Edited by: Sebastià Puig, University of Girona, Spain
Reviewed by: Ioannis Andrea Ieropoulos, University of the West of England, United Kingdom; Matteo Grattieri, University of Utah, United States
This article was submitted to Bioenergy and Biofuels, a section of the journal Frontiers in Energy Research
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Domestic and industrial wastewaters contain organic substrates and nutrients that can be recovered instead of being dissipated by emerging efficient technologies. The aim of this study was to promote bio-hydrogen production and carbon fixation using a mixed culture of purple phototrophic bacteria (PPB) that use infrared radiation in presence or absence of an electrode as electron donor. In order to evaluate the hydrogen production under electrode-free conditions, batch experiments were conducted using different nitrogen (NH4Cl, Na-glutamate, N2 gas) and carbon sources (malic-, butyric-, acetic- acids) under various COD:N ratios. Results suggested that the efficiency of PPB to produce biogenic H2 was highly dependent on the substrates used. The maximum hydrogen production (H2_max, 423 mLH2/L) and production rate (H2_rate, 2.71 mLH2/Lh) were achieved using malic acid and Na-glutamate at a COD:N ratio of 100:15. Under these optimum conditions, a significant fixation of nitrogen in form of single-cell proteins (874.4 mg/L) was also detected. Under bio-electrochemical conditions using a H-cell bio-electrochemical device, the PPB were grown planktonic in the bio-cathode chamber with the optimum substrate ratio of malic acid and Na-glutamate. A redox potential of −0.5 V (vs. Ag/AgCl) under bio-electrochemical conditions produced comparable amounts of bio-hydrogen but significantly negligible traces of CO2 as compared to the biological system (11.8 mLCO2/L). This suggests that PPB can interact with the cathode to extract electrons for further CO2 re-fixation (coming from the Krebs cycle) into the Calvin cycle, thereby improving the C usage. It has also been observed during cyclic voltammograms that a redox potential of −0.8 V favors considerably the electrons consumption by the PPB culture, suggesting that the PPB can use these electrons to increase the biohydrogen production. These results are expected to prove the feasibility of stimulating PPB through bio-electrochemical processes in the production of H2 from wastewater resources, which is a field of special novelty and still unexplored.
Typical wastewater systems entail the dissipation of the contamination. However, the high content of organics and nutrients in industrial and domestic wastewaters is a valuable resource for energy and products recovery (Puyol et al.,
Among the competing technologies, the biological accumulation of nutrients and their subsequent recovery, has received great attention as an environmental friendly and certainly cost-effective process (Batstone et al.,
PPB are extremely versatile organisms due to their complex metabolic system, involving major C, N, S, P, and Fe pathways, which absorbs the IR energy through their photosystem, composed by carotenoids and bacteriochlorophyls (Hunter,
In this sense, PPB can be used for the extraction of high value-added products from waste sources, such as biofuels like bio-hydrogen, bioplastics as PHA and single-cell proteins. The metabolic pathways to obtain the valuable bioproducts are catalyzed by variant enzymes (McKinlay and Harwood,
Finally, the internal electron recycling of PPB is a key issue, and an active modification of the electronic fluxes by means of artificial addition of electrons could drive toward different targeted bioproducts (Varfolomeyev,
Based on the above-mentioned grounds, the aim of the present work was the assessment of PPB to enhance the formation of valuable bioproducts, such as biohydrogen, using electric and light energy as the driving forces. This was accomplished by identifying the biological and electrochemical conditions that influence the process of bio-hydrogen production from PPB. The wise use of electric energy to decontaminate wastewater and to produce bio-hydrogen is undoubtedly an attractive and novel challenge, yielding substantial ecological and economic benefits.
All the chemicals compounds used were purchased from Sigma-Aldrich. The organic compounds that were used were: L-malic acid (C4H6O5), butyric acid (C4H8O2), acetic acid (C2H4O2), propionic acid (C3H6O2) and ethanol (C2H6O). Stock solutions of individual organic compounds (20 gCOD/L) were prepared in ultra-pure water and stored at 4°C. The nitrogen sources used were: ammonium chloride (NH4Cl) as inorganic N-source, L-glutamic acid monosodium salt monohydrate (Na-glutamate, C5H8NNaO4·H2O) as organic N-source and nitrogen gas (N2) as external gaseous source. Stock solutions of both organic and inorganic nitrogen sources (5 gN/L) were prepared in ultra-pure water and stored at 4°C.
