POME

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A novel pico-hydro power (PHP)-Microbial electrolysis cell (MEC) coupled system for

sustainable hydrogen production during palm oil mill effluent (POME) wastewater treatment

Abudukeremu Kadier

^a,b,1,^*

, Raghuveer Singh

^c

, Dongsheng Song

^b,d,1

, Farshid Ghanbari

^e

, Nur Syamimi Zaidi

^f,g

, Putu Teta Prihartini Aryanti

^h

, Dipak A. Jadhav

ⁱ

, M. Amirul Islam

^j,^**

, Mohd Sahaid Kalil

^k

,

Walid Nabgan

^l,m,^***

, Aidil Abdul Hamid

ⁿ

, Hassimi Abu Hasan

^k,o

, Peng-Cheng Ma

^a,b,^****

aLaboratory of Environmental Science and Technology, The Xinjiang Technical Institute of Physics and Chemistry, Key Laboratory of Functional Materials and Devices for Special Environments, Chinese Academy of Sciences, Urumqi, 830011, China

bCenter of Materials Science and Optoelectronics Engineering, University of Chinese Academy of Sciences, Beijing, 100049, China

cResearch Division, James R. Randall Research Center, Archer Daniels Midland (ADM) Company, Decatur, IL 62521, USA

dCollege of Resources and Environment, Xinjiang Agricultural University, Urumqi, 830052, China

eResearch Center for Environmental Contaminants (RCEC), Abadan University of Medical Sciences, Abadan, Iran

fSchool of Civil Engineering, Faculty of Engineering, Universiti Teknologi Malaysia (UTM), Skudai, Johor, Malaysia

gCentre for Environmental Sustainability and Water Security (IPASA), Universiti Teknologi Malaysia (UTM), Skudai, Johor, Malaysia

hChemical Engineering Department, Faculty of Engineering, Universitas Jenderal Achmad Yani, Jl. Terusan Jenderal Sudirman, Cimahi, West Java, 40531, Indonesia

iDepartment of Agricultural Engineering, Maharashtra Institute of Technology, Aurangabad 431010, Maharashtra, India

jLaboratory for Quantum Semiconductors and Photon-based BioNanotechnology, Department of Electrical and Computer Engineering, Faculty of Engineering, Universite de Sherbrooke, Sherbrooke, Quebec, J1K 2R1, Canada

kDepartment of Chemical and Process Engineering, Faculty of Engineering and Built Environment, National University of Malaysia (UKM), 43600 UKM Bangi, Selangor, Malaysia

lDepartament d’Enginyeria Quı´mica, Universitat Rovira i Virgili, Av Paı¨sos Catalans 26, 43007, Tarragona, Spain

mSchool of Chemical and Energy Engineering, Faculty of Engineering, Universiti Teknologi Malaysia, Skudai 81310, Johor, Malaysia

nSchool of Biosciences and Biotechnology, Faculty of Science and Technology, National University of Malaysia (UKM), 43600 UKM Bangi, Selangor, Malaysia

*Corresponding author. Laboratory of Environmental Science and Technology, The Xinjiang Technical Institute of Physics and Chem- istry, Key Laboratory of Functional Materials and Devices for Special Environments, Chinese Academy of Sciences, Urumqi, 830011, China

** Corresponding author.

***Corresponding author.

**** Corresponding author.

E-mail addresses:[email protected](A. Kadier),[email protected](M.A. Islam),[email protected](W. Nabgan), [email protected](P.-C. Ma).

1These authors contributed to this work equally.

Available online atwww.sciencedirect.com

ScienceDirect

journal home page: www.elsevier.com/loca te/he

https://doi.org/10.1016/j.ijhydene.2022.09.023

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oResearch Centre for Sustainable Process Technology, Faculty of Engineering and Built Environment, National University of Malaysia (UKM), UKM Bangi, 43600, Selangor, Malaysia

h i g h l i g h t s g r a p h i c a l a b s t r a c t PHP-MEC exceptionally transforms

environmental toxic waste to renewable energy.

PHP-MEC yielded >96% pure H2

during toxic industrial effluent (POME) treatment.

MEC's energy requirement was exceedingly met by a novel Pico- hydro power (PHP).

H2yield and COD removal by PHP- MECs are superior than non-PHP- MECs to-date.

PHP-MEC is economic and scalable option to reduce the carbon footprint.

a r t i c l e i n f o

Article history:

Received 10 March 2022 Received in revised form 23 August 2022

Accepted 3 September 2022 Available online 28 September 2022 Keywords:

Sustainable H2production Microbial electrolysis cell (MEC) Palm oil mill effluent (POME) Pico-hydro power (PHP) Industrial effluents Hydrogen production rate

a b s t r a c t

Due to accelerating global efforts toward decarbonization, a clean hydrogen (H2) producing technology, Microbial Electrolysis Cell (MEC), has garnered considerable attention. How- ever, MEC's external energy requirement has raised concerns about its sustainability, scalability and application costs. The objective of this research was to build a renewable energy generating system for MECs'performance enhancement during the treatment of Palm oil mill effluent (POME). A novel integration of a pico-hydro-power generator (PHP) with single-chambered MECs exceptionally improved its performance. The performance boost was observed as 1.16 m³-H2/m³d H2and 113 A/m³current production in concomitant with 73% organics removal from Palm Oil Mill Effluent (POME) wastewater, which is higher than the previous single-chambered MECs studies. 78% H2recovery rcat(H2) along with 57%

coulombic efficiency (CE) corroborated the removal of a high percentage of electrons from POME organic materials to generate>96% pure H2. The MEC nourished POME wastewater degrading communities while stimulating growth of electroactiveGeobacterin the anodic biofilm which produced H2. The overall H2recovery, COD removal rate and energy efficiency of PHP-MEC are superior than other MECs powered by other external renewable energy sources reported to date. The PHP-MEC prototype paves the path of scale up studies to build a renewable energy dependent future.

Introduction

The rapidly growing world's population, economy and urbani- zation not only have resulted in an increased energy demand but also have caused more pollution of air and water resources [1,2]. Water pollution caused by industrial effluents, including waste oil [3], textile waste [4], synthetic azo dye [5,6], pharma- ceutical compounds [7], metals [8] and chemicals and are of major health concern. Air pollution is primarily being caused due to our reliance on fossil fuels to produce energy. A

substantial increase in the levels of CO2and other Greenhouse Gases (GHGs) has caused global warming which is an imminent threat to our environment. Due to a regulatory requirement on the discharge of pollutants, wastewater treatment to hydrogen research has gained a wider scope. Hydrogen (H2), among numerous energy sources, is a future green fuel due to its superior energy content per unit weight (142 kJ g¹or 61, 000 Btu lb¹), however its production from non-renewable sources (fossil fuels) is not eco-friendly. Therefore, higher H2yielding cleaner technologies must be explored to meet reasonable ex- pectations of global decarbonization efforts [9,10].

