Strains and plasmids
Strains and plasmids used in this study are provided in Table 3. E. cloacae WL1318, a bacterium reported to utilize cotton stalk hydrolysate for hydrogen production [30], served as the wild type strain from which recombinants were constructed. A previously described [30] growth medium was used for the wild strain; glucose 10 g/L, xylose 10 g/L, beef extract 5 g/L, peptone 10 g/L, NaCl 5 g/L, KH2PO4 0.5 g/L, and MgSO4·7H2O 0.5 g/L. E. coli DH5α served as the host strain for plasmid construction. Recombinant strain E. cloacae WL1318 – fhlA and E. coli DH5α were grown in LB medium and the antibiotics, 50 µg/mL ampicillin and 10 µg/mL kanamycin, were prepared and added into the media when required.
Preparation of cotton stalk hydrolysate
Cotton stalks were harvested from a cotton field in Xinjiang Alaer, China, dried, milled and sifted to 20-mesh size before being hydrolyzed. The fermentable sugar solution of the cotton stalk hydrolysate was prepared following optimum hydrolysis, then detoxified and decolorized as previously described [34, 35, 39]. The main sugar components in the hydrolysate solution were glucose and xylose [35, 40], which were modified to a previously determined optimum concentration and used as the substrate medium in the following fermentation experiments.
Cloning of the fhlA gene in Enterobacter cloacae WL1318 and sequence analysis
The total DNA of E. cloacae WL1318 was extracted using an Ezup Column Bacterial Genomic DNA Purification Kit (Sangon, Shanghai, China). Strains and plasmids used in this study were listed in Table 3. The sequence of the E. cloacae WL1318 fhlA gene was obtained via PCR (polymerase chain reaction) using the primer pair, fhlA-fw and fhlA-rv (Table 4), which was designed based on the open reading frames (ORF) of FHLA proteins from related facultative anaerobic bacteria. Related DNA and protein sequences were retrieved using the BLAST program in the NCBI database, multiple sequence alignments of amino acid sequences were performed using ClustalW program, and a phylogenetic tree was constructed via the MEGA 6.0 software using the neighbor-joining algorithm with a bootstrap support value of 1,000 replicates.
Sub-cloning and overexpression of the fhlA gene in Enterobacter cloacae WL1318
The gene fhlA was purified, and sub-cloned via PCR amplification using the primers, PfhlA-fw and PfhlA-rv, containing EcoR I and Xho I sites (underlined), respectively. The PCR product was purified and ligated to the pUCm-T vector, the purified plasmid, pUCm-T-fhlA, was digested with EcoR I and Xho I, and inserted into the multiple cloning site of the prokaryotic expression vector, pET28a, forming the recombinant plasmid, pET28a-fhlA (Kanr). The recombinant plasmid with the fhlA insert was purified and confirmed by double enzyme digestion and sequencing, then transformed into the E. cloacae WL1318 competent cells by electroporation (2500 V, 200 Ω, 25 µF) to obtain the recombinant strain E. cloacae WL1318- fhlA.
Cells of the recombinant strain were induced with 1 mmol/L IPTG for 6 h, harvested, and lysed using an ultrasonic cell disruptor (3 s, 1 s, 15 min) (Scientz-IID, SCIENTZ, Ningbo, China). Proteins were collected by centrifugation, and analyzed via 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to primarily confirm FHLA protein expression. Western blots were performed via the Novel One Step Western Blot Kit I (Sangon, Shanghai, China) and the specific fhlA encoded protein was examined using the W-TMB chromogenic kit (Sangon, Shanghai, China).
