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Biohydrogen production beyond the Thauer limit by precision design of artificial microbial consortia

Microorganisms and medium composition

C. acetobutylicum DSM 792 and E. aerogenes DSM 30053 were used for all experiments. A modified Clostridium-specific medium without yeast extract was used for growth of mono-culture C. acetobutylicum as previously described in detail elsewhere71. The medium was prepared containing (per L): 0.5 g of KH2PO4, 0.5 g of K2HPO4 and 2.2 g of NH4CH3COO and glucose or cellobiose were added at a concentration of 999 C-mmol. The pH was arranged with 1 mol L1 NaOH to 6.8. Trace elements solution was prepared as stock 100× solution containing (per L): 0.2 g of MgSO4·7 H2O, 0.01 g of MnSO4·7H2O, 0.01 g of FeSO4·7H2O, 0.01 g of NaCl. Vitamin solution was prepared as stock 200× solution containing (per L): 0.9 g of thiamine, 0.002 g of biotin and 0.2 g of 4-aminobenzoic acid. The trace elements solution and the vitamin solution were used for all experiments. Mono-culture of E. aerogenes was grown in a defined Enterobacter-specific medium, as described elsewhere72. The Enterobacter-specific medium was prepared containing (per L): 13.3 g K2HPO4, 4 g (NH4)2HPO4, 8 mg EDTA and trace elements (2.5 mg CoCl2·6H2O, 15 mg MnCl2·4H2O, 1.5 g CuCl2·4H2O; 3 mg H3BO3; 2.5 mg Na2MoO4·2H2O, 13 mg of Zn(CH3COO)2·2H2O). Glucose and cellobiose were prepared as stock solutions. Media, trace element solution, glucose and cellobiose solutions were flushed with sterile N2 to make the solutions anaerobic and sterilized separately at 121 °C for 20 min. Sterile anaerobic solutions of glucose or cellobiose, trace elements solution and filter sterilized vitamin solution were added into the media before the inoculation inside the sterilized biological safety cabinet (BH-EN 2005, Faster Srl, Ferrara, Italy).

Design of experiments

A mutual medium accommodating the nutritional requirements of both organisms was designed by using the DoE approach. The buffer compositions of two species specific media described above were analysed and the optimum concentrations of AC (NH4Cl), SA (Na+ acetate) and PB (KH2PO4/K2HPO4) capacity were investigated. The setting of DoE for concentration effect of AC, SA and PB capacity was based on 29 randomized runs within concentration range from 3–30 mmol L−1 of AC, 3–150 mmol L−1 of KH2PO4 and 10–120 mmol L−1 of SA (Table 1). Each experiment was performed in triplicates (n = 3), except for set E of the DoE experiment (centre points), which were performed in pentaplicate (n = 5). The DoE experiments were performed twice (N = 2). The end of the exponential growth phase of E. aerogenes and C. acetobutylicum was reached at 45 and 51.5 h, respectively. For modelling, these time points were used. The reason for providing an acetate source in the medium was due to the possibility to add an acetate oxidizing microorganism to the co-culture consortium, which was not performed in the context of this study.

Closed batch cultivations

Cultures of E. aerogenes and C. acetobutylicum were grown anaerobically at 0.3 bar in a 100 Vol.-% N2 atmosphere in a closed batch set-up33. Mono-culture and consortium closed batch experiments were conducted with the final volume of 50 mL medium in 120 mL serum bottles (Ochs Glasgerätebau, Langerwehe, Germany). Each serum bottle contained 45 mL Clostridium-specific medium, Enterobacter-specific medium or E-medium, 0.25 mL vitamin solution, 3.0 mL glucose or cellobiose stock solution, 0.5 mL trace elements solution and 1.25 mL inoculum. The serum bottles were sealed with rubber stoppers (20 mm butyl ruber, Chemglass Life Science LLC, Vineland, USA). For consortium experiments, different inoculum ratios were tested and initial cell concentrations were arranged with the ratios of (E. aerogenes : C. acetobutylicum) 1:2, 1:10, 1:100, 1:1000, 1:10,000 and 1:100,000 at a temperature of 37 °C. Pre-culture of E. aerogenes was diluted in DoE E-medium (Table 1) to inoculate the organism at cell densities of aforementioned ratios. The pressure in the headspace of the serum bottles were measured individually using a manometer (digital manometer LEO1-Ei,−1…3 bar, Keller, Germany). After each measurement, the pressure was released completely from the headspace of serum bottle by penetrating the butyl rubber stopper with a sterile needle. The pressure values were added up to reveal total produced pressure (cumulative pressure). Experiments were performed three times (N = 3) and each set was performed in quadruplicates (n = 4).