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One such emerging technology of cleaner H₂production is known as the microbial electrolysis cell (MEC). The MEC primarily uses carbon-rich materials, including wastewater and various types of renewable materials to produce high- quality H2. In an MEC, electrons (e) extracted from organic materials by electrochemically active bacteria are trans- ferred to the positive terminal (anode) while the protons (H^þ) are freed into the aqueous environment. The migration of e to the positive terminal and subsequent reaction with H^þ yields gaseous H2. This process requires external energy for continued hydrogen production [11]. An outside electricity supplying source energizes the MECs and this property dif- ferentiates MECs from bioelectrochemical systems (BESs). A traditional direct current (DC) or alternating current (AC) can provide constant potential to the MEC system. To replace the DC power source, a few reports have suggested the use of a potentiostat as a source of input voltage for MEC systems [12]. A potentiostat is an instrument that is used to modulate the potential between an electrode and a reference electrode for performing electrochemical analysis. Even though the energy needed for MECs is significantly lower than energy needed for the water electrolysis (1.8e2.0 V) process [13], it is yet high enough to recover from the underdeveloped areas [14,15]. Because of this concern, MECs have experienced a pushback for its industrial scale applications. Moreover, due to a lack of reliable energy providing source, the performance of MECs continue to deteriorate over time which limits its further applications. In the context of reliable energy generating sources, renewable energy sources are promising due to their scalability. Numerous attempts have been made to find a suitable alternative to the conventional DC power supply to reduce the input energy [13,16,17].

MECs can be a self-sustained system when these are integrated with a power generating system. Microbial fuel cells (MFCs), a well-known and acceptable electricity generating BES from wastewater [17,18], successfully energized a two-chamber MEC [19]. In this system, the hydrogen production rate (HPR) from MEC was 0.0149 m³H2/m³d. In a similar report, one to two MFCs were paired with a MEC to generate a MFC-MEC coupled system to improve the voltage supply [20]. The MFC coupling with the MEC has been sought as a viable strategy for combating environmental issues, especially climate change.

The integration of MFCs with MECs has been shown to sequester CO₂[21]. However, many challenges associated with the field application of MECs or MFCs-MECs need to be overcome. For instance, the voltage extracted from MFCs is lower than the actual maximum voltage value (0.8 V) that has been observed in typical MECs [22]. One remedy to improve the voltage would be joining several MFCs in a series however the voltage reversal phenomenon caused by stacking of MFCs may defeat the purpose [23,24]. Additionally, a lack of harmony between the MFCs and MECs could cause instability resulting in diminished performance. To tackle the above-mentioned re- strictions, Hatzell et al. [25] showed an innovative way to enhance the voltage output along with the proficiency of electricity transfer by employing a capacitor. Here, the linked MFCs in parallel charged the capacitors that, upon discharge, in series, enhanced the output voltage flowing into the MECs. This system improved energy recoveries (9%e13%) and HPR (0.31e0.72 m³H2/m³d).

In recent years, renewable energy sources have also been used to power MECs. Sunlight, a renewable energy source, has been used to energize MECs via a dye-sensitized solar cell (DSSC) [26]. The DSSC generates adequate voltage (0.6 V) in an open circuit system to energize MECs. As a result, the MEC system can evolve 0.4 mol H2in 5 h with remarkable cathode recovery efficiency (78%). To make the entire process economic, a Pt catalyst-free cathode has been considered in an MEC [26]. No compromise in H2production was noticed when an ordinary cathode replaced the Pt-rich cathode at>0.7 V.

Similarly, Chae et al. [26] also avoided a Pt cathode but used a nanopowder-shielded electrode to achieve significant improvements in H2synthesis. To sustainably produce H2from a DSSC-MEC hybrid system, extensive investigations are required. Thermal heat, as another form of renewable energy source, has gained a respectable rank in power generation.

Chen et al. [27] has used thermoelectric microconverters, which converted vented heat energy to electric power as an attractive energy producer for MECs. Apart from the above- mentioned power producers, a few well-documented renewable energy producing sources, including wind power, geothermal resources, and hydropower (HP) are solid alterna- tives to feed energy into MECs. While these systems can generate an enormous amount of energy, it can only be used during high load phases and most of it remains unused in low load phases. Strategically, this unused power can be shuttled into MECs for H2and other value aided chemical production.

In summary, here, a tactical use of energy can yield cleaner gaseous or fluid fuels (Hydrogen, ethanol) to satisfy the global demand. However, many challenges need to be overcome before operating such integration.

Compared to wind, solar vented heat, and geothermal energy sources, the hydropower (HP) is exceptionally economic, carbon-neutral, and mature renewable energy generating source [28,29]. HP is one of the critical and efficient energy generating systems as 20% of the global energy (electricity) is derived from HP [30e32]. HP-based energy production can be classified as large, medium, small, mini, micro and pico [29,33].

The pico HP systems have several selective advantages over larger HP systems regarding operational expenses, ease of design, plan and installation. The present work mainly focused on the development of the pico-hydro power PHP-MEC coupled design to improve the HPR, overall functioning of the technology and sustainable production of hydrogen, while effectively removing organic materials from the POME wastewater.

Although MECs integration with renewable energy source is a logical approach, such studies are still in their infancy. Not all renewable energy sources are suitable to power MECs due to inefficient energy conservation and scalability issues, the choice of an energy-generating partner has to be rationalized on cost, scalability and efficiency. Water-based energy- generating technologies have shown tremendous success, especially due to their ease of design and scalability. The objective of the current study was to develop a stable hydropower-based energy-generating system to energize the MECs treating POME wastewater for performance enhancement while addressing common energy related issues of MECs. To the best of our knowledge, the PHP generator has not been employed as the prime powerhouse for the MEC technology to produce energy, primarily H2 during industrial

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waste treatment. The PHP generator coupled with MECs has the potential to substantially increase the performance of MECs that can effectively turn wastewater into a cleaner fuel.

The fundamental understanding of PHP, MEC and PHP-MEC described in the current study would guide researchers to design a suitable PHP-MEC model to remediate a wide variety of industrial effluents. Additionally, the present study is going to be helpful in overcoming the common MEC issues such as overpotential losses associated with MEC, proliferation of hydrogenotrophic methanogens, formation of a thick layer of wastewater on electrodes that reduces electrons and the mass transfer rate, etc. Finally, the communities identified in the present study, that either effectively removed COD or produced H2, will provide a theoretical basis and technical guid- ance to design effective biological strategies to recover energy from complex industrial effluents.

Materials and methods

Palm Oil Mill Effluent (POME) wastewater collection

MEC was inoculated with the POME anaerobic sludge sample that was obtained from a pre-established reactor as described in the previous work [3]. Gas-tight and autoclaved DURAN® laboratory bottles (2 L) were used to store the POME sludge samples at 4C which minimized the deterioration and acidification caused by native microbes. The sample processing and analyses were performed as per the accepted standard water and wastewater methodology [34]. Prior to using the inoculum (seed), the POME sludge was thawed to room temperature followed by filtration using a Whatman filter paper (Grade 1, 11 mm, Whatman, USA) to eliminate undesirable materials (solid particles and debris) [35,36]. Because the POME wastewater is rich in carbon, no supplementation was needed or added. The feedstock samples collected from the pre-established reactor/pond are better suited because its native microorganisms can effectively acclimatize and adapt under a new or changing environment [3]. The temperature of raw POME ranged from 80C to 90C near the discharge point.

The samples were subjected to physicochemical analysis of important parameters immediately after transportation to the laboratory. Extensive chemical analysis of POME was carried out using the accepted standard water and wastewater methodology [34]. The POME wastewater was acidic (pH¼4.6) and had high concentrations of organics (chemical oxygen demand (COD) of 42.4 g/L) along with some significant levels of ions. The samples prior to analysis were preserved at 4C under anaerobic condition. This storage prevented any contamination and limited the activity of the biodegradation process to maintain the quality of the samples. The nature of POME was stable and this storage technique had no observ- able effects on its composition [37,38].

Reactor construction for the PHP-MEC hybrid system The PHP-MEC integrated system, employed in the present study, is comprised of one laboratory-scale PHP for supplying external electrical energy to MEC, and an MEC reactor for H2

production. The laboratory-sale PHP generator system was

designed and manufactured along with its components as shown inFig. 1.

