Strains and cultivation medium
Acinetobacter baylyi ADP1 (DSM 24193) devoid of glucose dehydrogenase gene ACIAD2983, (referred here as ADP1-g) and Clostridium butyricum, isolated from a hydrogen-producing bioreactor , were used in the study. Unless otherwise indicated, ADP1-g cells cultivated in low-salt Lysogeny broth (LB) (tryptone, 10 g/l; yeast extract, 5 g/l; sodium chloride, 1 g/l) at 30 °C and 300 rpm, were used as pre-inoculums. The growth kinetics of ADP1-g in a three stage bioprocess was studied in in MA/9 medium (g/l; 5.518 Na2HPO4·2H2O, 3.402 KH2PO4, 1 NH4Cl, 0.008 nitrilotriacetic acid, 0.487 FeCl3, 0.25 MgSO4·7H2O, 0.02 CaCl2·2H2O, 0.2% casein amino acids and 2 ml/l of SL7 trace element solution). ADP1-g acetate and butyrate utilization experiments were conducted in modified minimal medium referred as JM media  ((g/l): 1.5 K2HPO4, 2.0 (NH4)2SO4, 0.2 MgSO4*7H2O, 0.015 CaCl2*2H2O, 0.005 FeSO4*7H2O, 0.3 yeast extract). C. butyricum precultivations were conducted in anaerobic Reinforced Clostridial Medium (RCM, Sigma Aldrich, US) at 37 °C and 150 rpm. The co-cultivation experiments, using ADP1-g and C. butyricum, were conducted in JM medium and the cultures were grown at 30 °C and 300 rpm. Chloramphenicol (25 µg/ml, diluted in ethanol) was used as an antibiotic in experiments conducted by ADP1-g alone.
The growth test to investigate ADP1-g viability in the three stage aerobic–anaerobic–aerobic bioprocess was conducted in tubes containing 10 ml of sterile aerobic MA/9 medium. Upon inoculating (initial optical density 0.02), the cultivation tubes were closed with sterile septum rubber stoppers, tightened with aluminum crimps and incubated at 30 °C and 300 rpm. A blank cultivation (devoid of inoculant) was included as control to determine ‘false positive’ results from contamination and as analytical control. The experiment was conducted in triplicates and the cell growth data measured at specific intervals using a spectrophotometer (Ultrospec 500 pro, Amersham Biosciences, UK) for 120 h, were averaged.
Batch experiments to investigate ADP1-g capacity to utilize organic acids from C. butyricum fermentation was performed in 120 ml serum bottles with a working volume of 50 ml sterile JM medium. Acetate and butyrate were supplemented at concentrations 20 mM and 15 mM, respectively. The inoculated cells (initial cell density 0.02) were cultured aerobically at 30 °C and 300 rpm. The cells were also inoculated to a substrate blank, i.e. cultivation medium devoid of acetate and butyrate. Samples to analyze cell growth, organic acid utilization, lipid analysis and medium pH were collected in 3-h interval for 24 h.
The co-cultivations of ADP1-g and C. butyricum were conducted in triplicate 500 ml batch bottles containing 300 ml of sterile aerobic JM medium. To initiate ADP1-g and C. butyricum growth, the medium was supplemented with 10 mM acetate and 20 mM glucose, respectively. Two millilitre of precultivated ADP1-g with an optical density at wavelength 600 nm (OD600nm) of 3.2 was inoculated alone and co-inoculated with 2 ml of C. butyricum precultivation (OD600nm 3.1). ADP1-g cultivated in similar medium and a blank medium were used as the controls in this experiment. Following inoculation, the batch bottles were capped (rubber stoppers), tightened (aluminum crimps). Initial samples to determine cell growth, substrate utilization, liquid metabolites and lipids were collected and the cultures were incubated at 30 °C and 200 rpm. The gaseous end products were analyzed from the bottle headspace after a 24 h cultivation period and the rubber stoppers were removed. Opening the bottle caps ensured the end of anaerobic phase and culture samples for analysis were collected. The medium pH, after the anaerobic phase, from the coculture, ADP1-g alone and blank cultivations were measured aseptically and adjusted to pH 7.4 (initial pH) with 5 M sterile sodium hydroxide solution. The cultures were divided to sterile 50 ml serum bottles and incubated under aerobic conditions at 30 °C and 200 rpm. The experiment was conducted in triplicates and samples were collected every 3 h during 20-h cultivation for analysis.
Additional cocultivation experiments to determine the hydrogen production as a function of time were conducted in 500 ml bottles as above, except samples were collected for cell growth, gaseous product analyses, substrate consumption and metabolite formation periodically during the anaerobic phase and the experiment was ended before the second aerobic phase. C. butyricum alone and ADP1-g alone cultivations were used as controls. Cell growth of the ADP1 alone cultivation was monitored to determine the starting point for sampling (OD, pH, GC and HPLC analysis) from the cocultivation.
