Supplementary information
Bioelectrochemical treatment of table olive brine processing wastewater for biogas production and phenolic compound removal
A. Maronea, A.A. Carmona-Martíneza, Y. Sireb, E. Meudecc, J.P. Steyera, N. Berneta,*, E. Trablya
a LBE, INRA, 102 Avenue des Etangs, 11100, Narbonne, France;
b INRA, UE999 Unité Expérimentale de Pech-Rouge, 11430, Gruissan, France;
c INRA, UMR1083 Sciences pour l’œnologie, Plateforme Polyphénols, Montpellier, France
* Corresponding author, email:
Table S1. Physico-chemical characteristics of raw TOPW; TOPW plus sediments and MES buffer, pH adjusted to 7 (t0-TOPW); treated TOPW in reactor R3, operated in fed-batch mode at the end of cycle III (with sediments addition) and cycle IV (without sediments addition), respectively tTOPW cycle III and tTOPW cycle IV.
Parameter / Raw TOPW / t0-TOPW / tTOPW cycleIII / tTOPW cycleIVpH / 5.42 / 7.05 / 7.59 / 7.51
Total Solids (g L-1) / 76.7 / 84.47 / 67.59 / 56.86
Volatile Solids (g L-1) / 10.20 / 15.51 / 10.68 / 11.09
COD (g L-1) / 23.82 / 28.80 / 19.60 / 21.15
Total Sugars (g L-1) / 12.03 / 12.03 / 0.00 / 0.00
Acetate (g L-1) / 1.17 / 1.17 / 0.00 / 0.00
Lactate (g L-1) / 2.63 / 2.63 / 0.00 / 0.00
Formate (g L-1) / 0.39 / 0.39 / 0.36 / 0.34
Propionate (g L-1) / 0.01 / 0.01 / 0.53 / 0.598
Ethanol (g L-1) / 0.96 / 0.96 / 0.00 / 0.00
Electrical Conductivity (mS/cm) / 55.3 / 88.20 / 68.30 / 63.90
Phenolic compounds (mg L-1)* / 1894 / 1894.00 / 1104.40 / 1451.15
* Determined with Folin-Ciocalteu method; n.a. = not available.
Analytical procedures for TOPW characterization. Analyses of Total (TS) and Volatile Solids (VS) of TOPW were performed according to standard methods (APHA, 2005). The chemical oxygen demand (COD) analysis was performed using analytical test kits for high chloride content waters (MERCK 117059.0001). Total Kjeldahl Nitrogen content (TKN) was analyzed using a Büchi digestion unit K438 and a Büchi 370-K distillator/titrator. Total phenolic compounds content was determined with Folin-Ciocalteu method (Boizot and Charpentier, 2006). The volatile fatty acids (VFA) composition was determined with a gas chromatograph (GC-3900 Varian) equipped with a flame ionization detector according to Quéméneur et al. (2012). Concentrations of other soluble organic molecules such as glucose, ethanol, lactate, and formate were measured by HPLC analysis and refractometric detection as previously described (Monlau et al., 2012). Ionic content was determined by ion chromatography (ICS 3000, Dionex, USA) as described in Uggetti et al. (2014).
Fig. S1. Bioelectrochemical reactor (R0) containing four planar graphite working electrodes (WE) used to select the most suitable applied potential for treating TOPW using an N-Stat (N) configuration. Each WE was connected to a separate potentiostat channel sharing the same RE and CE. In an N-Stat configuration the multi-channel potentiostat individually controls each anode with respect to a single RE. Applied potentials were +0.2; +0.4; +0.6. +0.8 (V vs. SCE).
Fig. S2. Chronoamperometric batch cycle of electrochemically derived biofilm from the two replicate reactors R1 and R2.
Peak / Retention time (min) / λ max (nm) / [M-H]- / Fragment ion / Neutral loss / IdentificationA / 7.1 / 281 / 153 / 123 / 30 / Hydroxytyrosol
B / 9.5 / 276 / ND / Tyrosol
C / 7.9 / 281 / 167 / 123 / 44 / 3,4-dihydroxyphenyl acetic acid
D / 8.2 / 281-309 / 137 / 3,4-dihydroxybenzaldehyde
E / 10.5 / 274 / ND / p-hydroxyphenyl acetic acid
Fig. S3. UPLC profiles at 280 nm of TOPW at the START and the END of the batch cycle and after further incubation for 1 and 4 days, UV-visible and MS data of the major peaks detected.