Design and manufacturing: major parts of the PHP generator system

As per the PHP manufacturer's recommendations, the main materials and parts of the PHP systems were designed and fabricated (Fig. 1). The details of each PHP component as follow- (1) Curved spoon-shaped water turbine blades or cups - the 12 blades of the water turbine at an angle of 30were fixed on ribs welded to the shaft. These blades are made up of steel and the average length of each blade is 11 cm. (2) Alternator - the alternator was designed to provide an output ~230 V at 1500 rpm. The alternator has a regulator to adjust the electric current to prevent oversupply. (3) Generator e rotational power is transformed into electric power. Flowing water in a small stream can result in electricity production. (4) Turgo turbine runner - the turbine is estimated to last 20 years with little maintenance. (5) Stopcock (5 cm) or water flow control valve e it was installed to pause the system either in an emergency or for maintenance. (6) Water inflow or inlet from HDP penstock system (7) Single nozzle (turbo turbine) (8) Control box or Diodes (9) Voltage regulator or meter (10) In- dicator lamp (11) Consumer's load (12) Main switch (13) The housing component - the turbine, generator, and trans- mission lines were integrated into a housing component for portability. The housing was framed with a steel cylinder (14).

Surrounding caseeused to protect the PHP parts.

Installation and construction of the PHP system

The proposed PHP was manufactured at Suneco Micro Hydro Power Supplier and Manufacturer, China (“www.micro- hydro-power.com”).Fig. 2outlines the PHP generator operation used in the present study.

Multiple components ensured a consistent performance of the nozzle, turbine and generator. A small digital multimeter was used to measure the voltage and current generated by the PHP generator. The shaft speed was measured via a Laser tachometer (Model 461,920 l, Extech Instruments, Waltham, MA). A photograph of the laboratory-scaled PHP system installation and configuration setup is shown inFig. 3. The PHP generator system included a water tank to hold water (150 L - Black, Deluxe Polyethylene Round Type Water Tank).

Water stored in the tank was pumped through the flow loop by using a home booster water pump (Grundfos 1.0 HP Home Booster Water Pump: CM5-4PM2). Other components such penstock (high density polythene (HDPE)), cross-flow turgo turbine or runner, induction generator, alternator, charge controller, dump resistor also called dump load to keep the voltage steady, and a set of batteries were placed in place.

To minimize the cost of the PHP system, some of the components of the PHP were self-made, or made with used parts gathered from other laboratories in our department. The generated electricity from the PHP generator was stored in car batteries, which are inexpensive and portable.

Water head and water flow rate measurement methods According to Basar et al. [39] the critical elements for a PHP system are the water head and water flow. The head refers to the pressure that is developed due to the elevation difference

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Fig. 1eThe layout of the main materials and the principle components of the PHP system.

Fig. 2eSchematic diagram of the developed PHP generator system.

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between the intake pipeline and the water turbine. It is sometimes referred as a vertical water fall source. The quantity of water determines the water flow or water flow rate.

Generally, three popular methods are used for measuring water flow: (1) container fill method, (2) float method, and (3) weir method. The bucket method or container fill method is the easiest method used for measuring the water flow rate for small water streams [29,40]. This method measured the flow rate of water in the present study. According to this methodology, a defined volume container/bucket is used to collect water and the time that is taken to fill the container is de noted as t. The total volume of the bucket or container is V (L or m³), therefore, the water flow rate, Q (m³/s or L/s) can be calculated as shown in (Eq. 1):

Q¼ Bucket volume Time to fill the bucket¼V

t (Eq.1)

Calculation of efficiency and productivity of the PHP system Eq. 2 can be used to determine the efficiency of the PHP system [29,32,41,42].

EfficiencyðhÞ ¼ Pout*100 Pin

(Eq.2) The peak power delivered test (PDT) is useful for calculating the output power (Pout). The open circuit test (OCT), when performing the max PDT, achieves the potential input power (Pin). The power input and output can be represented by Eq. 3 and 4.

Pin¼r g H Q (Eq.3)

Pout¼ h r g H Q (Eq.4)

Here, r is the density (kg/m³) of the water source;

g¼9.81 m/s²; H is Effective head or pressure (m);his the hydraulic effectiveness of the water turbine (%); Q is the Water flow rate (m³/s or L/s).

Construction of an MEC reactor and materials for electrodes The MEC design was based on the previously published study by Kadier et al. [3]. A clear wide-mouth 101 mm diameter and

152 mm tall bottle (DURAN®) was used to construct the membrane-less single-chamber reactor. The final reactor possessed a total capacity of 0.8 L with an effective volume of 0.5 L. The remaining headspace of 0.3 L served as the hydrogen collection space. The top of the reactor was fitted with an outlet and an inlet. (Fig. 4).

A 6.4 mm thick isomolded graphite plate (Grade GM-10, IL, US) served as an anode for each MEC. The anode surface was dipped into a mixture of 1 N HCl and 1 N NaOH for 2 h followed by rinsing with deionized water prior to constructing the MEC.

The dimensions of each anode were 70 mm60 mm x 6.4 mm which made a total of 1000 mm²surface area. Here, the Ni electro-formed mesh cathode was used because of its excellent catalytic activities resulting in hydrogen evolution in MECs [43].

The overall dimensions of the cathode made a total of 840 mm² surface area. To prevent diminishing performance, majorly because of fouling, the cathode was changed twice a year. The anode was kept 25 mm apart from the cathode. The electrodes were joined together using a titanium wire with a constant 10U external resistance. The V through the outside resistance (Rex) became the basis of calculating the current. Typically, some voltage is lost because of a Rextherefore the final voltage on the cathode and anode is lower than the provided voltage. Further, to minimize the waste of voltage, a small Rex(1e10U) has been proposed [44e46]. Taking into consideration these earlier attempts, a Rex ¼ 10 U was installed as a peripheral load throughout the MECs operation in the present study. A viton tube was attached to the outlet and was inserted into an inverted 2 L graduated cylinder containing DI water for gas collection. The MEC inlet was then sealed and the N2sparging was paused to allow the accumulation of H2.

The PHP system and the MEC were connected in a circuit with an automatic DC power source through an electric wire and a 10 U resistor, creating a new PHP-MEC system. The positive (þ) end of the power source was joined to the anode of MEC and the cathode was linked to the negative end of the MEC. A high-precision resistor (Rex¼10U) joined theþand the - terminal of the power supply for current measurement (Fig. S1). All connections were checked constantly to prevent any fire hazard.

Fig. 3eA schematic (A) and actual laboratory scale PHP prototype (B) to generate electric energy.

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Operation of the combined PHP-MEC system

After being connected, the way to start up the PHP-MEC was similar to that of the MEC, as described previously by Kadier et al. [3]. Prior to inoculation, the MEC reactor was autoclaved.

The MEC reactors were filled (0.450 L) with the pre-treated POME wastewater at an optimum initial COD concentration of 50.71%. Afterwards, the MEC reactor was inoculated with 0.1 L POME anoxic seed sludge containing enriched culture as described by Kadier et al. [3]. Meanwhile, to maintain anaerobiosis in MECs a pure N2 gas was sparged in each reactor for 15 min. 2-bromoethanesulfonate (50 mM) acted as an inhibitor for any methanogenic activity [47]. After the MEC was seeded, a voltage of 1.1 V was supplied to test the MEC system. The voltages (V) and current generated by MECs through theRexwere measured in every 10 min using a small digital multimeter. For the current calculation, the Ohm's law I¼^V_R;R_ex¼10UðEq:5Þwas used. MEC operations were moni- tored in every 10 min by current measurement.

