant electricity production. Animal manure treatment has been poorly explored using MFCs, and its implementation at full-scale primarily relies on the bacterial distribution and activity within the treatment cell. This study reports the bacterial community changes at four positions within the anode of two almost identically operated MFCs fed swine manure. Changes in the microbiome structure are described according to the MFC fluid dynamics and the application of a maximum power point tracking system (MPPT) compared to a fixed resistance system (Ref-MFC). Both external resistance and cell hydrodynamics are thought to heavily influence MFC performance. The microbiome was characterised both quantitatively (qPCR) and qualitatively (454-pyrosequencing) by targeting bacterial 16S rRNA genes. The diversity of the microbial community in the MFC biofilm was reduced and differed from the influent swine manure. The adopted electric condition (MPPT vs fixed resistance) was more relevant than the fluid dynamics in shaping the MFC microbiome. MPPT control positively affected bacterial abundance and promoted the selection of putatively exoelectrogenic bacteria in the MFC core microbiome (Sedimentibacter sp. and gammaproteobacteria). These differences in the microbiome may be responsible for the two-fold increase in power production achieved by the MPPT-MFC compared to the Ref-MFC.

Low Fermentation pH Is a Trigger to Alcohol Production, but a Killer to Chain Elongation.

Gasification of organic wastes coupled to syngas fermentation allows the recovery of carbon in the form of commodity chemicals, such as carboxylates and biofuels. Acetogenic bacteria ferment syngas to mainly two-carbon compounds, although a few strains can also synthesize four-, and six-carbon molecules. In general, longer carbon chain products have a higher biotechnological (and commercial) value due to their higher energy content and their lower water solubility. However, de-novo synthesis of medium-chain products from syngas is quite uncommon in acetogenic bacteria. An alternative to de-novo synthesis is bioproduction of short-chain products (C2 and C4), and their subsequent elongation to C4, C6, or C8 through reversed ß-oxidation metabolism. This two-step synergistic approach has been successfully applied for the production of up to C8 compounds, although the accumulation of alcohols in these mixed cultures remained below detection limits. The present work investigates the production of higher alcohols from syngas by open mixed cultures (OMC). A syngas-fermenting community was enriched from sludge of an anaerobic digester for a period of 109 days in a lab-scale reactor. At the end of this period, stable production of ethanol and butanol was obtained. C6 compounds were only transiently produced at the beginning of the enrichment phase, during which Clostridium kluyveri, a bacterium able to carry out carbon chain elongation, was detected in the community. Further experiments showed pH as a critical parameter to maintain chain elongation activity in the co-culture. Production of C6 compounds was recovered by preventing fermentation pH to decrease below pH 4.5-5. Finally, experiments showed maximal production of C6 compounds (0.8 g/L) and alcohols (1.7 g/L of ethanol, 1.1 g/L of butanol, and 0.6 g/L of hexanol) at pH 4.8. In conclusion, low fermentation pH is critical for the production of alcohols, although detrimental to C. kluyveri. Fine control of fermentation pH to final values around 4.8 could allow sustained production of higher alcohols.