Microorganism and growth medium
Clostridium tyrobutyricum, strain DSMZ 2637, was obtained from Deutsche Sammlung von Microorganismen und Zellkulturen (DSMZ) and it was adapted to Pretreated and Hydrolyzed Wheat Straw (PHWS) by adaptive laboratory evolution technique as described in Baroi et al. (2015a). The adapted stain was stored at −80 °C in growth medium with 10 % glycerol and used throughout this study. The growth medium used has been described by O’brien and Morris (1971) and also applied in the studies of Baroi et al. (2015a, b). Experiments were carried out using either glucose or xylose alone or a mixture of both as carbon source.
Analytical methods
Sugars were quantified by HPLC-RI as described in Baroi et al. (2015b). Acetic and butyric acids were quantified by gas gas chromatography with a flame ionization detector, FID and quantification of hydrogen gas was carried out by gas chromatography with a thermal conductivity detector, TCD as described in Baroi et al. (2015b). Total volatile suspended solids (VSS) were quantified according to standard methods (APHA 2005).
Reverse electro enhanced dialysis—REED technology
The REED technology applied for in situ acid removal is a membrane separation process that combines elements from reversed electrodialysis and Donnan dialysis operations. Detailed descriptions of REED can be found in Garde (2002), Rype and Jonsson (2002) and Prado-Rubio et al. (2011). The REED membrane stack was built of 1 cell pair (915 cm2) and was equipped with anion-exchange (AX-REED) membranes to transport anions. The REED system was provided by Jurag Separation A/S (Denmark).
Acids separated by REED were collected as Na-salts. Dialysate and electrolyte were NaOH solutions at an initial concentration of 0.1 M. Fermentation broth and dialysate were recirculated at a flow rate of 400 and 200 mL min−1, respectively. Disinfection of the REED system and pipes was performed by circulating 400 ppm peracetic acid solution for 60–90 min followed by circulation of 10 l of sterile de-ionized (DI) water to wash out the disinfectant from the system. pH, maximum current and voltage were set for 7, 5A and 10 volt, respectively. The REED extraction efficiency was calculated as following (Eq. 1) (Baroi et al. 2015b):
$$REED\;extraction\;efficiency,\;\% = \frac{Butyric\;acid\;extracted}{Total\;butyric\;acid\;produced} \times 100$$
(1)
Batch and continuous fermentations
Batch and continuous experiments were performed in a 3-L Applikon® autoclavable glass reactor equipped with a controller for pH, temperature and agitation as described in Baroi et al. (2015b). The fermentation was carried out at 37 °C, 150 rpm and pH was maintained at 7 with 4M KOH when the REED system was not coupled to the fermentor. The fermentor was connected to the REED membrane unit as shown in Fig. 1, during experiments with in situ acid removal.
Inhibition experiments and calculations
Three series of batch experiments were performed in order to investigate possible inhibition of the growth of C. tyrobutyricum from increasing concentrations of glucose, xylose and potassium ions.
Experiments with glucose as carbon source were carried out in triplicate in 117-ml serum vials sealed with rubber stoppers and aluminium crimps. The vials contained 50 ml of growth medium and the initial glucose concentration was in the range of 9–50 g L−1. The vials were inoculated with 10 % of a fully grown culture of C. tyrobutyricum on 5 g L−1 glucose. Glucose consumption was followed and initial glucose consumption rates were calculated.
Experiments with xylose as carbon source presented the challenge of a rather long and unpredictable lag phase after inoculation when the experiments were performed in serum vials, which made the calculation of initial rates difficult and non-accurate. Therefore, four xylose experiments were performed in the 3-L glass reactor with a pre-activation step of C. tyrobutyricum at a glucose and xylose mixture (1:1) with a total concentration of 2.25 g L−1. Subsequently and when the culture had reached the late exponential state it was spiked with a concentrated xylose solution to establish an initial xylose concentration in the range of 5–40 g L−1. Xylose consumption was followed and initial xylose consumption rates were calculated.
The set-up of the experiments with glucose and xylose allowed for the same initial concentration of microbial cells in all experiments with the same carbon source. Considering Monod kinetics (Eq. 2), comparison of initial rates for the carbon source consumption allowed for direct deduction of the effective maximum specific growth rate µmax,eff, in each case, as the substrate consumption rate becomes zero-order (Eq. 3) when Ks ≪ S. Therefore, comparison of initial rates under the conditions mentioned before, allows also for drawing conclusions on the existence or not of substrate inhibition, as substrate inhibition negatively affects the effective maximum growth rate (Rittmann and McCarty 2001).
$$\left( {\frac{dS}{dt}} \right)_{eff,in} = - \frac{1}{{Y_{X/S}}} \cdot \frac{{\mu_{{\rm max}, eff}\cdot S_{in} }}{{K_{S,eff} + S_{in} }} \cdot X_{in}$$
(2)
where Sin and Xin is the initial substrate and microbial biomass concentration respectively, µmax, eff is the effective maximum growth rate, KS,eff is the effective saturation constant and YX/S is the microbial cell yield.
$$\left( {\frac{dS}{dt}} \right)_{eff,in} = - \frac{1}{{Y_{X/S}}} \cdot \mu_{{\rm max} ,eff} \cdot X_{in}$$
(3)
Four batch experiments with potassium ions concentration of 5, 10, 15 and 20 g L−1 were performed in the 3-L glass reactor. Potassium ions were supplied in the form of the K2HPO4 and KH2PO4 buffering system, while the concentration of other nutrients in the growth medium was as described previously. A mixture of glucose and xylose at a mass ratio of 1.3:1 was used as carbon source at a low initial concentration (<4 g L−1) in order to ensure a neutral pH in all experiments. The growth of the microbial cells was monitored by measuring the optical density, OD, at 660 nm. Microbial biomass concentrations were calculated in g L−1 using Eq. 4, which represents a calibration curve of OD660 versus Volatile Suspended Solids (VSS) obtained for C. tyrobutyricum grown on a 5 g L−1 glucose and xylose-based medium.
$$VSS = 0.5251 \cdot OD_{660} - 0.0235$$
(4)
Estimation of the µmax,eff was performed for each experiment at the early exponential phase where KS ≪ S by integrating Eqs. 5 and 6. The yields of microbial biomass were calculated based on the experimental measurements at the specific time period according to Eq. 7.
$$\left( {\frac{dX}{dt}} \right)_{eff} = \mu_{{\rm max} ,eff} \cdot X$$
(5)
$$\left( {\frac{dS}{dt}} \right)_{eff} = - \frac{1}{{Y_{{X/S}}}} \cdot \mu_{ {\rm max} ,eff} \cdot X$$
(6)
$$Y_{X/S} = \frac{\Delta X}{\Delta S}$$
(7)
where S and X is the substrate and microbial biomass concentration respectively, µmax, eff is the effective maximum growth rate, and YX/S is the yield of the microbial cells.