Finally, macro- and micro-nutrient solutions were prepared following the recipe proposed by Ormerod et al. (
All experimental tests were inoculated with a mixed culture of PPB. These bacteria were enriched from a wastewater influent taken from the pilot-scale WWTP located at the Rey Juan Carlos University (Mostoles, Madrid, Spain). Enrichment was performed by inoculating a 1 L suspended growth reactor (SGR) with sludge liquor, and subsequent incubation under near infra-red (NIR) light illumination and anaerobic conditions using a synthetic wastewater (SW) as growth medium. The SW (prepared with tap water) contained the 5 different organic carbon sources (acetic acid, malic acid, propionic acid, butyric acid and ethanol) with a total COD concentration of 2 gCOD/L, 0.26 gN/L as NH4Cl and 1 and 100 mL/L of micro- and macro-nutrient solutions, respectively. After the addition of SW the bioreactor liquor was flushed with argon gas in order to remove any presence of oxygen. The bioreactor was illuminated with LED lamps (850 nm) as IR light source. The reactor's surface was covered with UV-VIS absorbing foil (ND 1.2 299, Transformation Tubes, Banstead, UK). The foil absorbed around 90% of the wavelength below 750 nm. The average light intensity measured on the outside reactor's surface was 13 W/m2. The PPB mixed culture was continuously stirred and incubated at room temperature (25 ± 1°C). The liquor of the reactor was refreshed every week with fresh SW (99% volume exchange) to achieve final concentrations of 2 gCOD/L and 0.26 gNH4-N/L. The pH was weekly adjusted to 6.8 ± 0.1. The enrichment of PPB was evaluated by the detection of Bacteriochlorophylls (
The ability of the PPB enriched culture to produce bio-hydrogen using different carbon and nitrogen sources was evaluated in batch assays. Initially, the capacity of the PPB culture to produce hydrogen using different organic and inorganic nitrogen sources was examined. The first set of experiments were conducted by using 2 gCOD/L of L-malic acid as the carbon source. Malic acid was chosen as a suitable carbon source that could favor hydrogen production by PPB (Assawamongkholsiri and Reungsang,
Experimental runs of PPB biological experiments under different nitrogen and carbon sources.
1 | Malic acid | 2,000 | NH4Cl | 75 | 100:3.75 |
2 | Malic acid | NH4Cl | 150 | 100:7.5 | |
3 | Malic acid | NH4Cl | 300 | 100:15 | |
4 | Malic acid | 2,000 | Na-glutamate | 75 | 100:3.75 |
5 | Malic acid | Na-glutamate | 150 | 100:7.5 | |
6 | Malic acid | Na-glutamate | 300 | 100:15 | |
7 | Malic acid | 2,000 | N2 gas | 8.8 |
– |
8 | Butyric acid | 2,000 | Na-glutamate | 300 | 100:15 |
9 | Acetic acid | 2,000 | Na-glutamate | 300 | 100:15 |
All the experiments were conducted in 160 mL serum bottles with a working volume of 100 mL. The reactors contained 99 mL of SW medium (prepared as described above) with the corresponding COD and N contents and were inoculated with PPB enriched culture (1% v/v inoculum). The initial pH of the medium was adjusted to 6.8 ± 0.1 using NaOH or H2SO4. The liquid medium of each reactor was flushed with argon for 10 min. Thereafter, the bottles were closed with rubber stoppers and capped with aluminum seals. Subsequently, the headspace of the reactors was flushed again with argon for 2 min except from the reactors where nitrogen gas was used as nitrogen source that were flushed with N2 gas. The bottles were continuously shaken horizontally at 120 rpm at 25 ± 1°C (Orbital shaker, optic ivymen system) and illuminated at an average light intensity of 20 W/m2 using LED lamps for 7 days. The performance for H2 production using identical conditions but without PPB enriched culture was studied by conducting control experiments under sterilized conditions (all the glassware and media used were autoclaved). During these control experiments, no biomass growth as well as no H2 production or acid assimilation were detected. Both the liquid and the gas media were sampled periodically to evaluate, the carbon and nitrogen assimilation, the PPB growth and the hydrogen production. All the experiments were conducted in duplicate.