Batch fermentation for biohydrogen production
Single colonies of the recombinant strain E. cloacae WL1318 – fhlA were selected and inoculated in 3 mL LB medium (including 10 µg/mL kanamycin) and cultured overnight at 37 °C, 180 r/min, then 3-mL broth was inoculated with 30 mL fresh seed medium (glucose 10 g/L, xylose 10 g/L, beef extract 5 g/L, NaCl 5 g/L, peptone 10 g/L, KH2PO4 0.5 g/L, MgSO4·7H2O 0.5 g/L, and 10 µg/mL kanamycin), which was then shake-cultivated at 37 °C and 180 r/min until the optical density value at 600 nm (OD600) reached 0.4 ~ 0.6 (after 2 ~ 3 h). Next, sterilized IPTG was added to a final concentration of 1 mmol/L to induce expression at 37 °C for 6 h. The entire 30 mL seed broth was introduced into the fermentation medium composed of the following: cotton stalk hydrolysate (reducing sugar concentration 40 g/L) 1000 mL, beef extract 5 g/L, peptone 10 g/L, NaCl 5 g/L, KH2PO4 0.5 g/L, MgSO4·7H2O 0.5 g/L, kanamycin 10 µg/mL, and IPTG at a final concentration of 1 mmol/L. The fermenter was well sealed to maintain anaerobic conditions for hydrogen production and cultivation was performed at 37 °C for 120 h. The fermentation method for hydrogen production was also applied to the wild strain without the addition of kanamycin and IPTG. All the experiments were carried out in triplicates. The volume of the fermentative hydrogen was examined thrice every 24 h and summed to obtain the daily hydrogen production, which was measured at each fermentation time point, was accumulated and calculated as the cumulative hydrogen production in the corresponding fermentation period. The concentration of the main soluble metabolites, glucose, and xylose in the cotton stalk hydrolysate were measured at 24-h intervals.
Analytical methods and calculations
The hydrogen gas volume was measured via 1 mol/L NaOH displacement in an inverted burette, and the gas concentration was examined using a hand-held hydrogen detector (KP810H20, Henan Zhong’an Electronic Detection Technology Co Ltd, Zhengzhou, China). At each sampling time, bacterial growth was measured via the OD600 using a UV–visible spectrophotometer (7230G, Jinghua Instruments Co. Ltd., Shanghai, China). The rest of the aqueous samples were centrifuged at 8000 × g for 10 min and filtered through syringe filters with 0.22 µm membranes before analysis. The total concentration of reducing sugars in the broth was determined via the 3,5-dinitryl-salicylic acid reagent (DNS) method [41]. Glucose concentration was measured using a glucose detection kit (Comin Biotechnology, Suzhou, China) via the principle of glucose oxidation and colorimetry, using a UV–visible spectrophotometer (7230G, Jinghua Instruments Co Ltd, Shanghai, China). Xylose concentration was measured using a xylose detection kit (ZZStandard, Shanghai, China) via the xylose dehydrogenation reaction and detection of NADH formation using a UV–visible spectrophotometer (7230G, Jinghua Instruments Co. Ltd., Shanghai, China). The concentrations of soluble metabolites, such as succinate, lactate, acetate, pyruvate, formate and ethanol were measured using detection kits (ZZStandard, Shanghai, China) according to the manufacturers’ instructions.
The reducing sugar (glucose or xylose) consumption (%, w/w) in the cotton stalk hydrolysate was calculated as a percentage of the initial sugar concentration in the fermentative medium. The difference between the OD600 value in the final fermentation broth (120 h) and that in the initial fermentation broth (0 h) was described as the increment in OD600 (ΔOD600). The hydrogen yield Y(H2/S) (mol H2/mol sugar) was defined as hydrogen concentration generated from the consumed reducing sugar in the cotton stalk hydrolysate.
The kinetic parameters of the cumulative hydrogen production in batch fermentation experiments were calculated using the modified Gompertz model (Eq. 1) [42] as follows:
Where H is the cumulative hydrogen production (mL/L), P is the hydrogen potential (mL/L), Rm is the maximum hydrogen production rate (mL/(L·h)), e is 2.71828, λ is the lag phase time (h), and t is the culture time (h). The kinetic parameters (P, Rm, and λ) were estimated via Sigmaplot software 12.
Assay of FHL activity and specific related metabolic enzyme activity
FHL activity was assayed as described by Yoshida et al. (2005) [21]. The specific hydrogen production rate was measured as the rate of hydrogen produced at 37 °C from a stirred cell suspension at an OD600 of 1.0 in 50 mL PBS in the presence of 100 mmol/L sodium formate. The volumetric hydrogen production rate was measured at 37 °C by injecting 25 mmol/L formic acid into 100 mL cell suspension in a mixing reactor. The hydrogen concentration was examined using a hand-held hydrogen detector (KP810H20; Henan Zhong’an Electronic Detection Technology Co Ltd, Zhengzhou, China).
LDH activity was examined using an LDH kit (Comin, Suzhou, China) based on the principle of lactic acid oxidation and colorimetry. ALDH activity was determined using an ALDH kit (Comin, Suzhou, China) based on the principle of aldehyde oxidation and colorimetry.