Environmental Engineering and Management Journal
November 2013, Vol.12, No. S11, Supplement, 101-104
http://omicron.ch.tuiasi.ro/EEMJ/
“Gheorghe Asachi” Technical University of Iasi, Romania
PRODUCTION OF VOLATILE FATTY ACIDS FROM CHEESE WHEY WITH IMMOBILIZED CELLS Extended abstract Joana M.B. Domingos1,2, Gonzalo A. Martinez1, Alberto Scoma1, Lorenzo Bertin1, Maria A.M. Reis2, Fabio Fava1 1
DICAM, University of Bologna, via Terracini 28, I-40131, Bologna, Italy REQUIMTE/CQFB, Chemistry Dpt., FCT, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal
2
Background Dairy industry is practised all over the world for the production of milk, butter, yogurt, ice cream, cheese, and other milk derivates. This activity generates big amounts of highly COD containing effluents. Since their disposal requires a treatment step in dedicated plants aimed at abating the wastes organic content, their valorization within biorefinery schemes represents a valuable opportunity. In that frame, there is a strong interest on producing volatile fatty acids (VFAs) for the subsequent biotechnological production of biopolymers (namely, polyhydroxyalkanoates (PHAs)). Some investigations, which employed cheese whey (CW) as the PHA process feedstock, were reported in the literature (Pais et al., 2009). Such a waste is one of the dairy industry effluents exerting higher organic contamination, mainly due to its high lactose content. Thus, it represents a good raw material candidate for biorefinery processes. Few experiences about CW anaerobic acidogenic digestion for the production of ethanol and lactic acid with immobilized cells by employing a membrane bioreactor or packed-bed column were already described (Kosseva et al., 2009). However, almost no experiments for VFAs production with immobilization, from CW, have been reported. Objectives The present work proposes the production of VFAs from cheese whey powder (CWP) by employing a packed-bed biofilm reactor (PBBR) filled with a ceramic support, which was operated continuously under anaerobic acidogenic conditions. Outline of the work This work is divided in two main parts: Preliminary batch experiment in small scale, 100-mL Pyrex bottles, was performed in order to study and define the main process parameters. The second part is the operation of a PBBR in batch and continuous state with a HRT of 9 days. Methods The inoculum used in the present work was an acidogenic mixed culture, which was recovered from a membrane bioreactor that was also producing VFAs from CWP. A concentrated sample of the consortia was cultured in a 500 mL-Pyrex bottle with a solution of CWP prepared according to what reported below, in order to have enough active biomass for all experiments. CWP was kindly provided by Lactogal (Portugal). Technically, it is
Author to whom all correspondence should be addressed: e-mail:
[email protected]
Domingos et al./Environmental Engineering and Management Journal 12 (2013), S11, Supplement, 101-104
lyophilized cheese whey used for animal feed. Its main composition was (w/w CWP): proteins, 13.6%; lactose, 78.4% and fat, 1.21%. All experiments were done with the same culture media: a CWP solution (20 g/L) prepared with distilled water, corresponding to 15 g/L of lactose. All anaerobic microcosms were prepared in quadruplicate in 100-mL Pyrex bottles and had a packed and working volume of 45 and 55 mL, respectively. They were filled with a ceramic material supports (Vukopor S10, Lanik, CZ) and inoculated at 10% of the liquid volume. The incubation conditions were 37°C and 150 rpm; pH was maintained at 6 by adding drops of NaOH 10 M. The process was monitored every 2-3 days for VFAs and metabolites concentrations, pH, biogas production and composition. Three sequential batches were performed for favouring biofilm formation, while a consecutive fourth batch test was lasted until VFAs accumulated. Each subsequent process was started, after lactose complete consumption, by taking out the liquid phase and by refilling the microcosms with an equal volume of the CWP solution. An anaerobic packed-bed biofilm reactor (PBBR), filled with Vukopor S10 ceramic cubes, was developed. It Consisted of 1L-hermetically closed glass column wrapped with a silicon tubing serpentine continuously recycling thermostated water (37 °C) and equipped with down flow recycle line. It was packed with 118.36 g of support, and inoculated with the acidogenic mixed consortium 20% of the liquid volume of the reactor, which was filled with the experimental solution of CWP (20 g/L). The whole reactor working volume was 830 mL. The liquid and the gas effluents were collected in a 1-L bottle hydraulically connected to a 2.5-L “Marriotte” system. A preliminary batch experiment was performed for favouring biofilm formation. Thereafter it was turn to continuous state operation with a hydraulic retention time (HRT) of 9 days. The VFAs concentrations were determined by gas chromatography using a GC (Agilent Technologies, Milano, Italy), which is coupled to a Flame Ionization Detector (GC-FID model 7890A) and equipped with a HP-INNOWAX column (length 30m, diameter 0.250 mm film 0.25 μm) (Bertin, 2010), The samples were centrifuged at 14,000 RPM for 10 minutes; supernatant was diluted with oxalic acid solution (60 mM) and filtered (0.45 μm membrane). The lactose and lactic acid concentrations were determined by HPLC using IR as detector and a Varian Hi-Plex H 300 x 7.7 mm column. 0.01 N sulfuric acid was used as eluent, with an elution rate of 0.6 mL/min and a 65°C operating temperature. The samples were centrifuged at 14,000 rpm for 10 minutes and filtered (0.45 μm membrane). The produced biogas volume was determined by using a glass syringe of 50 mL for microcosm experiments while for the bioreactor was by a “Mariotte” system. The composition was measured applying gas chromatography using a μGC-FID, either microcosms as for the bioreactor (Scoma et al., 2011). Results and discussion The VFA production trend obtained in the fourth microcosm batch experiment is represented in Fig. 1. It was observed a typical behaviour, according to which lactose was converted to lactic acid, with a yield of 91% (C-mol lactic acid/C-mol lactose). When the latter reached its maximal concentration, VFAs started to be produced; therefore, their concentration increased till lactic acid was completely depleted. The total VFAs yield (9 days) was 77% (C-mol VFAs/C-mol lactose) (Table 1). The experimental time is in concordance with the results obtained in a previous work (Bengtsson et al., 2008), but in this study the organic load was 4 times higher. The final VFAs mixture composition was also different with respect to the one obtained by Bengtsson and co-workers (Bengtsson et al., 2008), in particular, the concentration of propionic acid which in this study was higher with 3 g/L as maximum obtained. Since such parameter is directly linked to the type of PHA, which can be obtained within the subsequent PHA biotechnological production process, these evidences are of interest in the perspective of producing defined PHAs having specific chemical and mechanical features. From the profile obtained within microcosms and reported in Fig. 1, a HRT of 9 days was selected for the PBBR.