Bio-electrochemical experiments were performed in an H-cell device as shown in Figure
Experimental set-up of the foto-bio-electrochemical H-cell device.
As shown in Figure
Experiments were conducted by setting the potential of bio-cathode at −0.5 V in order to force the PPB culture to be adapted to the electrochemical conditions. The reaction period among the PPB culture and the bio-cathode was chosen to be 1 week, similar to the biological experiments. Control electrochemical (abiotic) experiments were conducted using the same experimental conditions without PPB biomass. In order to determine whether the PPB culture interacted with the cathode or not by means of electron acceptance from PPB, cyclic voltametry (CV) in the range of −0.8 to 0.8 V was performed during the weekly reaction process.
All parameters except total chemical oxygen demand (COD) and total Kjeldahl Nitrogen (TKN) were determined after filtering with a 0.45 μm nylon filter (Chrodisc filter/syringe, CHMLab, Barcelona, Spain). Total and soluble COD were determined using a dichromate-reflux colorimetric method (APHA,
The following section include all results generated after exploring the physiology of PPB for selecting those culture conditions, including nitrogen and carbon sources, for achieving an optimal conversion of an extracellular source of electrons into hydrogen production and CO2 fixation.
The enrichment process was performed aiming to enhance the growth and acclimation of a mixed PPB culture from domestic wastewater, using a specific environment of NIR radiation. The organic mixture used for the enrichment was chosen on the basis that PPB can efficiently produce hydrogen from wastes that contain mixed VFAs (Wu et al.,
Figure
Performance of PPB culture during a weekly operating cycle:
As shown in Figure
Enriched PPB biomass was used for inoculum purposes in order to study the hydrogen formation from wastewater in presence and absence of an electrode as electron donor.
Hydrogen production under nitrogen fixation conditions is described by Equation (1) where molecular nitrogen (N2) is converted to ammonia (NH3) and protons (H+) to hydrogen (H2) (Rey et al.,
Biological experiments were conducted in order to extract the optimum biological conditions to maximize hydrogen production while minimizing CO2 emission. Our first approach was to analysis how biohydrogen production depended on nitrogen substrate at different concentrations by using three different N sources (ammonium, glutamate and nitrogen gas) and malic acid as a model substrate of organic carbon. Interestingly, glutamate increased PPB growth rate by 2-fold in comparison with ammonium or nitrogen gas (see Figure
Comparison of H2 production under different nitrogen and carbon sources.
1 | Malic/NH4Cl | 100:3.75 | 3.67 ± 0.82 | 0.21 ± 0.05 | 451.0 ± 2.1 | 2.63 ± 0.13 | 0.13 ± 0.01 | 0.70 ± 0.04 |
2 | Malic/NH4Cl | 100:7.5 | 4.74 ± 0.84 | 0.19 ± 0.04 | 2.2 ± 2.3 | (1.35 ± 1.1) × 10−2 | (0.59 ± 0.58) × 10−3 | (3.15 ± 3.18) × 10−3 |
3 | Malic/NH4Cl | 100:15 | 5.47 ± 1.01 | 0.20 ± 0.04 | 13.7 ± 14.5 | (1.30 ± 0.7) × 10−2 | (0.50 ± 0.63) × 10−3 | (2.55 ± 2.76) × 10−3 |
4 | Malic/Na-glutamate | 100:3.75 | 3.39 ± 0.97 | 0.21 ± 0.06 | 300.2 ± 85.0 | 2.06 ± 0.90 | (8.75 ± 2.89) × 10−2 | 0.47 ± 0.16 |
5 | Malic/Na-glutamate | 100:7.5 | 6.26 ± 2.07 | 0.20 ± 0.06 | 416.1 ± 148.2 | 2.57 ± 1.03 | 0.12 ± 0.05 | 0.66 ± 0.27 |
6 | Malic/Na-glutamate | 100:15 | 7.56 ± 1.89 | 0.19 ± 0.04 | 423.0 ± 40.9 | 2.71 ± 0.27 | 0.12 ± 0.01 | 0.67 ± 0.05 |
7 | Malic/N2 gas | – | 5.57 ± 1.68 | 0.21 ± 0.04 | 12.2 ± 11.3 | 0.12 ± 0.05 | (0.36 ± 0.39) × 10−2 | (1.97 ± 2.12) × 10−2 |
8 | Butyric/Na-glutamate | 100:15 | 3.23 ± 0.18 | 0.06 ± 0.01 | 214.2 ± 7.2 | 1.21 ± 0.12 | 0.22 ± 0.02 | 0.79 ± 0.08 |