Bioreactor experiment was conducted in a sterile 1-l vessel (Sartorius Biostat B plus Twin System, Germany) with an online pH and pO2 monitoring system. To a medium volume of 795 ml, 50 ml of ADP1-g precultivated in JM medium supplemented with 25 mM acetate (OD600nm 3.9) and 5 ml of C. butyricum precultivated in RCM (OD600nm 2.3) were inoculated, totaling the cultivation volume to 850 ml. To prevent gas leakage, the fermentor outlets were closed and a gas collection bag (Supelco, USA) was connected to the exhaust. After initial sample collection, the cultures were grown at 30 °C and 350 rpm for 24 h. At the end of anaerobic phase, the collection bag was removed, samples were collected for analysis and the medium pH was adjusted at 7.3 with sterile 5 M NaOH solution. The aerobic phase was initiated with air supply. The medium pH and partial O2 (pO2) profiles were obtained from the bioreactor. From the bioreactor, 45 ml of culture was removed every hour from which duplicate technical repeats to monitor cell growth and organic acid utilization were collected and the remaining culture (~ 40 ml) was used for quantitative lipid analysis.
For qualitative lipid analysis, 4 ml of culture samples were analyzed using thin layer chromatography as described in . Briefly, the samples were centrifuged and the cell pellet was suspended in methanol and chloroform. After centrifugation, the lower chloroform phase was applied on the TLC plates (10 × 10 cm Silica Gel 60 F254 HPTLC glass plates with 2.5 × 10 cm concentrating zone, Merck, USA) and visualized with iodine. For dry cell weight (DCW) calculations in bioreactor experiment, 40 ml of original cultures were pelleted in pre-weighed tubes at 30,000g (4 °C), freeze-dried and weighed. The tube weights were subtracted from the freeze-dried cell weights to determine the DCW (g/l). The lipids extracted from the freeze-dried cells were quantitatively analyzed using NMR as described in . Briefly, the WE content of the cells was quantified from extracted total lipids by 1H NMR measurements (Varian Mercury spectrometer, 300 MHz). The spectra were taken from samples dissolved in chloroform using trifluorotoluene as an internal standard. The obtained data were processed using ACD NMR processor program and interpreted accordingly. NMR allows quantitative detection of α-alkoxy methylene protons of alcohols specific for wax esters.
Glucose, acetate and butyrate concentrations were analyzed, in triplicates, using high-performance liquid chromatography (HPLC) (LC-20AD, Shimadzu, Japan) equipped with Shodex SUGAR (SH1011) column (300 × 8 mm), refractive index detector (RID, RID-10A) and 0.01 N H2SO4 as mobile phase. One millilitre of culture samples were centrifuged at 15,000g for 7 min (4 °C), filtered through 0.2 µm polycarbonate filter (Chromafil® PET-45/25, Macherey–Nagel, Germany) and diluted tenfold with ultrapure water in 1 ml HPLC vials. The injection volume, column temperature and mobile phase flowrate was set to 100 µl, 40 °C and 0.6 ml/min, respectively. Identification and quantification of carbon substrate and liquid fermentation metabolites were based on chromatography using external standards.
A gas chromatograph (GC-2014, Shimadzu GC) fitted with a thermal conductivity detector, PORAPAK column (2 m × 2 mm) and N2 as carrier gas was used to analyze the gaseous end products as described in . In brief, the GC maintained with column, oven and detector temperatures of 80, 80 and 110 °C, respectively, 200 µl of sample from the collection bag was injected to the sampling port. The overpressure, i.e. gas volume exceeding the headspace volume of the vessel, was analyzed using water displacement method. The H2 and CO2 concentrations (mM) were calculated by dividing the cumulative gas (H2/CO2) volume (ml, obtained from the GC data, injection volume and gas overpressure values) with the product of ideal gas law constant (for room temperature) and cultivation volume (l). Hydrogen yields (mol H2/mol glucoseconsumed) were calculated by dividing the H2 concentration (mM) with the utilized sugar (mM). In H2 productivity studies, the cumulative hydrogen production (ml) profile was calculated as described previously . Hydrogen productivities (mmol/l/h) were calculated by converting the cumulative hydrogen volume to concentration and dividing it with sampling time interval. For batch experiments, each experimental repeats were measured twice and in bioreactor experiment, triplicate technical repeats were included.
Carbon balances of C. butyricum gaseous and liquid metabolites and utilized substrate were calculated by multiplying the concentrations with the respective number of carbon in the molecular formula. The CO2 in the liquid phase was ignored during the carbon balance calculations. Electron balances were calculated by multiplying the carbon in utilized glucose and each metabolite with the corresponding degree of reduction (mol electrons per C-mol). For carbon and electron mass calculations, the chemical formula of C. butyricum biomass was assumed to be CH1.624O0.456N0.216P0.033S0.0047 . The biomass concentrations (mM) after anaerobic cultivation was calculated by dividing the DCW with the total mass from the formula (g/mol). Both carbon and electron recoveries were calculated by determining the percentage of sum total of carbon and electron mass of liquid and gaseous metabolites divided with the respective masses calculated for the utilized sugar.