MEC reactors in duplicates, situated in water baths, were operated in the batch mode under optimum conditions obtained in this study; an optimum temperature of 30C, start- ing pH of 6.63, and 50.71% COD. Hydrogen production paused when the current fell below 10% and this occurred at the end (1 cycle) [48,49]. At the end of each cycle, air was circulated in the reactor to restrict methanogens’ growth [50] and the electrodes were treated as described previously [3]. The new cycle was started by adding 0.4 L previously treated POME substrate with 0.1 L inoculum. The anaerobiosis was established by headspace replacement of the MEC reactors with pure N2for 15 min.

Analysis of evolved gases

The gases accumulated in the headspace of the MEC were measured by the water column replacement method as described previously [3]. CO2absorption was carried out using

~95% NaCl (pH¼0.5) as described previously [43]. Gaseous samples were collected in gas collection bags (Tedlar Gas

Sampling Bag, CA, USA). Gas bags were deflated via a vacuum pump before use. Gas components and volumetric fractional percentages of hydrogen, methane and nitrogen were estimated via a gas chromatograph fitted with a helium ionization detector (HID) and a thermal conductivity detector (TCD).

Helium (>99.999% pure), at a constant flow rate of 25 mL/min, served as the carrier gas. The gas injections into the GC were conducted via a 250mL gas leak proof syringe. The gas production was evaluated based on the experimental conditions.

The aggregated volume for a specified gas (Vi) including hydrogen, methane and carbon dioxide was calculated using Eq. 6 mentioned below:

Vi¼ ðVtþVhÞ*X (Eq.6)

where, Vt¼ mL of quantified gas,Vh¼ reactor's headspace volume (mL), t¼gas sampling pipe at sample time, and the particular proportion of gas (%)¼Xi.

Analytical techniques

A laboratory pH meter was used for pH measurements at different stages. An automatic conductivity meter was employed for electric conductivity (EC) measurement. An NaCl-saturated Ag/AgCl reference electrode measured the half-cell potentials of the anode and cathode at each cycle.

The chemical oxygen demand (COD), BOD, total suspended solids (TSS), volatile suspended solids (VSS) of the influent and effluent were estimated as described previously [3,34]. The Hanna instruments test kit was used for overall N2, NH4eN, P, oil/grease and turbidity measurement.

Microbial community analysis

The diversity of the microbial community in the MEC-PHP hybrid system was analyzed using the 16s metagenomics library preparation method recommended by the Illumina MiSeq Sequencing. The 0.1 L biological samples of the initial inoculum and the anodic microbial community (biofilm and suspension) from the MEC anode were used for analyses. The Fig. 4eA schematic of MEC chamber equipped with electrodes and a laboratory scale membrane-less single chamber MEC reactor constructed from a glass bottle.

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recovered samples were then sent to the 1st Base laboratory (1st BASE, Next Generation Sequencing (NGS) team) for high- throughput sequencing analysis. This included the isolation of genomic DNA and the amplification of specific gene areas in the 16S ribosomal RNA (rRNA).

Total community genomic DNA from each sample was extracted by the Fast DNA purification kit (A1123, Promega, US) as per the supplier's instructions. The isolated DNAs were preserved at20C before use. At 1st BASE, the concentration or quality of the extracted DNA was measured using three methods; Microvolume spectrophotometer (NanoDrop), Fluorometer (Biotek FLX800), and agarose gel electrophoresis (AGE) ensuring the high-quality of the isolated genomic DNA.

Polymerase chain reaction (PCR) was started immediately after the DNA was extracted. The universal primers for bacterial samples (”341F(5⁰-CCTACGGGAGGCAGCAG-3⁰) and 805R(5⁰-GACTACCAGGGTATCTAATC-3⁰)”) and for archaeal sample (”515F(5⁰-GTGCCAGCMGCCGCGG-3⁰) and 907R (5⁰- CCGTCAATTCMTTTRAGTTT-3⁰)”) were used that focused the variable V3eV4 region (~455 bp) of 16S rRNA. After amplification, the quantity and quality of the PCR products or li- braries were measured using tapestation 4200 (Library Size), picogreen (Concentration, ng/mL) and qPCR method (Molarity, nM) methods. QC passed samples were used to construct li- braries. Standard Illumina sequencing method was used to sequence the amplicon library via the Illumina MiSeq system with 2301PE format (Illumina MiSeq, USA) according to the manufacturer's instructions.

Raw reads obtained from the different samples were sorted using a unique barcode for each PCR product. Reads were matched to the known 16S rRNA databases of the National Center for Biotechnology Information (NCBI), Sequence Read Archive or GenBank through the nucleotide-nucleotide Basic Local Alignment Search online tool (BLASTn) and the Ribo- somal Database Project II (RDP) using the web-based sequence match finder tool. The presence of two or more ambiguous nucleotides in any reads or an overall low-quality score (<25) or reads<300 bp were filtered out. The operational taxonomic units (OTUs) were classified using a 97% identity level to the 16S rRNA gene sequence as a cutoff using UPARSE. The Shannon diversity index and Chao1 estimator were calculated at a 97% similarity level using the RDP to find species abundance and diversity. The phylogenetic affiliation of each sequence was assessed using the RDP classifier at a confi- dence level of 80%.

Results and discussion

Performance evaluation of the PHP generator

To achieve a steady performance, PHP was operated for more than a week (10 days) prior to connecting it with the MEC.

Analysis of the results showed that the OCV is directly pro- portional to the generator speed and water flow rate. Notably, the stable and maximum power output or OCV produced with the PHP generator system was 176 V when the water pressure and water flow rate was 4 bars and 2.40 L/s, respectively. The performance of the PHP generator was stable throughout the PHP-MEC operation because fixed water pressure by a water

pump was maintained. Theoretically, H₂can be produced in the cathodic chamber of MECs when>0.11 V is applied in a circuit, however, in practice, due to the overpotential existence, greater voltages are needed. Liu et al. [51], described H2synthesis in the cathodic compartment of MECs when using >0.250 V.

Furthermore, previous MEC studies reported that a typical voltage of 0.6 V or more [22,50,52e54] is needed for high- efficiency H2production in MECs. The PHP offered an advan- tage because the output voltage was stable and high enough to power the MEC reactors. The electricity requirement of the MEC reactors can be easily fulfilled by the PHP system that allows the MEC to produce H₂while remediating wastewater.

Performance evaluation of PHP-MEC integrated system The PHP-MEC systems fitted with a Ni mesh cathode were operated under optimal conditions as mentioned previously [3]. The functioning of the PHP-MEC coupled system was accessed in terms of measuring Coulombic efficiency (CE, %), cathodic hydrogen recovery (rcat(H2), %), overall hydrogen recovery (rH2, %), hydrogen production rate (Q), volumetric current density (Iv), COD removal efficiency (COD, %) and hydrogen yield (YH2). The summary of the PHP-MEC results with Ni mesh cathode and Eap of 1.1 V are tabulated inTable 1.

PHP-MEC showed an effective treatment of POME by removing 73% of organics concomitant with the production of cleaner fuel (1.16 m³-H2/m³d H2) and electricity (113 A/m³). These results are superior to those of previous similar MECs studies [55e59] because the PHP integration with MECs fully satisfied the increasing energy demand during POME or wastewater treatment.