9 | Acetic/Na-glutamate | 100:15 | 3.96 ± 0.09 | 0.16 ± 0.0 | 320.4 ± 82.5 | 2.49 ± 0.36 | 0.21 ± 0.05 | 0.50 ± 0.13 |
10 | Bio-electrochemical Malic/Na-glutamate |
100:15 | 5.91 | 0.17 | 390 | 2.32 | 0.11 | 0.60 |
Biohydrogen analysis revealed an interesting correlation of hydrogen with the ratio COD:N. So, hydrogen production was enhanced (451 ± 2.1 mLH2/L) when NH4Cl was used as inorganic nitrogen at a COD:N ratio of 100:3.75. In contrast, very low amount of hydrogen was produced when higher concentrations of NH4Cl (COD:N of 100:7.5 and 100:15) were tested (Figure
Hydrogen
However, it was observed that the use of N2 gas as nitrogen source did not efficiently produce H2 (Figure
Finally, the results indicated that PPB culture produced large amounts of hydrogen when Na-glutamate was used as an organic nitrogen source (300–423 mLH2/L, Table
Considering Na-glutamate concentration, results showed that H2 production (mLH2/L) as well as its production rate was increased as the organic nitrogen concentration increased, achieving a maximum H2 production at a COD:N ratio of 100:15 (Figure
It should be highlighted that reducing the NH4Cl, levels (100:3.75 ratio) resulted in a high hydrogen production (Figure
Considering the importance of the internal electron recycling, an active modification of the electron fluxes through artificial addition of electrons by applying electrochemical technology may potentially enhance the PPB activity and drive toward an optimum H2 production process. In this sense, the bio-electrochemical capability of interaction between PPB and graphite-electrode has been explored, specially emphasizing the situation when graphite electrode behaves as an electron donor to PPB (setting graphite-electrode potential at −0.5 V) aiming to increase PPB metabolic paths activity by supplying electric current. Therefore, this study focused on the analysis of a bio-electrochemical device based on PPB, using malic acid as organic source and Na-glutamate as nitrogen source, compared to electrode-free biological systems.
Figure
Cyclic voltammograms at different time intervals during bio-electrochemical (red line) and control electrochemical operation (blue line).
After 48 h of polarizing the electrode at −0.5 V (vs. Ag/AgCl), the cyclic voltammograms revealed the electroactivity of the PPB biofilm interacting with the graphite-electrode surface. These results suggested that PPB started to interact with the working electrode when sufficient amount of biomass (0.1 gVSS/L) and malic acid as carbon source were present in the cathode chamber (Figure
Culture of PPB under bio-electrochemical conditions at −0.5 V (vs. Ag/AgCl)
The biological production of hydrogen by PPB was studied by testing different organic carbon sources, as malic acid, butyric acid and acetic acid, using Na-glutamate as optimum nitrogen source at a COD:N ratio of 100:15. Results indicated that the PPB culture was able to assimilate all the organic acids tested toward biomass growth as well as hydrogen production. Maximum PPB growth rate (7.56 mgVSS/Lh) was obtained when malic acid was used as compared to butyric (3.23 mgVSS/Lh) and acetic acid (3.96 mgVSS/Lh; Table
The experimental results obtained from the biological study (electrode-free) of hydrogen production indicated that the combination of malic acid and Na-glutamate was the optimum for maximizing the hydrogen production by PPB. The efficiency of H2 production from the PPB mixed culture enriched in this study is comparable to those of previous studies where pure or mixed PPB cultures were used (Table
Comparison of hydrogen production rates by different cultures and systems with the one studied in the present work.