Cell counting, absorption measurements, DNA extraction and qPCR

A volume of 1 mL of liquid sample was collected by using sterile syringes at regular intervals for monitoring biomass growth by measuring the absorbance (optical density at 600 nm (OD600)) using a spectrophotometer (Beckman Coulter Fullerton, CA, USA). Every sampling operation was done inside the sterilized biological safety cabinet (BH-EN 2005, Faster Srl, Ferrara, Italy).

E. aerogenes and C. acetobutylicum cells were counted using a Nikon Eclipse 50i microscope (Nikon, Amsterdam, Netherlands) at each liquid/biomass sampling point. The samples for cell count were taken from each individual closed batch run using syringes (Soft-Ject, Henke Sass Wolf, Tuttlingen, Germany) and hypodermic needles (Sterican size 14, B. Braun, Melsungen, Germany). Ten microlitres of sample were applied onto a Neubauer improved cell counting chamber (Superior Marienfeld, Lauda-Königshofen, Germany) with a grid depth of 0.1 mm.

DNA for qPCR was extracted from 1 mL culture samples by centrifugation at 4 °C and 13,400 r.p.m. for 30 min. The following steps were applied for DNA extraction; (1) cells were resuspended in pre-warmed (65 °C) 1% sodium dodecyl sulfate (SDS) extraction buffer and (2) transferred to Lysing Matrix E tubes (MP Biomedicals, Santa Ana, CA, USA) containing an equal volume of phenol/chloroform/isoamylalcohol (25:24:1). (3) Cell lysis was performed in a FastPrep-24 (MP Biomedicals, NY, USA) device with speed setting 4 for 30 s and the lysate was centrifuged at 13,400 r.p.m. for 10 min. (4) An equal volume of chloroform/isoamylalcohol (24:1) was added to the supernatant of the lysate, followed by centrifugation at 13,400 r.p.m. for 10 min and collection of the aqueous phase. (5) Nucleic acids were precipitated with double volume of polyethylenglycol (PEG) solution (30% PEG, 1.6 mol L−1 NaCl) and 1 μL glycogen (20 mg mL−1) as carrier, incubated for 2 h at room temperature. (6) Following centrifugation at 13,400 r.p.m. for 1 h, nucleic acid pellets were washed with 1 mL cold 70% ethanol, dried at 30 °C using a SpeedVac centrifuge (Thermo Scientific, Dreieich, Germany), eluted in Tris-EDTA buffer and stored at −20 °C until further analysis. Nucleic acid quantification was performed with NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). qPCR assays were developed for quantifying E. aerogenes and C. acetobutylicum in consortium. The primer pairs were designed by targeting species specific genes (Supplementary Table 6) to prevent false-positive amplification and sequences of genes were compared for identifying optimal primer using the ClustalW2 multiple sequence alignment programme (http://www.ebi.ac.uk/Tools/clustalw2/). qPCR assays were performed in Eppendorf Mastercycler epgradientS realplex2 (Eppendorf, Hamburg, Germany). The PCR mixture (20 μL) contained 10 μL SYBR Green labelled Luna Universal qPCR Master Mix (M3003L, New England Biolabs), 0.5 μL of forward and 0.5 μL reverse primer, 8 μL sterile DEPC water and 1 μL of DNA template. Negative controls containing sterile diethyl pyrocarbonate (DEPC) water as a replacement for the DNA templates and DNA template of the non-targeted species were included separately in each run. The amplification protocol started with an initial denaturation at 95 °C for 2 min, followed by 45 cycles of denaturation at 95 °C for 30 s, annealing and fluorescence acquisition at 60 °C for 30 s and elongation at 72 °C for 30 s. A melting-curve analysis (from 60 °C to 95 °C at a transition rate of 1 °C every 10 s) was performed to determine the specificity of the amplification. All amplification reactions were performed in triplicates. A standard curve was generated as described elsewhere29. Culture samples of each organism were collected at different time intervals for cell count and genomic DNA extraction cell density of each strain were determined by cell counting under microscope during growth and subsequent gDNA extraction was applied to reflect absolute quantification. Six tenfold dilution standards were prepared and a linear regression analysis was performed between qPCR reads and cell counts and OD600 measurements.