The performance of MECs in terms of hydrogen production is also dependent on the pH and salinity because it will directly impact the microbial communities involved in the degradation of complex organic compounds and electroactive bacteria producing H2[60,61]. In addition, the redox potential of H2 evolution reaction and methanogenesis could be affected by the catholyte pH [62]. The first study investigating the effects of catholyte pH on H2evolution by MEC demonstrated that catholyte pH and voltage applied influenced H2

evolution. An increase in pH from 5 to 9 decreased of H2

evolution (32.2±3.7 mL/day to 1.68±1.2 mL/day) suggesting that slightly acidic pH is suitable for optimum H₂evolution [62]. In the current study, higher H2evolution is further sup- ported by the slightly acidic pH conditions of the MECs.

Additionally, the hydrogen evolution rate (HER) on the anode is also influenced by electrolyte. Typically, wastewaters have very low ionic conductivity (0.2U/m) [63] and electrolytes are needed which is a major challenge in scaling up an electrochemical process [64]. A higher ionic conductivity increases the current density [65]. Sodium phosphate is a common choice in the MEC medium due to its lower chances of consumption by the microbes. Merrill et al. studied phosphate, carbonate, acetate and chloride electrolytes to observe their effects on MEC performance [66].

Composition of gaseous products evolving from the PHP-MEC system

Even though methane is an energy carrier, it is a potent greenhouse gas. Hydrogenotrophic methanogens are capable

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of utilizing hydrogen to produce methane that could significantly reduce hydrogen productivity or yield. Interestingly, methane was produced in traces while hydrogen remained the major product produced by PHP-MEC. The volume of total gas produced over the length of the batch experiments varied from 0.213 to 0.215 L. In terms of the gas composition, H2

content was higher (>96%) than the other gases (CO2(2.1%), N2

(1.4%), and CH₄(0.5%). These results revealed that the PHP- MEC integrated process primarily produced a cleaner fuel (H2) and these results are comparative to the previous reports showing the proportions of H2as>98% [49,56], 97e99% [67], 97% [68], 96% [50,69], 95% [70].

In addition to H2production through MEC, there exist other biological routes of hydrogen production such as hydrogen production using fermentative bacteria via dark fermentation [10]. These fermentative bacteria can oxidize fermentable sugars into hydrogen and CO2 [9,10,16]. These reactions in MEC are unlikely because, hypothetically, CO2in the cathode compartment cannot exist and the electric current used in MECs could negatively affect growth of hydrogen producing microbes. Traces of CO2existed, possibly due to the seepage of MEC's headspace environment after the extended operation.

Overall, the MEC process is superior than dark fermentation in terms of H2 production because in MEC a wide variety of complex molecules are transformed into H2that may either not be utilized by fermentative bacteria or could inhibit their growth.

Furthermore, no considerable amount of CH4was detected in batch tests, likely due to the operating conditions such as higher applied voltage, and exposure of the MEC cathode to oxygen or air in between batch cycles that would be lethal for strictly anaerobic methanogens. These findings are consistent with the previous reports showing methanogens suppression in MECs [69e74]. When MEC is operated under a higher voltage, the higher voltage increases the current density and thus shortens the reactor run-time. This shorter run-time helps to reduce the CH4production. Due to the superior ability of PHP to deliver higher current, the PHP-MEC system produced the highest proportion of H2 while suppressing fermentation H2/CO2and CH4production.

Hydrogen production rate (HPR)

Hydrogen gas was produced in the reactors with small varia- tions and it was evaluated in terms of HPR. HPR dropped at the end of each batch cycle due to complete substrate consumption. The HPR positively correlated with H2content and MEC's performance. The max HPR of 1.16± 0.08 m³ H2/m³d was achieved at 1.1 V with a 10 Uloading resistance (Table 1).

Similarly, earlier studies have reported an HPR of 1.11 m³H2/ m³d and 1±0.1 m³H2/m³d when synthetic effluent (5 g/L) with real fermentation product from vegetable sources [53]

and swine wastewater (2 g/L), respectively, were fed to the MEC [52]. Furthermore, the maximum HPR determined in the present study was comparatively superior than those of previously reported values obtained from MEC powered by other alternative renewable power sources. The boost in the MEC's performance in the current study is attributed to the higher voltage supplied by PHP and a use of an efficient Ni mesh cathode [43].

A higher HPR can be produced when simple or pure substrates such as acetate [50,75e78], glucose, P-Glycerol [22,79], real wastewater/synthetic or post-fermentation effluent, and anaerobic sludge as the feed-stock of MEC reactors [54,69,80,81] are used. While a respectable HPR was obtained during the POME wastewater treatment, which is comparatively higher than the similar studies conducted previously [52,53], a further improvement in HPR would require pre- treatment of POME because POME wastewater is highly complex in having amino acids, free organic acids, inorganic nu- trients (Na^þ, K^þ, Ca^2þ, and Mg^2þ), small fibers, microbial organelles, nitrogenous compounds and a blend of carbohydrates varying from complex hemicelluloses to hexoses.

Moreover, some parts of this cannot be utilized as being recalcitrant or inhibitory to the metabolic activities and growth of the EAB consortium [82,83]. Furthermore, unstable electron transfer in the anode might also result in low HPR, as MEC using POME establishes a thick film on the surface of the electrode that can block the migration of electrons from the microbes to the electrodes [51,84]. Similarly, the highly gelat- inous POME wastewater may block the electrode surface, which could reduce electrons and mass transfer rate. This could directly affect the current production and HPR [84].

In addition, the HPR is also dependent on the cathode performance which is generally seen as a limiting step for H2

production. Therefore, an ample number of efforts have led researchers to construct a highly efficient cathode [85]. Even though the PHP-MEC system has the potential to mitigate environmental problems by degrading toxic industrial wastewater, further optimizations to improve the catalytic reactions occurring on the cathode [86] and the utilization of biocathode [50,87,88] in MEC configuration are likely to overcome various challenges.

Hydrogen yield, hydrogen recovery, and the coulombic efficiency (CE)

COD represents the organic content and it is often regarded as a measure of water quality. POME that has high COD cannot be discharged into the environment due to regulatory re- strictions. Moreover, high COD is likely to deplete oxygen in an aquatic system that could threaten aquatic life. Fortunately, the MEC technology can effectively transform COD to H2. In the current study, the average hydrogen yield (YH2) of COD Table 1ePerformance of the PHP-MECs hybrid system using Nickel mesh at Eap of 1.1 V

Cathode CE (%) rcat(H2) (%)

rH2

(%)

Q(m³H2/m³d) IV(A/m³) COD removal(%) YH2mmol H2/g COD

Ni mesh 57±2.1 78±2.7 75±1.9 1.16±0.08 113±6 73±0.8 894.42±0.3

±is standard deviation of two biological replicates.

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was 0.894±0.3 L H₂/g COD or 894.42±0.3 mmol H₂/g COD (Table 1). The maximum YH2achieved in the present study is superior to a previous MEC study by Lu et al. [53,89] in which 21 mmol-H2/g COD was achieved through a single chamber MEC.

The key factors influencing the hydrogen yield include cathodic hydrogen recovery (rcat(H2)) and Coulombic efficiency (CE). Cathodic hydrogen recovery is the efficiency of the conversion of the current into H2. The rcat(H2) value reflects the H2recovery from current and is largely dependent on the proton reduction rate on the cathode of MECs. As shown in Table 1, the maximum rcat(H₂) was 78±2.7%. However, some studies using pure and simple substrates have shown higher values for rcat(H2) such as Butyric acid (1 g COD/L)e80% [22], Acetate (10 mM) - 98% [90], and simulated landfill leachatee 66e95% [67]. Considering the complex nature of POME wastewater, such a ceiling of rcat(H2) is expected.