Sequencing of bacterial 16s DNA gene indicates the microbial selection that occurred in NStat experiments. The microbial communities of all anodes (except +0.8V, which was unsuccessful to form an effective biofilm) showed closely related CE-SSCP (capillary electrophoresis single-strand conformation polymorphism) profiles with prominent peaks (data not shown). Those peaks belong to the most abundant sequences.
This finding is confirmed by the results obtained from the analysis of 16S ribosomal DNA bacterial gene sequences obtained from microbial communities of anodic biofilms. The microbial communities of biofilms developed at +0.2, +0.4 and +0.6V anode poised potentials (namely R0-N-0.2V, R0-N-0.4V and R0-N-0.6V respectively) were, in average, 99.7 ± 0.2%. On the other side, the similarity between those sequences and the ones obtained at +0.8V (namely R0-N-0.8V) was only 40.5 ± 0.5% in average. Some authors have reported that the microbial community composition seems to be unaffected by the applied anode potential (Zhu et al., 2014). Others have found a major influence of anodic applied potentials on the selection of anode respiring bacteria (ARB) (Commault et al., 2013; Torres et al., 2009). Actually, there is not yet a consensus regarding this question.
The phylum of Proteobacteria was always predominant with > 80% of the 16S rDNA bacterial sequences from the anodic biofilms belonging to the “δ” group, while sequences belonging to the phyla of Bacteroidetes and Firmicutes were found only in minor abundance. The ability of several Deltaproteobacteria to transfer electrons to an electrode material is widely known (Patil et al., 2012). Interestingly, the most abundant ARB species found in all electroactive biofilms belonged to the order of Desulfuromonadales, with the exception of the anode at +0.8V (R0-N-0.8V) applied potential in which most of the sequences (67%) were affiliated to the order of Desulfovibrionales, representative to a unique OTU belonged to Desulfovibrio genus from Desulfovibrionaceae family. All the sequences belonged to Desulfuromonadales order were representative of a unique OTU belonged to the Desulfuromonas genus of Desulfuromonadaceae family (Figure S3, Table 1). The present results are consistent with a previous study of our group showing a high ARB selection and a decrease of diversity from an inoculum of the same origin as the one used in this study (Pierra et al., 2015). In that study, the ARB species found in all electroactive biofilms were closely related to each other and were up to 97% DNA similar to either Desulfuromonas acetoxidans or Geoalkalibacter subterraneus in all reactors. The results obtained in this study showed in all the anodes a strong selection of ARB belonging to the Desulfuromonadales or Desulfovibrionales orders. However, the second group which dominated the microbial community developed at posed potential of +0.8V (R0-N-0.8V) seemed to be less effective in current production (Table 1).
Fig. S4. Microbial community composition (OTUs whit sequences abundance >5%) for anodic biofilm developed in reactor R0 at different potentials (+0.2V; +0.4V; +0.6V and +0.8V vs SCE) and in the sediment of origin.
S3. Methanogens in electroactive biofilms
As showed in figure 2 and figure S3, the fraction of Archaeal 16S rRNA genes was very low (< 0.5%) in all the electroactive biofilms when compared to the ones found in the sediment used as inoculum (11%). Nevertheless, the sequences showed a high variability between samples. Most of the archaea sequences (≥ 60%) found in the samples were affiliated to the phylum Euryarchaeota, followed by sequences affiliated to the phylum Crenarchaeota (Miscellaneous Crenarchaeotic Group). Among the phylum Euryarchaeota, most the sequences belonged to the class of Methanomicrobia, more precisely to two families, Methanomicrobiaceae and Methanosarcinaceae, indicating a strong selection of methane producing microorganisms in all the anodic archaeal communities. For the family of Methanomicrobiaceae, methanogenesis from H2/CO2 or from formate is the major energy-generating process (Rosenberg et al., 2014)
while, the family of Methanosarcinaceae, is known as the most versatile of all families of methanogenic Archaea with respect to the diversity of substrates used for energy generation including H2/CO2 and acetate. However, in a single chamber BES, methane production is more likely related to the hydrogenotrophic pathway. Indeed, in BES low contribution on methane production is expected from acetoclastic methanogens given the fact that their affinity for acetate is lower than for ARB (Ruiz et al., 2015). However, in the present study, due to the low Archaea content of the biofilms, the methanogenic activity was probably mostly attributable to the activity of planktonic Archaea.
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