Substrate and inoculum
The microalgae C. vulgaris (strain ESP-6, Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan) were grown photoautotrophically in Liquid Bold’s Basal Medium (BBM)[29] with 0.1 vvm CO2 (5% CO2 and 95% 0.45-μm filtered air) sparging. Light was provided by 8000–10000 lux LED lights (WD-TM-D35W, Widen Photodiode Technology Co., China). After 7 days of incubation, the microalgal biomass was harvested and concentrated by centrifugation at 3600?×?g for 15 min. The solid concentrate was subjected to anaerobic digestion. The concentrated algal biomass contained 12.9% (on wet weight basis) of total solid (TS), 93.5% (on dry weight basis) of volatile solid (VS), and 58, 11, and 14% (on dry weight basis) of proteins, lipids, and sugars, respectively.
C. thermocellum (strain DSM2360) was obtained from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures. Fresh cultures were maintained by routinely transferring 5% (v/v) inoculum into fresh medium containing 5 g/L of absorbent cotton. Other compounds contained in the fresh medium included (per liter of distilled water): KH2PO4, 0.50 g; K2HPO4?·?3H2O, 1.00 g; urea, 2.00 g; MgCl2?·?6H2O, 0.50 g; CaCl2?·?2H2O, 0.05 g; FeSO4?·?7H2O, 1.25 mg; morpholinopropane sulfonic acid, 10.00 g; resazurin, 1.00 mg; yeast extract, 6.00 g; glucose, 5.00 g; cysteine-HCl?·?H2O, 1.00 g. C. thermocellum was freshly harvested after 4 days of incubation when no more hydrogen production was detected.
Granular sludge, cultivated in a laboratory-scale (3.5 L) anaerobic sequenced batch reactor (ASBR), was added as the methanogenic inoculum. The ASBR was operated at 55°C, and glucose and acetate (80%:20%, calculated as COD) were utilized as the feedstock at an organic loading rate of 2 g COD/(L-day). The methanogenic sludge was taken after being acclimated for more than 50 days and rinsed with anaerobic preheated (55°C) buffer solution to remove the residual carbon. The buffer solution was the same as that used in the subsequent batch experiments. The TS of the granular sludge was 11.1% (w/w) and the VS was 77.8% (w/w) of the TS.
Experimental setup
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(1)
Series 1: One-step methane production with different inoculum ratios of C. thermocellum
Batch experiments were conducted at 55°C with 2 g VS/L of the algal biomass and 3 g VS/L of methanogenic sludge. The culture medium containing C. thermocellum was added at different inoculum ratios: 0%, 1%, 5%, and 10% (v/v). A control with only granular sludge and without microalgae was prepared to measure the endogenous activity of the sludge itself. Another control with granular sludge and culture medium containing 5% (v/v) of C. thermocellum was set up to determine the methane production potential of the culture medium. The bottles were filled up to 500 ml with buffer solution and flushed with nitrogen for 2 min to maintain anaerobic conditions. The composition of the buffer solution was as follows (per L): 1.0 g of NH4Cl, 0.4 g of K2HPO4?·?3H2O, 0.2 g of MgCl2?·?6H2O, 0.08 g of CaCl2?·?2H2O, 10 ml of trace element solution, and 10 ml of stock vitamin solution. The stock trace element and vitamin solutions were prepared according to Chen et al.[30].
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Series 2: Two-step co-production of hydrogen and methane
In the two-step experiment, 3 g VS/L of algal biomass was first incubated with 5% (v/v) of C. thermocellum containing culture medium at 55°C for 7 days in 500-ml buffer solution, as mentioned earlier, for hydrogen production. Subsequently, 2 g VS/L of methanogenic sludge was added to produce methane. As a reference, a one-step experiment with the same amount of algal biomass, granular sludge, and 5% (v/v) of C. thermocellum containing culture medium was set up. Furthermore, a control with granular sludge and 5% (v/v) of C. thermocellum containing culture medium, similar to that used in Series 1, was also included in this series. Two different inoculum to substrate ratios (methanogenic sludge to microalgal biomass: 3:2 in series 1 and 2:3 in series 2) were introduced to investigate the influence of inoculum to substrate ratio. All the experiments were carried out in duplicate and the results were expressed as means.