Quantification of gas composition

Gas chromatography (GC) measurements were performed from serum bottles that remained without any manipulation after inoculation until the first time point GC measurement. After every GC measurement, remaining gas was released completely from the serum bottles by penetrating the butyl rubber stopper using a sterile needle. The pressure of serum bottles headspace was determined to examine whether there was any remaining overpressure by using a manometer (digital manometer LEO1-Ei,−1…3 bar, Keller, Germany). The gas compositions were analysed by using a GC (7890 A GC System, Agilent Technologies, Santa Clara, USA) with a 19808 Shin Carbon ST Micropacked Column (Restek GmbH, Bad Homburg, Germany) and provided with a gas injection and control unit (Joint Analytical System GmbH, Moers, Germany) as described before73,74,75. The standard test gas employed in GC comprised the following composition: 0.01 Vol.-% CH4; 0.08 Vol.-% CO2 in N2 (Messer GmbH, Wien, Austria). All chemicals were of highest grade available. H2, CO2, N2, 20 Vol.-% H2 in CO2 and 20 Vol.-% CO2 in N2 were of test gas quality (Air Liquide, Schwechat, Austria).

Quantification of liquid metabolites

Quantification of sugars, volatile fatty acids and alcohols were performed with high-performance liquid chromatography (HPLC) system (Agilent 1100), consisting of a G1310A isocratic pump, a G1313A ALS autosampler, a Transgenomic ICSep ICE-ION-300 column, a G1316A column thermostat set at 45 °C and a G1362A RID refractive index detector, measuring at 45 °C (all modules were from Agilent 1100 (Agilent Technologies, CA, USA). The measurement was performed with 0.005 mol L−1 H2SO4 as solvent, with a flow rate of 0.325 mL min−1 and a pressure of 48–49 bar. The injection volume was 40 µL.

Data analysis

For the quantitative analysis, the maximum specific growth rate (µmax [h−1]) and mean specific growth rate (µmean [h−1]) were calculated as follows: N = N0·eµt with N, cell number [cells ml−1]; N0, initial cell number [cells ml−1]; t, time [h] and e, Euler’s number. According to the delta cell counts in between sample points, µ was assessed. The Y(H2/S) [mol mol−1], HER [mmol L−1 h−1], CER [mmol L−1 h−1] and the specific H2 production rate (qH2) [mmol g−1 h−1]32 were calculated from the intervals between each time point and the gas composition in the headspace of serum bottle was determined using the GC. The elementary composition of the corresponding biomass59 was used for the calculation of the mean molar weight, carbon balance and the DoR balance. Yields of byproducts were determined after HPLC measurement. Values were normalized according to the zero control. Moreover, the Shannon diversity index (H) was calculated to interpret the changes in microbial diversity, accounting for both richness (S), the number of species present and abundance of different species. Relative abundance of two species was evaluated according to the calculated evenness (EH) values76. Global substrate uptake rate, byproduct production rates and the mass balance analyses of the mono-cultures and consortium on glucose and cellobiose were calculated between the first and last time point.