60 W/m2 | Batch | Succinate/glutamate | – | 21 4.3 | – | Melnicki et al., |
|
100 W | Batch | Malate/glutamate Malate/NH4Cl Acetate/NH4Cl | 0.541 0.224–0 0.467–0.135 | 5.1 4.6–0 5.8–3.3 | – | Akkose et al., |
|
5 klux |
Continuous | Mixture of VFAs |
0.185 | 1.125 | – | Ozmihci and Kargi, |
|
2,400 lux |
Batch | Acetate/nitrate Malate/nitrate Succinate/nitrate Succinate/N2 Succinate/NH4C | – | 2.7 2.5 3.3 0.5 1.25 | – | Merugu et al., |
|
Mixed culture–dominant |
190 W/m2 | Continuous | Mixture |
0.97 | 121 | – | Tawfik et al., |
10 W/m2 | Batch | Succinate/(NH4)2SO4 | – | 31 | – | Ryu et al., |
|
2,500 lux |
Batch | Malate/glutamate | – | 6.8 | – | Assawamongkholsiri and Reungsang, |
|
2,000 lux |
Batch | Lactate/glutamate Butyrate/glutamate | – | 8.4 19.9 | 2.57 4.92 | Hu et al., |
|
Mixed culture | 20 W/m2 | Batch | Malic acid/glutamate | 0.12 | 2.71 | 0.67 | This study |
Results suggested that bio-electrochemical process of PPB resulted to similar H2 production rate and hydrogen yields (Table
Current evolution during bio-electrochemical and abiotic electrochemical reactions.
Figure
It is well-reported that graphite electrodes, and generally carbon electrodes, exhibit a high overpotential for hydrogen evolution and carbon dioxide reduction (Sullivan et al.,
In contrast, at −0.5 V, the capability of hydrogen production of the bio-electrochemical system was comparable to the exhibited by PPB in absence of electrode, and no electrode potential was observed in this process. The origin of the second bio-electrochemical process developed below −0.6 V, that can be tentatively assigned to hydrogen production, will require further investigation beyond the scope of this work. The capability of PPB for using graphite-electrode as electron donor was demonstrated. The extra electron donor source can be used in more than one metabolic pathway. Polarizing the electrode at −0.5 V, allows PPB to use electrode for carbon fixation reaching almost no carbon dioxide accumulation in contrast to the electrode-free biological system. This is the first study indicating that electroactive capture of CO2 by PPB is feasible.
Finally, the SCP production achieved by PPB during the bio-electrochemical process (81% mgSCP/mgVSS) was similar to this observed by PPB growing in absence of electrodes. Therefore, the bio-electrochemical process did not seem to affect proteins yields under the experimental conditions tested.
This work analyzed the optimum culturing conditions for maximizing the hydrogen production by a mixed culture of purple phototrophic bacteria. In addition, the effect of a negatively polarized bio-electrochemical device on the modification of the behavior of the culture in terms of metabolic shifts and current consumption was explored. The main conclusions extracted from this work are shown below:
- Among all the conditions tested in absence of electrodes, best results on the hydrogen production have been achieved by using malic acid as a carbon source (instead of acetic and butyric) and Na-glutamate as a N source (instead of ammonium and dinitrogen gas), in a COD/N relationship of 100/15. Under these conditions, the production of CO2 was also minimized. - Cyclic voltammograms of the bio-electrochemical system shown the appearing of at least three potentials (two negative and one positive) with clear interaction between the PPB culture and the electrode. This makes evident the high electroactivity of PPB cultures and their potential as a MET microbial candidate. - Negative polarization of the electrode at −0.5 V caused a detectable consumption of electrons associated with a depletion of the produced carbon dioxide, which indicates that the PPB culture was capable of using electrons from the cathode to capture the excess of C released as CO2 during the CBB cycle. This behavior was not observed before in an indigenous (non-genetically-modified) PPB culture. - Results presented herein have shown that further in-depth research using different conditions (other polarization of the cathode) will be of extreme benefit and may enhance the H2 production rate.
IV designed and performed the experiments and wrote the manuscript, AB critically reviewed the manuscript, CM helped in the experimental stage, JM critically reviewed the manuscript, FM critically reviewed the manuscript and supervised the work, AE-N and DP designed the experiments, supervised the work, and corrected the manuscript. Both DP and AE-N are corresponding authors.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
IV thanks the International Excellence Campus Smart Energy Program (CEISEP) for a Post-doctoral Fellowship. Financial support of Regional Government of Madrid provided through project REMTAVARES S2013/MAE-2716 and the European Social Fund as well as Spanish Ministry of Economy are acknowledged.
The Supplementary Material for this article can be found online at: