Microbial community characterization and the dynamics of the continuous biohydrogen fermentation process

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Biogas analysis in batch reactors

Total volume of biogas produced in batch BHP tests was daily measured by using a water displacement method determined by the volume of acidified water displaced (pH=2). Acidified water at pH 2 (with HCl 37 %) was used to avoid the dissolution of CO2 in water. Biogas measurement system is showed in Figure II-4. All the measurements of the porduced biogas volume had been performed at 25 °C and 1 atm. The factor to calculate to standard condition (0 °C, 1 atm) is 0.9161. After having determined the total volume of the biogas produced, 1 mL of biogas was directly sampled from headspace to meeasure the biogas composition using a gas chhromatograph (Shimadzu GC-8A) connectted to a C-R8A integrator and equipped with a CTRI Alltech column. The following gases were measured: CO2, H2, O2, N2 and CH4. Thhe column was made up of 2 concentric collumns. The 3.175 mm-diameter inner column was filled with Sillicagel. It allowed the separaation of CO2 from the other gases. The other gaases were separated in the 6.350 mm-diameeter outer column filled with a molecular sieve. The carrier gas was argon at 2.8 bars. The temperatures were 30 °C for the oven and 100 °C for the injector and the detector. The detection of gaseous compounds was done using a thermal conductivity detector and the intensiity of current was 80 mA. The calibration was done with a standard gas composed of 25 % of CO2, 5 % of H2, 2 % of O2, 10 % of N2 and 58 % of CH4.
Figure II-4. Measurement system of the biogas produced in batch reactor.
The cumulative hydrogenn gas production was calculated using a masss balance on each step which led to the following equation (II-1):
where VH2,I corresponds to the cumulative hydrogen volumes (in mL) calculated at time “i” according to the cumulative hydrogen volume at time “i-1”, i.e. VH2,i-1; Vp the biogas volume measured by the water displacement method at time “i”(ml); Vh,i and Vh,i-1 the total volumes of bottle headspace at days “i” and “i-1”,respectively, i.e. considering the volume of liquid sampled over the experimental time; and CH2,i and CH2,i-1 the hydrogen percentages in bottle headspace at days “i” and “i-1”, respectively (in %).

Assessment of biogas production in continuous reactors

When biogas was produced in CSTR, the increased pressure values were measured by the manometer (Keller Mano2000, Swiss) connected to the reactors, and the data were acquired via TES to the on-line controlled computer. The pressure regulation was fixed at 1 bar and a peristaltic pump was activated to keep constant the pressure and evacuate the extra biogas produced in the reactor. According to the pump calibration, the total biogas volume and therefore productivities were calculated based on the rates of use of the pressure pump. Since the room temperature was constantly at 25 °C, the volume of produced biogas was therefore assessed at this temperature. The factor to calculate to standard condition (0 °C, 1 atm) is 0.9161.
A multiplexed micro-GC (R3000, SRA instruments, France) was directly connected to the gas outlet of the reactors for measuring online the biogas composition (Figure II-5). The Micro-GC R3000 was equipped with two columns: one micro-MolSieve 5Ǻ column (10 m × 0.32 mm) with argon as carrier gas was used to detect CO2 and the other Q PLOT column (8 m × 0.32 mm) with helium as carrier gas was used to detect O2, H2, N2 and CH4. The injector temperature was fixed at 90 °C. The temperature of the columns was maintained at 80 °C and the carrier gas pressure was fixed at 30 psi in both columns. The detector corresponded to a thermal conductivity detector (TCD). Each analysis was carried out over 180 seconds. The data were analyzed through the Soprane software (SRA Instruments) to assess the composition of the injected gas in mmol based on an external calibration. All data were collected in an internal mySQL database called SILEX, for any further analysis.

Metabolic end-product analysis

Volatile fatty acids (VFAs) analysis

The liquid samples were collected in 2 mL Eppendorf® tubes and were then centrifuged at 11 337g for 15min. Afterwards, 500 L of the supernatant were transferred in analytical vials where 500 L of standard internal solution (1 g.L-1 of diethylacetic acid (C6H12O2) acidified to 5 % with H3PO4) was added. These samples were then analyzed by gas chromatography coupled to flame ionization detection (GC-FID) in a Varian GC 3900 chromatograph equipped with an auto-sampling system (Middelburg, The Netherlands)
Figure II-6). Basically, the VFAs compounds of the samples are volatilized in the injector and then separated by affinity to the stationary phase materials within the column. The elution was carried out in a semi-capillary column FFAP of 15 m and 0.52 mm in diameter (Phase ECTM 1000). The carrier gas was nitrogen (LindeTM, Nitrogen gas 5.0). The detection was performed with a FID (flame ionization detector) with H2 (LindeTM, hydrogen gas 5.0) as burning gas. The conditions of elution were as follows:
• Carrier gas: nitrogen, P = 20 psi
• Injector temperature: 210 °C
• Detector temperature: 280 °C
• Gas flow rate: 6 mL min-1
• Range of oven temperature: 80 to 120 °C with a ramp rate of 10 °C per minute after 1 min of elution Data acquisition was performed with the software Varian Galaxy Work Station (version 1.9.3.2).