Additionally, consumption of electrons by methanogens to form methane or the overpotential increase due to the characteristics of the cathode material [91] could be another reason for a slightly lower rcat(H2) obtained from the PMP- MEC system. Furthermore, this could be attributed to the electrons that may engage in side reactions with alternative electron acceptors such as O2 which might enter via the installed ports [19,27]. A reduction in the number of electroactive microbes might be a reason of incomplete decom- position of POME wastewater [84] that could decrease rcat(H2). It is also possible that the native microbial community of the MEC anode, inherited from POME wastewater, may consume electrons [92]. These results suggest that further research is required to reduce overpotential and to clarify where the main electron, H2, and energy loss occur within the PHP-MECs system.

From the viewpoint of MEC efficiency, the Coulombic efficiency (CE) represents the extraction of electrons from POME wastewater [44]. The highest CEand overall hydrogen recovery (rH2) of 57±2.1% and 75±1.9, respectively at 1.1 V (Table 1), indicate that a relatively high percentage of electrons from the organic materials of POME wastewater was converted to the current output. The maximum CEestimated in this study is in the same range of CE reported in the previous MEC studies using wastewater or composite substrates such as winery wastewater (50± 8%) [93], domestic wastewater (55%) [94], mixture of furanic and phenolic compounds (58±1%) [95].

While it is common to get a lower CEin the MECs fed with real or complex wastewaters, a little higher CEhas been reported in earlier studies not using complex wastewaters [69,92,96,97].

Typically, CEinforms which microbial community is degrading COD. When CEis low then majority of COD is targeted by non-electrogenic microorganisms rather than the EAB. The relatively lower CEin this study might have been caused by the complexity of the POME wastewater, which might have sup- ported competition or harmonious interplay between fermentative microbes and EAB in electron-consuming reactions in MECs [98,99]. Moreover, electrons resulting from the oxidation of the substrate can either be consumed for gas production or lost to alternative electron acceptors (Sulphur or nitrogenous compounds) which could further lower the CE

[44]. Additionally, the CE value is significantly impacted by loading resistance, the complexity of the feed, cellular

biomass production, transformation of substrates into polymers, fermentation, methane evolution, electrode type and various electrons removing routes, which have higher ten- dencies to occur in the MEC [20,100e102]. To improve CEand rH2, a few measures need to be taken into consideration, such as inhibiting non-EAB pathways and pre-treating POME to enhance the biodegradability of the POME wastewater entering into the PHP-MEC system.

Organic matter removal by the PHP-MEC integrated system POME is extremely rich in organic pollutants which increase the COD and BOD to 55.7 g/L and 25.5 g/L, respectively [103].

Extraction of its higher organic content, conventionally, takes laborious routes of treatments to meet the discharge standard set by the regional authority. A detailed analysis of raw and pre-treated POME was performed as described previously [3]

and total/dissolved/suspended solids, COD, BOD, total N/

NH3eN/PO4eP, oil/grease, turbidity, color, conductivity, pH and temperature were estimated. Raw POME is acidic (pH¼4.6) in nature but rich in organics (COD 42.482 g/L) with considerable amounts of ions. The COD removal rates or efficiencies in this study were all>60% at a supplied voltage of 1.1 (Fig. 5). Under optimum conditions, the 73±0.8% COD was removed (21,326±37 mg/L influent COD and 5758±21 mg/L effluent COD), which follows the similar trend as mentioned in the previous reports [84,104] and is comparatively higher than the previous studies [55e59]. Effective biodegradation of POME wastewater in the current study is likely because of the comprehensive effects of diverse microbial consortium in the sludge inoculum [105e107] and the operation mode affecting the performance of MECs and MFCs [48,85].

On the contrary, the maximum COD removal rate in this study is significantly lower than the COD removal rate values (over 90%) from two-stage integrated MFC-immobilized biological aerated filter (IBAF) systems [108], MFC-anaerobic membrane bioreactor (AnMBR) [109] and continuous stirred tank reactor (CSTR)-mesophilic MECs fed with POME [110].

This might be due to the complex nature of POME wastewater

Fig. 5eThe key performance indicator of the PHP-MEC reactor shown as current density (C) and COD removal (◼).

Maximum volumetric current density from the PHP-MEC system treating POME wastewater initiated with the addition of anaerobic seed sludge and 50% of initial COD.

The progressive removal of % COD from POME wastewater by PHP-MEC starts quickly and attains maximum value.

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which contains high amounts of complex lignocellulosic compounds and longer chain fatty acids that are recalcitrant toward microbial degradation and inhibitory to the metabolic power and growth of microorganisms [82]. The nature of wastewater can significantly affect COD removal as only 29.8%

COD removal was achieved when household wastewater was processed through a dual-compartment continuous MEC [56].

Electrochemical analysis of the PHP-MEC system

The MEC anodic microbial community was enriched for 30 days to demonstrate stable performance and reproducible maximum voltage generation. A stable output voltage or OCV of 0.75e0.80 V was achieved after overnight enrichment. The anodic half-cell potential was 0.476 V (versus Ag/AgCl).

Theoretically, in biocatalyzed acetate oxidation the negative pole potential0.480 V (Ag/AgCl) of the MEC operated at pH 7 is likely [44]. These results confirmed that the EAB biofilm was successfully formed on the anode of MECs. It is documented that the natural microflora of the POME anaerobic sludge contains various microorganisms whose ability to form the biofilm on the anode and produce enzymes or structures to transfer electrons vary [44]. Generally, the diverse microbial community in the POME anaerobic sludge is best suited for a steady current production than pure cultures [106].

Fig. 5shows steady current density (IV) over a week by PHP- MEC when treating POME wastewater. Initially, the current exponentially generated and reached its peak on the 5th day thereafter it dropped slightly and this trend is consistent with the previous current generation trends described in various studies [84,104,106]. The continuous current generation can be attributed to the action of POME microbes that are feeding on the organic matter of POME wastewater to convert COD into current [84]. Furthermore, since the MEC reactors were operated in a batch mode, the available organic compounds in POME wastewater were depleted with time, thus a decline in further generation of the current was expected at the end.

Based on the data obtained, the circuit current decreases with an increase in the loading resistance. The maximum circuit IV of 113 ± 6 A/m³ was obtained at the minimum loading resistance of 10Uand Eap¼1.1 V (Table 1). Note that the average IV estimated in our studies was found to be comparative to the previous reports employing swine wastewater e 106e112 A/m³ [52], diluted municipal solid waste (LPW) - 115.9±7.2 A/m³[97] or higher than previously reported results by Montpart et al. and Sun et al. [20,47]. Furthermore, the experimental observations suggested that the IVmight be more reliant on Rin rather than the supplied voltage in the MEC and these findings were in-line with the study by Loagan et al. [111]. Moreover, the past studies reported that the IVwas raised by ~10% when the Rinreduced by 18% [88] and the Rin

was influenced by the migration of electrons, and MEC setup [112]. Thus, additional fine-tuning of the present MEC setup is required to minimize the R_in. What's more, the results exhibited that the solution conductivity was partially dependent on the ionic strength. A higher ionic strength has been observed to enhance the current production by reducing the Ohmic resistance of the electrolyte [113]. The ionic strength of the crude POME was adjusted to further optimize/maximize the current production. Thus, in order to raise the ionic

strength, the raw POME was diluted with a higher concentration of the phosphate buffer (50 mM) as suggested previously [113,114].

