Inoculum
The microbial inoculum used in this work was a mixed culture issued from a long-term continuous dark fermentation lab-scale reactor operated at pH 6.5 under micro-aerobic conditions for the production of H2 from glycerol [12]. It was stored at 4 °C for 1 month before use.
Fermentation medium
The composition of the fermentation medium (per liter of water) was modified from Dietz et al.'s as follows: 1.66 g glycerol, 1 g NH4Cl, and 0.5 g NaCl for pH-buffered experiments or 23.50 g glycerol, 2.5 g NH4Cl and 1.0 g NaCl for pH-regulated experiments (Sigma-Aldrich, ≥99 %). In all experiments, 20 mL of a trace element solution (1.5 g/L nitrilotriacetic acid; 3.0 g/L MgSO4·7H2O; 0.50 g/L MnSO4·H2O; 1.0 g/L NaCl; 0.10 g/L FeSO4·7H2O; 0.18 g/L CoSO4·7H2O; 0.10 g/L CaCl2·2H2O; 0.18 g/L ZnSO4·7H2O; 0.01 g/L CuSO4·5H2O; 0.02 g/L KAl(SO4)2·12H2O; 0.01 g/L H3BO3; 0.01 g/L Na2MoO4·2H2O; 0.03 g/L NiCl2·6H2O; 0.30 mg/L Na2SeO3·5H2O; 0.40 mg/L Na2WO4·2H2O) and 150 mM phosphate buffer were added.
pH-buffered fermentation set-up
Batch experiments were performed in triplicates in glass bottles containing 200 mL of solution and around 300 mL of headspace. Bottles were sealed with butyl rubber septa and aluminum caps. Initial biomass was obtained after centrifugation of 33 mL of the inoculum (volatile solids = 0.40 ± 0.01 %total mass) at 12,000g for 15 min. The pellet was then suspended in the culture medium. Anoxic conditions were assured just after inoculation by flushing the media with high-purity N2 (>99.995 %) for at least 30 min. The temperature was controlled at 37 °C. Initial pH was adjusted at 4, 5, 6, 7, 8, 9, or 10 using 150 mM phosphate buffer and hydrochloric acid. Final pH values were, respectively, 3.9 ± 0.2, 4.2 ± 0.2, 5.7 ± 0.2, 6.9 ± 0.1, 7.7 ± 0.2, 8.0 ± 0.2, and 9.9 ± 0.2.
pH-regulated fermentation set-up
Glycerol fermentations under pH regulation were conducted in four replicates in glass reactors containing 1 L of solution and about 500 mL of headspace. The temperature was controlled at 37 °C and the pH was regulated at 7.0 by adding 2 M NaOH (pH probe InPro 4260i, Mettler Toledo). Bottles containing pH 7 from the pH-buffered experiments were used as inoculum after storage at 4 °C. Initial biomass was obtained after centrifugation of 100 mL of the inoculum at 12,000g for 15 min. The pellet was then suspended in the culture medium. Anaerobic conditions were assured just after inoculation by flushing the media with high-purity N2 (>99.995 %) for at least 30 min.
Analytical methods
Concentrations of glucose, glycerol, 1,3-PDO, and organic acids were measured by HPLC with a refractive index detector (Waters R410). Samples were first centrifuged at 12,000g for 15 min and then supernatants were filtered with 0.2 µm syringe filters. HPLC analysis was performed at a flow rate of 0.4 mL/min on an Aminex HPX-87H, 300 × 7.8 mm (Bio-Rad) column at a temperature of 35 °C. H2SO4, 4 mM was used as the mobile phase. Biogas composition was determined using a gas chromatograph (Clarus 580, Perkin Elmer) equipped with a thermal conductivity detector. The columns used were a RtQbond column (for H2, O2, N2, and CH4) and a RtMolsieve column (for CO2), and the gas vector was argon at a pressure of 3.5 bar.
The COD balances were established based on the number of electrons per mol of each fermentation product and for microbial biomass, assuming an elemental composition of C4H7O2N [13]. Biomass was estimated from the metabolites produced considering a Y
X/ATP of 10.5 g/mol [14].
Microbial community analysis
DNA was extracted with the QIAamp fast DNA stool mini kit in accordance with the manufacturer’s instructions (Qiagen, Hilden, Germany). Extractions were confirmed using Infinite 200 PRO NanoQuant (Tecan Group Ltd., Männedorf, Switzerland). The V4 and V5 regions of the 16S rRNA genes were amplified using the primers 515F (5′-GTGYCAGCMGCCGCGGTA-3′) and 928R (5′-CCCCGYCAATTCMTTTRAGT-3′), which captures most of the bacterial and archaeal diversity [15]. Adapters were added for multiplexing samples during the second amplification step of the sequencing. The PCR mixtures (50 µl) contained 0.5 U of Pfu Turbo DNA polymerase (Stratagene) with its corresponding buffer, 200 mM of each dNTP, 0.5 mM of each primer, and 10 ng of genomic DNA. Reactions were performed in a Mastercycler thermal cycler (Eppendorf) as follows: 94 °C for 2 min, followed by 35 cycles of 94 °C for 1 min, 65 °C for 1 min, and 72 °C for 1 min, with a final extension at 72 °C for 10 min. The amount and size of PCR products were determined using a Bioanalyzer 2100 (Agilent). A capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) method was used for PCR product's diversity characterization. Samples were heat-denatured at 95 °C for 5 min and re-cooled directly in ice for 5 min. CE-SSCP electrophoresis was performed in an ABI Prism 3130 genetic analyzer (Applied Biosystems) in 50 cm capillary tubes filled with 10 % glycerol, conformation analysis polymer and corresponding buffer (Applied Biosystems). Samples were eluted at 12 kV and 32 °C for 30 min, as described elsewhere [16]. CE-SSCP profiles were aligned with an internal standard (ROX) to consider the inter-sample electrophoretic variability. CE-SSCP profiles were normalized using the StatFingerprints library [17] in R software version 2.9.2 (R. Development Core Team 2010). The community composition was also evaluated using the MiSeq v3 chemistry (Illumina) with 2 × 300 bp paired-end reads at the GenoToul platform (www.genotoul.fr). Sequences were retrieved after demultiplexing, cleaning, and affiliating sequences using mothur [18]. Sequences have been submitted to GenBank with accession No. KT287117–KT288056.