Fig.1. Lactose, lactic acid and VFAs concentrations in microcosms during the batch experiment
102
Production of volatile fatty acids from cheese whey with immobilized cells
Interestingly, the preliminary batch experiment, which was carried out with the PBBR for favouring the biofilm formation, has shown the same behaviour in terms of sequential bioreactions trend than that observed within microcosm experiment. Nevertheless, it was obtained a different final VFAs mixture composition, whereas the lactic acid and VFAs yields were similar to the previous ones obtained at the smaller scale (Table 1). The VFA concentrations as a function of the time for the continuous mode operation are shown in Fig 2. It can be said that an almost stationary state condition was reached after 9 days, which means after the bioreactor changed 1 time its liquid phase. From day 9, and during the whole stationary state, the VFAs yield was 79% (C-mol VFAs/C-mol lactose), which is similar to what previously obtained under batch conditions (Table 1).
Fig. 2. VFAs concentrations observed during PBBR operation
Anyway, the VFAs mixture composition was not definitely constant: in particular, propionic acid concentration decreased drastically from 6 g/L at day 0, 1.3 g/L after 9 days of operation, until 0.8 g/L at stationary state. This could be attributed to a wash out of the microorganisms responsible for propionic acid accumulation. Thus, the process still needs to be optimized, with the aim of avoiding the loss of specific metabolic activities. Table 1. Yields obtained in microcosm experiment and in the PBBR Microcosm Y lactic acid/lactose % (C-mol/C-mol) Y VFAs/lactose % (C-mol/C-mol)
91 77
Bioreactor batch 93 84
Bioreactor HRT 9 79
Concluding remarks An anaerobic acidogenic immobilized-cells process for the production of VFAs from CWP was developed by employing a PBBR filled with Vukopor S10. All the lactose present in the solution of CWP was bioconverted producing a VFA mixture with a yield about 80%. Despite it was obtained a good VFAs yield, during the operation with the PBBR a loss of capability of producing propionic acid was observed. Since the latter represents an interesting substrate in the perspectives of producing PHAs with specific features, and the employed inoculum presented the capacity to produce it in considerable amounts, the optimization of the biotechnological process for the obtainment of a VFA mix rich in propionic acid has to be persecuted. Keywords: Acidogenic mixed culture, Ceramic support, Cheese Whey, Packed-bed bioreactor, Volatile Fatty Acids References Bengtsson S., Hallquist, J.m Werker A., Welander T., (2008), Acidogenic fermentation of industrial wastewaters: Effects of chemostat retention time and pH on volatile fatty acids production, Biochemical Engineering Journal, 40, 492–499. Bertin L., Lampis S., Todaro D., Scoma A., Vallini G., Marchetti L., Majone M., Fava F., (2010), Anaerobic acidogenic digestion of olive mill wastewaters in biofilm reactors packed with ceramic filters or granular activated carbon, Water Research, 44, 4537-4549. Kosseva M.R., Panesar P. S., Kaur G., Kennedy J. F., (2009), Use of immobilized biocatalysts in the processing of cheese whey, International Journal of Biological Macromolecules, 45, 437-447.
103
Domingos et al./Environmental Engineering and Management Journal 12 (2013), S11, Supplement, 101-104
Pais J., Serafim L.S., Farinha I., Prieto M.A., Arévalo-Rodríguez M., Reis M.A.M., (2009), Bioplastics production from cheese whey by recombinant E. coli, New Biotechnology, 25, S220. Scoma A., Bertin A., Zanaroli G., Fraraccio S., Fava F., (2011), A physicochemical–biotechnological approach for an integrated valorization of olive mill wastewater, Bioresource Technology, 102, 10273-10279.
104