Energy recovery and efficiency

In an MEC system, the energy input comprises of the electricity and substrates consumed, while the energy outputs are the amounts of H2or other biofuels produced. Energy recovery and efficiency of the PHP-MEC coupled system are presented inTable 2. The maximum consumed substrate energy or energy content of the removed substrate (WS), electrical supplied energy (WE), energy of collected H2or the recovered H2(WH2) of 2.6±0.3 KJ, 1.41±0.9 KJ, 1.89 ±0.7 KJ, respectively were obtained with the Ni mesh cathode and Eap of 1.1 V. Regarding energy efficiency, as shown inTable 2, the energy efficiency for the PHP-MEC system based on substrate (hS) and electricity (hE) were calculated to be 93.6 ± 2.9% and 156 ± 4.4%, respectively. The hE is more conservative due to the fact it only considers the energy input from electrical energy, but not from the substrate. Because of this, its value is higher than 100%. In contrast,hEþS considers both, thus its value is al- ways under 100%. The electrical energy input combined with substrate energy (hEþS) of 71.69±1.14% was obtained with the Ni mesh cathode that represents the overall energy efficiency.

Table 2 demonstrates substrate energy contribution (eS) and electrical energy contributions (eE) for a PHP-MEC hybrid system equipped with a Ni mesh cathode. TheeSandeEfor the PHP-MEC system were 38.71% and 61.29%, respectively. The results of the current study pointed out that when a higher Eap is applied to the MEC system, MECs obtain more energy from electricity than from substrates-to-energy captured in the H2. Similar findings have been observed in the previous studies [44,50]. In summary, the integrated PHP-MEC system is highly effective in terms of energy recovery and efficiency.

Microbial community analysis

The eubacterial and archaeal communities of the initial inoculum (POME anaerobic seed sludge), used to inoculate all MECs, and the community in the anodic biofilm, at the end of the experiment, were examined by pyrosequencing. A detailed analysis of the archaeal and eubacterial community structure was performed as mentioned elsewhere in the manuscript. Illumina MiSeq sequencing data showed that massive bacterial populations are present and are involved in the anode biofilm formation. For extensive characterization of the phylogenetic distribution of bacterial OTUs is shown in Figs. 6e9.

At the phylum level (Fig. 6A), taxonomic analysis of the communities demonstrated that the bacterial community in the initial inoculum was composed primarily of Chloroflexi (15.1%),Bacteroidetes(13.7%),Actinobacteria(11.5%),Thermoto- gae (11.2%), Proteobacteria (10.4%), Firmicutes (10.1%), Spiro- chaetes(8.3%),Planctomycetes(6.2%),Verrucomicrobia(5.4%), and Acidobacteria (5.1%) along with other low-abundance phyla (Fig. 6A). It is worth mentioning that Chloroflexiwas found abundant in the anodic biofilm in the earlier MFC study [115].

Chloroflexi phyla include important microbes that degrade

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carbohydrates and remove nitrogen during wastewater treatment [116]. A huge shift occurred in the microbial community present in the anodic biofilm owing to the enrichment of EAB (Fig. 6B), with the phyla belonging mainly toProteo- bacteria(66.2%), followed by Bacteroidetes(12.8%), and Firmi- cutes(5.8%),Actinobacteria(4.2%),Spirochaetes(3.7%),Chloroflexi (3.3%) in addition to several low-abundance and unclassified phyla. These results are consistent with the studies concluding the dominance of Proteobacteria, Firmicutes, and Bacteroidetesphyla in MECs. Furthermore, the results are in good agreement regarding the high abundance ofProteobac- teriawith the previous MEC report [55,117,118]. This observa- tion is plausible because Proteobacteria species can produce and consume H2 [68,119]. Moreover, it was reported that Actinobacteriais the major lignocellulose degrader whileFir- micutes degrade simpler organic compounds [120,121]. In addition, fermentativeBacteroidetesthat thrived in the anoxic environment of the anode have been known for the degradation of organic pollutants [122].

FromFig. 7, it can be seen that dominant microbial communities in the initial inoculum were Anaerolineae (13.9%), Bacteroidia(13.5%),Actinobacteria(10.8%),Thermotogae(10.5%), Clostridia(10.3%), Deltaproteobacteria(8.8%), Betaproteobacteria (7.1%), Spirochaetes (5.8%), Alphaproteobacteria (5.3%), Plancto- mycetia(5.3%),Gammaproteobacteria(4.6%). After a shift in microbial communities (Fig. 7), the microbial community of the MEC anode biofilm was dominated by the classesDeltaproteo- bacteria(38.5%),Bacteroidia(10.3%),Clostridia(9.6%),Gammap- roteobacteria (8.1%), Actinobacteria (7.4%), Betaproteobacteria (6.1%), and Alphaproteobacteria (5.8%), Epsilonproteobacteria (5.5%), Anaerolineae (3.9%) in addition to several low-

abundance and unclassified classes. Deltaproteobacteria are EAB that produce H^þand efor energy production [123]. The role of classClostridiahas been defined in power generation as these bacteria convert fermentable substrates into simple organics [124].

On the basis of genus-level analysis (Fig. 8), the most abundant genus found in the anodic biofilm of MEC was the species ofGeobacter(30.8%).Geobactera is well-document EAB reported in MFCs or MECs [118,125]. GeneraGeobacteris well- known for transporting electrons between the bacterial cells and electrodes. A previous study found that the microbial consortium structure depends on the prevalence of a type of nutrient in the feed, catabolic pathways and supplied voltage [126]. In our studyGeobacterwas the major part of the anodic biofilm which remained pivotal for current production. In the anodic biofilm communities, the second and third most abundant genera were Pseudomonas sp (11.1%), Klebsiella (9.4%), respectively. Several previous studies have reported that both PseudomonasandKlebsiellasp. can produce electricity and treat POME wastewater in MFC by producing electron shuttles to enhance the electron-transfer rate [36,106,127e129].

Other dominant groups wereBacteroides(8.3%) andDesul- fovibrio(6.7%).Bacteroidessp e.g.Bacteroides graminisolvensis a xylanolytic anaerobewhich produces acetate, propionate and succinate [130].Desulfovibriois a well-known sulfate reducing bacterium (SRB) and belongs to the Deltaproteobacteriaclass that has the tendency to remove sulfate in neutral and even acidic conditions [131]. Kim et al. [132] described hydrogen synthesis by Desulfovribio sp. that reduces protons (H^þ) by accepting electrons from the cathode and this is known as Table 2eEnergy yields, efficiencies, and contribution for the PHP-MEC system with Eap¼1.1 V.

Cathode WS(KJ) WE(KJ) WH2(KJ) hS (%) hE (%) hEþS (%) eS (%) eE

(%) Ni mesh 2.5±0.3 1.31±0.9 1.89±0.5 93.6±2.9 156±4.4 71.69±1.14 38.71±0.5 61.29±0.8 Note: WS: the utilized substrate energy; WE: electrical input energy; WH2: energy of collected hydrogen;hS: substrate energy efficiency;hE:

electrical energy efficiency;hEþS: Overall energy efficiency; (eS): Energy contribution; eE: electrical energy contributions.

Fig. 6eThe phylum-level taxonomic classification of the communities present in the inoculum (A) and anodic biofilm (B).

The phylum level was determined by Illumina Miseq sequencing. The phyla that were<3% of the total bacterial community were denoted as“others”.

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hydrogen cycling. Moreover, Liu et al. [80] reported that current generation along with organic material degradation is also possible.