High Pressure Liquid Chromatography (HPLC) analysis of the metabolic end-products

Expect of VFAs, other fermentative products such as organic acids (e.g. lactate and formate), alcohols (e.g. ethanol) were quantified by high pressure liquid chromatography. After centrifugation of the reactor samples in Eppendorf® of 2 mL, 800 l of supernatant were transferred to a vial prior to the analysis by high-pressure liquid chromatography (HPLC). A 0.2-µm filtration step (Nylon membrane, Acrodlsc ®) was performed when supernatant was not clear since the HPLC column was highly sensitive to micro particles. The analytical chain was composed of an automatic sampler (Water 717plus), a pumping system (DIONEX UltiMate 3000), an oven (DIONEX ultimate 3000RS) equipped with a protective precolumn (Microguard cation H refill cartbridges, Bio-Rad), a separation Aminex column (HPX-87H, 300×7.8mm), and a refractometer as detector (Waters996) (Figure II-7). The pre-column aimed to filter out the residual particles before flowing into the separation column. The compounds were thus separated using an Aminex HPX-87H column, 300 x 7.8 mm (Bio-Rad). The column was placed in an oven maintained at 35 °C. The isocratic elution consisted of H2SO4 6 mmol.L-1, pumped at a rate of 0.4 mL min-1. The refractometric detector temperature was fixed at 45 °C. The elution retention time of the different compounds are presented in Table II-1.

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Reactor sampling procedure

Duplicate liquid samples of 2 mL were collected from continuous reactor outlets twice a day and were centrifuged at 11 337 g for 20 min in 2 mL Eppendorf® tubes. The supernatant and the pellet were conserved separately at -20 °C for further metabolite (see section II-3.2.2) and microbial community analysis, respectively.

Molecular fingerprinting of microbial community

An overview of the different microbial ecosystems involved in H2 metabolic networking was obtained by molecular analysis which consisted of (i) DNA extraction and purification from the environmental samples,(ii) a PCR (Polymerase Chain Reaction) amplification of the V3 region of 16S rDNA genes followed by (iii) a DNA PCR product separation by CE-SSCP (Capillary Electrophoresis-Single Strand Conformation Polymorphism). This technique provides the structure of the microbial community that could be further used to evaluate the diversity and the relative abundance of the present microbial population. Furthermore, CE-SSCP analysis provides accurate assessment of the microbial dynamics over experimental time.

Table of contents :

CHAPTER I. LITERATURE REVIEW 
I.1 OVERVIEW ON HYDROGEN PRODUCTION FROM SOLID WASTE
I.1.1 Hydrogen as an ideal energy carrier
I.1.2 Hydrogen production by biological ways, so-called biohydrogen
I.1.2.1 Direct biophotolysis processes
I.1.2.2 Indirect biophotolysis process
I.1.2.3 Photofermentative processes
I.1.2.4 Dark fermentation processes
I.1.3 Main microbial pathways producing biohydrogen by dark fermentation
I.1.4 Involvement of hydrogenases as electron and proton flux regulators
I.1.4.1 Structures, localizations and functions of hydrogenases
I.1.4.2 Factors influencing the hydrogenase activity
I.1.5 Potentiality of available feedstock for bioH2 production
I.1.5.1 Crop Residues
I.1.5.2 Animal manure – livestock waste
I.1.5.3 Food waste
I.1.5.4 A model substrate: the Jerusalem artichoke plant
I.2 DARK FERMENTATION PROCESS ENGINEERING WITH TARGET OF HYDROGEN PRODUCTION
I.2.1 Operation parameters
I.2.1.1 pH
I.2.1.2 Biohydrogen Partial Pressure
I.2.1.3 Temperature
I.2.1.4 Hydraulic Retention Time
I.2.1.5 Organic loading rate (OLR)
I.2.2 Bioreactor configuration
I.2.2.1 The dark fermentation processes within the biorefinery concept
I.2.2.2 Coupling dark fermentation to anaerobic digestion (methane production)
I.2.2.3 Coupling dark fermentation to photofermentation
I.2.2.4 Coupling dark fermentation to microbial electrochemical cells
I.3 MICROBIOLOGY FUNDAMENTALS OF BIOHYDROGEN PRODUCTION FROM AGRICULTURAL WASTE
I.3.1 The biohydrogen producers
I.3.2 H2 consumers and metabolic competitors
I.3.2.1 Homoacetogenic bacteria
I.3.2.2 Sulfate-Reducing Bacteria
I.3.2.3 Methanogens
I.3.2.4 Lactic Acid Bacteria
I.4 CONCLUSION
CHAPTER II. MATERIALS AND METHODS 97
II.1 ORGANIC SUBSTRATE SOURCES AND PREPARATION
II.1.1 Substrates used in the Biochemical Hydrogen Potential (BHP) tests
II.1.2 Preparation of substrate feeding solution for continuous hydrogen production in CSTR
II.2 BIOLOGICAL PROCESSES USED FOR BIOHYDROGEN PRODUCTION
II.2.1 Biological Hydrogen Potential (BHP) test
II.2.2 Operation of Continuous Stirred Tank Reactor (CSTR) for bioHydrogen production
II.2.2.1 Instrumentation of CSTR bioreactors
II.2.2.2 Start-up and operation in CSTR bioreactors
II.3 ANALYTICAL METHODS
II.3.1 Physico-chemical characterization of the organic substrate
II.3.1.1 Total solids (TS) and volatile solids (VS)
II.3.1.2 Determination of carbohydrate composition
II.3.1.3 Determination of protein composition
II.3.1.4 Substrate fractionation according to the Van Soest Method
II.3.2 Bioprocess parameters analysis
II.3.2.1 Biogas volume measurement and composition analysis
II.3.2.2 Metabolic end-product analysis
II.4 CHARACTERIZATION OF THE MICROBIAL COMMUNITIES
II.4.1 Reactor sampling procedure
II.4.2 Molecular fingerprinting of microbial community
II.4.2.1 DNA extraction and purification
II.4.2.2 DNA amplification by Polymerase Chain Reaction
II.4.2.3 CE-SSCP (Capillary Electrophoresis-single Strand Conformation Polymorphism)analysis
II.5 PROCESS MODELING
II.5.1 Hydrogen performance modeling of the BHP test
II.5.2 Multivariate analysis of experimental data and prediction of biological
hydrogen potential with PLS regression
CHAPTER III. RESULTS AND DISCUSSION 
III.1 IMPACT OF ORGANIC SOLID WASTE COMPOSITION AND STRUCTURE ON BIOHYDROGEN PRODUCTION
III.1.1 Optimization of the biochemical hydrogen potential (BHP) test
III.1.1.1 Impact of the type of buffer
III.1.1.2 Effect of MES buffer concentration
III.1.1.3 Effect of oligo-nutrients
III.1.1.4 Effect of organic load
III.1.1.5 Conclusion
III.1.2 BHP determination from organic solid substrates of various compositions and structures
III.1.2.1 Biochemical characterization of organic complex substrates
III.1.2.2 Biohydrogen production potentials from the organic solid substrates
III.1.2.3 End-products produced by different type of substrates
III.1.2.4 Substrate mapping considering biochemical composition, end-products and Hmax
III.1.2.5 Prediction model for biological hydrogen potentials
III.1.2.6 Fine characterization of carbohydrate composition by fractionation of lignocellulosic substrates and relationship with hydrogen potentials
III.1.3 Conclusion
III.2 CONTINUOUS FERMENTATIVE HYDROGEN PRODUCTION AND DYNAMICS USING A
SOLID SUBSTRATE: THE JERUSALEM ARTICHOKE TUBERS
III.2.1 Start up in batch of the CSTR
III.2.2 Hydrogen production at different organic loading rates
III.2.2.1 Results from stage 1 -reactor A
III.2.2.2 Results from stage 1 -reactor B
III.2.2.3 Results from stage 2-reactor A
III.2.2.4 Results from stage 2 – reactor B
III.2.2.5 Discussion on reactor A and B performances according to the organic loading rates
III.2.2.6 Relationship between hydrogen yields and metabolic pathways at different organic loading rates
III.2.3 Microbial community characterization and the dynamics of the continuous biohydrogen fermentation process
III.2.3.1 PCA analysis of CE-SSCP profiles
III.3 CONCLUSION

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