Analysis of gaseous and liquid samples
Gas production was measured by manometric methods. The pressure in the headspace of the serum bottles was measured by a Testo 512 pressure meter (Testo, Germany). The concentrations of hydrogen, methane, and carbon dioxide in the biogas were analyzed using a gas chromatograph (GC112A, Shanghai Precision & Scientific Instrument Co., China) equipped with a thermal conductivity detector (TCD). The gas volumes were corrected to standard temperature and pressure conditions (STP: 0°C and 1013 kPa). Methane production from the culture medium and methanogenic sludge was deducted in the reported data. Gompertz modeling (Eq.1) was used according to Lü et al.[31] to fit the curve of the cumulative methane production, and the values of three parameters (P, Rmax, and λ) were determined.
(1)
M(t) is the cumulative methane production (ml/g VS added) at time t (days), P is the highest methane yield (ml/g VS), Rmax is the maximum methane production rate (ml/g VS/day), and λ is the lag phase (days). Lag phase refers to the initial adaptive phase, during which methane production remains relatively constant prior to rapid growth.
The liquid samples were centrifuged at 16,000?×?g for 10 min. Subsequently, the supernatants were collected and analyzed for pH, volatile fatty acids (VFAs), alcohols, dissolved organic carbon (DOC), total inorganic carbon (TIC), dissolved nitrogen (DN), and three-dimensional fluorescent intensity. The pH was tested with a pHS-2 F Digital Meter. The DOC, TIC, and DN were analyzed on a TOC-VCPH Analyzer (Shimadzu, Japan). The concentrations of VFAs (including acetic, propionic, isobutyric, butyric, and isovaleric acids) and alcohols in the supernatant were determined using an Agilent 6890 N gas chromatography (GC) system equipped with a flame ionization detector (FID). The fluorescence excitation-emission matrixes (EEM) were recorded for the supernatant in a 10-mm quartz cuvette in a Varian Cary Eclipse fluorometer (Agilent, Santa Clara, CA, USA). The emission was scanned from 220 to 750 nm at 2-nm intervals and 10-nm bandwidth, while the excitation was produced with a Xenon flash lamp in 10-nm bandwidth at 10-nm intervals from 200 to 700 nm. The EEM signals were processed and subjected to parallel factor analysis (PARAFAC), as described in the study by Lu et al.[32].
Transmission electron microscopy observation of the microalgal cell
The cells of C. vulgaris were observed using transmission electron microscopy (TEM). Samples were prepared according to the procedure developed by Yamamoto et al.[33], and examined with a transmission electron microscope (JEM-1230, JEOL, Japan).
Multiple fluorochrome staining of the microalgal cell and spectral microscopy observation
The cells of C. vulgaris were stained successively by FITC for proteins, Con A for α-polysaccharides and calcofour white for β-polysaccharides according to Chen et al.[34]. The samples were then examined with a Leica DMI 4000B spectral microscope imaging system.
DNA manipulation
Both the liquid samples and granules corresponding to different sampling dates were used for DNA extraction. The total DNA was extracted from the pellets using PowerSoil DNA isolation kit (MoBio Laboratories Inc., CA), according to the manufacturer’s protocol. The fingerprint technique of Automated Ribosomal Intergenic Spacer Analysis (ARISA) was used to monitor the microbial dynamics. The extracted DNA was amplified using primers 1389 F and 71R for archaea, and primers ITSF and ITSReub for bacteria, respectively. Polymerase chain reaction (PCR) and ARISA of the PCR product were carried out according to the method described by Qu et al.[35]. Shannon diversity index was used to analyze the ARISA profiles and H value was calculated using the software PAlaeontological STatistics (PAST) version 2.17b, according to the procedure proposed by Hammer et al.[36].