For the Archaeal communities at the phyla and class levels, no distinct differences were observed in either the initial inoculum or anodic biofilm of MEC. The three dominant phyla

wereEuryarchaeota(45.7% and 44.9%),Crenarchaeota(30.6% and 33.3%) and Parvarchaeota(18.8% and 16.7%) and at the class levels Methanomicrobia (39.4% and 38.2%), Thermoplasmata (30.1% and 32.8%), MiscellaneousCrenarchaeotalGroup (MCG) crenarchaeota (15.6% and 14.3%) and Parvarchaea(11.5% and 9.9%) were abundant in the initial inoculum and anode biofilm of MEC. This genus-level categorization provided a comprehensive understanding of the microbial community structure.

Interestingly, at the genus level the most dominant Archaea in both initial inoculum and anodic biofilm of MECs were acetate-utilizing methanogens (Methanosaeta) with its proportions as 32.5% and 30.7%, respectively (Fig. 9). Taxo- nomically, these archaea belong to the Family: Meth- anosaetaceae; Class:Methanomicrobia; Phylum: Euryarchaeota;

Domain: Archaea, and are commonly detected in anaerobic conditions. Similar findings were described by Kim et al. [133]

that the dominant methanogenic population was aceto- clasticMethanosarcinalesin the anoxic breakdown of thermo- alkaline-simplified waste-activated sludge. In MECs, Meth- anosaetacompetes with EAB for both substrate and product as well as electron loss for non-electrogenic reactions. For instance, acetoclastic methanogens sp like Methanosaeta convert acetate to CH4and hydrogenotrophic methanogens consume H2to produce CH4[13]. BecauseMethanosaetahas a high affinity towards acetate, proliferation ofMethanosaetais Fig. 7eThe class-level classification of the microbes present in the inoculum and anodic biofilm of MEC. Classes that were

<3% of the total bacterial community were denoted as“others”.

Fig. 8eThe genus-level categorization of microbes present in the inoculum and anodic biofilm of MEC. Genera that were

<3% of the total bacterial community were denoted as“others”.

Fig. 9eThe genera-level categorization of Archaeal microbes present in the inoculum and anodic biofilm of MEC.

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dependent on the concentration of acetate. In order to sup- port the hydrogen production by MECs methanogen inhibitors are often used. Even with the use of methanogen inhibitors in the inoculum, at an initial seeding stage, there is a possibility of growth of a methanogenic population in the MEC over long-term operation and hence, intermittent dosing of methanogen inhibitors is recommended for the future operations [15,134]. In addition, scientists at the Uni- versity of Massachusetts, Amherst discovered an interesting phenomenon thatMethanosaetahave the ability to reduce CO2

to CH4 through electrical connections with other microorganisms [135].

The second most abundant Archaea was Methanoculleus with the percentages of 26.2% and 28.4% in the inoculum and anodic biofilm of MEC, respectively. In taxonomy, this genus belongs to the Methanomicrobiaceae family. The species related to the genusMethanoculleusare found in marine environments (brackish water) though these are commonly found in bioreactors and wastewater.Methanoculleusand a few species that grow on ethanol can accept electrons from ethanol to produce methane.

In summary, the microbial community analysis indicated that the microorganisms in MEC anodic biofilm can be func- tionally categorized into two groups such as the EAB (Geo- bacter, Pseudomonas, Klebsiella and Rhodoferax) and the fermentative or complex compounds-degrading hydrolytic bacteria (Clostridium, Bacteroides, Syntrophomonas, Desulfovi- brio). These dominant bacterial communities found in the present study can efficiently degrade the organic content rich POME (cellulose, lignin and residual oil) to produce bio-H2by EET directly or indirectly. The presence and growth of diverse eubacterial and archaeal populations in the anodic biofilm of POME-fed MEC was determined by the highly complex nature of POME wastewater [136]. Typically, high molecular weight complex polymers are not the best substrates for electrogenic bacteria present in the anodic compartment of MECs [118,137].

FirmicutesandBacteroidetespresent on the anode may play a role in metabolizing and breaking down the complex organic materials present in POME into easily degraded simple sub- stance, such as glucose and acetate and other fermentation by-products [138] [45,139,140], which would serve as a suitable substrate for EABs to produce bio-H2. Therefore, the complex syntrophic interactions or the co-existence of various functional microbial communities such as EAB and fermentative bacteria in POME wastewater was crucial for both bio-H2

production and POME wastewater treatment in the PHP-MEC hybrid system.

Implementation of the study, prospective and challenges

Sustainable energy-development systems have been widely accepted to reduce the carbon footprint. In the context of waste management and waste to energy generation, MECs have proven to be an innovative and sustainable system. The major issue of MEC implementation is associated with its external power requirement and a performance loss with time. MECs can be powered by alternative power sources that are not based on the DC power supply for H2 production.

Renewable energy sources have the potential in terms of scalability and meeting the energy requirements of MECs.

Table 3presents a comparative analysis of the current work with the published work employing renewable energy sources. As presented inTable 3, Sun et al. [19] proposed an auto- powered MEC-MFC paired system in which electricity produced by a MFC energized the MEC to yield H2. Similarly, the MFC with a capacitor-generated electricity for H2production via the MEC. This system improved energy recovery (9%e13%) and HPR [25].

Sunlight is a viable renewable energy source that can sustainably produce energy. In earlier conceptions, a dye- sensitized solar cell (DSSC) harvesting renewable energy from Sun energized MECs [26]. It generated enough voltage (0.6 V) in an open circuit system to energize MECs. As a result, the MEC system evolved 0.4 mol H2in 5 h with remarkable cathode recovery efficiency (78%).

Interestingly, a combination of photoatalytic material with a microbially catalyzed bio-anode has been shown to produce H2however H2can be produced only in the presence of Sun- light. These bio-anodes include TiO2[141], MoS3modified p- type Si nanowire [142], p-type Si nanowires [143], and p-type Cu₂O nanoparticles [144]. More recently, MFC-Dark Fermen- tation-MEC [145], the thermoelectric microconverter-MEC [27]

and Al-air battery-MEC [146] coupled systems have been constructed for H2production during wastewater treatment (Table 3).

Besides the above-mentioned power sources, the PHP has demonstrated its potential as being an effective energy source for MECs in the present study. The maximum HPR and Iv of 1.16 ±0.08 m³H2/m³d and 113 ± 6 A/m³, respectively are comparatively higher than the MECs energized by alternative power sources. However, one system (pyrolysis-MEC) [153]

managed to achieve HPR and Iv of 4.3 m³H2/m³d and 202 A/

m³, respectively. This was most possibly because a much smaller MEC reactor (~16 times smaller (0.029 L) was used than our present MEC, which could over-estimate the productivity.

Additionally, the PHP-MEC hybrid system is stable, inexpensive and has a longer life span, which makes it a sustainable technology for energy production. In summary, PHP can sustainably produce renewable energy and the PHP-MEC integrated process holds great potential for the production of bio- H2during wastewater treatment. Integration of MEC with PHP leads to improvements in the complexity of the system and further advancement in research needed to address scaling- up challenges.

A major limitation of the MEC is associated with the low conductivity of wastewater especially low conductivity of domestic wastewater (0.2 S m¹). Scale up of this process could be challenging [155,156]. The addition of phosphate buffer [157], synthetic medium supplemented with acetate [49] and use of 35 g/L NaCl [158] in the medium has resulted in an increase in current density. However, high salts could alter the buffering capacity of the electrolyte especially exacer- bating the acidification of the anodic biofilm. Deposition of wastewater materials and ions on the electrode could inhibit electrode catalysis which decrease the performance of a MEC.

In scale up, the surface area needs to be kept small to avoid the likelihood of clogging and maintenance. Overpotential losses are another stumbling block of a MEC and bubbling of CO2