Fluorescence in situ hybridization

For FISH, samples of 2 mL were collected for cell fixation. The samples were centrifuged in micro-centrifuge (5415-R, Eppendorf, Hamburg, Germany) for 10 min at 13,200 r.p.m. and pellets were resuspended in 0.5 mL phosphate-buffered saline (PBS) (10 mmol L−1 of Na2HPO4/NaH2PO, 130 mmol L−1 of NaCl, pH of 7.2–7.4). After repeating this procedure twice, 0.5 mL ice-cold absolute ethanol was added to the 0.5 mL PBS/cell mixture. The ethanol fixed samples were thoroughly mixed and then stored at −20 °C. Poly-l-lysine solution (0.01 % (v/v)) was used for coating the microscope slides (76 × 26 × 1 mm, Marienfeld-Superior, Lauda-Königshofen, Germany) containing ten reaction wells separated by an epoxy layer. After dipping the slide into the solution for 5 min, residual poly-l-lysine from the slides was removed by draining the well, followed by air-drying for several minutes. Cells were immobilized on prepared slides by adding samples (1–10 µL) on each well and air-drying. For cell dehydration, the slides were impregnated with ethanol concentrations of 50% (v/v), 80% (v/v) and 96% (v/v), respectively. The slides were dipped into each solution for 3 min, starting from the lowest concentration.

The EUB338 probe77 was used to target specific 16S rRNA found in almost all organisms belonging to the domain of bacteria78. The GAM42a probe specifically binds to target regions of gammaproteobacterial 23S rRNA79 (Supplementary Table 7). Both probes were diluted with DEPC water to a certain extent depending on the fluorescence label. Cy3-labelled EUB338 was diluted to a probe concentration of 30 ng DNA μL−1, whereas FLUOS-labelled GAM42a was adjusted to a final concentration of 50 ng DNA μL−1. For hybridization of the probe, 20 µL of hybridization buffer (900 mmol L−1 NaCl, 20 mmol L−1 Tris/HCl, 30% formamide (v/v), 0.01% SDS (v/v)) and 2 µL of diluted probe solution were added into each well. The hybridization reaction (46 °C, overnight) was facilitated using an airtight hybridization chamber (50 mL centrifuge tube) to prevent dehydration.

A stringent washing step was performed at 48 °C for 10 min in pre-warmed 50 mL washing buffer (100 mmol L−1 NaCl, 20 mmol L−1 Tris/HCl, 5 mmol L−1 EDTA). Afterwards, the slides were dried up and a mounting medium (Antifade Mounting Medium, Vectashield Vector Laboratories, CA, USA) was added to each well. The slides were sealed with a cover glass and examined under phase-contrast microscope (Nikon Eclipse Ni equipped with Lumen 200 Fluorescence Illumination Systems) using filter sets TRITC (557/576) (maximum excitation/emission in nm) for cy3-labelled EUB338 probe and FITC (490/525) for FLUOS-labelled GAM42a probes by a 100 × 1.45 numerical aperture microscope objective (CFI Plan Apo Lambda DM ×100 Oil; Nikon Corp., Japan).

Statistics and reproducibility

DoE experiments were designed and analysed using Design Expert version 11.1.2.0 (Stat-Ease, Inc. USA). Analysis of variation was performed at α = 0.05. The p-values for each test are indicated in the ‘Results’ section. All closed batch experiments were reproduced three times (N = 3) and each replication contained quadruplicate (n = 4). qPCR and FISH experiments, which applied all of the mentioned replicates, were performed in technical triplicates (n = 3). DoE experiments were conducted twice (N = 2) and each replication contained triplicate experiments for corner points (n = 3), except the set E (centre points), which was performed in biological pentaplicates (n = 5).

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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