Quantitative PCR (qPCR)
PCRs were prepared using 96-well real-time PCR plates (Eppendorf, Hamburg, Germany) and Mastercycler ep gradient S (Eppendorf, Hamburg, Germany). Then, 6.5 μl of Express qPCR supermix with premixed ROX (Invitrogen, France), 2 μl of DNA extract with three appropriate dilutions, 100 nM forward primer F338-354 (5′-ACTCC TACGG GAGGC AG-3′), 250 nM reverse primers R805-785 (5′-GACTA CCAGG GTATC TAATC C-3′), 50 nM TaqMan probe, and water were added to obtain a final volume of 12.5 μl for all analyses.
An initial incubation of 2 min at 95 °C and 40 cycles of denaturation (95 °C, 7 s; 60 °C, 25 s) were performed. One standard curve was generated from each assay by using tenfold dilutions in sterilized water (Aguettant Laboratory, Lyon, France) of a target plasmid (Eurofins Genomics, Germany). The initial DNA concentrations were quantified using the Infinite 200 PRO NanoQuant (Tecan, France). The average number of bacterial cells was estimated by dividing the average number of 16S rRNA gene copies per cell by a factor 4.1 [19].
Theoretical yield calculations
Metabolic pathways of glycerol fermentation were assumed to be similar as in [20]. In particular, the biochemical routes leading to lactate, acetate, and ethanol without formate production were written as follows:
$${\text{Glycerol}} + {\text{ADP}} + {\text{P}}_{\it{i}} + {\text{NAD}}^{+} \to {\text{Lactate}} + {\text{ATP}} + {\text{H}}_{2} {\text{O}} + {\text{NADH}}_{2}$$
$${\text{Glycerol}} + 2\left( {{\text{ADP}} + {\text{P}}_{{i}} } \right) + 3 {\text{NAD}}^{+} \to {\text{ Acetate}} + \text{{CO}}_{ 2} + 2{\text{ATP}} + {\text{H}}_{ 2} {\text{O}} + 3 {\text{NADH}}_{ 2}$$
$${\text{Glycerol}} + {\text{ADP}} + {\text{P}}_{{i}} +{\text{NAD}}^{+} \to {\text{Ethanol}} + {\text{CO}}_{2} +{\text{ATP}} + {\text{H}}_{ 2} {\text{O}} + {\text{NADH}}_{ 2}$$
$${\text{Glycerol }} + {\text{ NADH}}_{ 2} \to \, 1,3{\text{-}}{\text{propanediol }}+ {\text{NAD}}^{ + } +{\text{ H}}_{ 2} {\text{O}}.$$
The conversion of formate into hydrogen was assumed as follows:
$${\text{Formate}} + {\text{H}}_{ 2} {\text{O}}_{{}} \to {\text{ HCO}}_{ 3}^{-} +{\text{ H}}_{ 2}$$
The elemental constitution of biomass was assumed to be C4H7O2N with a biomass production yield of 10.5 g/molATP [14], leading to the following equation:
$$4_{{}} {\text{Glycerol}} + 3 {\text{NH}}_{3} + 30{\text{ATP}}+24 {\text{H}}_{ 2} {\text{O}} + 4 {\text{NAD}}^{ + }_{{}} \to_{{}} 3 {\text{ C}}_{ 4} {\text{H}}_{ 7} {\text{O}}_{ 2} {\text{N}} + 4 {\text{NADH}}_{ 2} +30 \left( {{\text{ADP}} + {\text{P}}_{{i}} } \right)$$
Pearson correlation matrix
A Pearson correlation matrix was calculated from metabolite profiles after 3 days of fermentation (n = 15) and the bacterial community composition obtained after sequencing (n = 5, only one per triplicate). The correlation and significance calculations were made with the R 3.1.3 software (R Development Core Team 2010) and the function “rcorr” of the package Hmisc. The hierarchical clustering was made with the function “corrplot” of the package corrplot using the centroid method.
Principal component analysis (PCA)
In order to analyze and compare the microbial consortia, a principal component analysis (PCA) was performed on the microbial community compositions obtained from CE–SSCP with the R 2.12 software (R Development Core Team 2010), the vegan 2.12.2 package.