*Manuscript Click here to view linked References 1 Toluene biodegradation in an algal-bacterial airlift 1 2 2 photobioreactor: influence of the biomass concentration and 3 4 3 the presence of an organic phase 5 4 6 7 8 5 Raquel Lebrero*, Roxana Ángeles, Rebeca Pérez, Raúl Muñoz 9 10 11 12 6 Department of Chemical Engineering and Environmental Technology, University of 13 14 7 Valladolid, Dr. Mergelina, s/n, 47011, Valladolid, Spain. Tel. +34 983186424, Fax: 15 8 +34983423013. 16 17 9 18 19 20 10 *Corresponding author: [email protected] 21 22 11 Keywords: Airlift bioreactor, algal-bacterial photobioreactor, toluene biodegradation, 23 24 12 two-phase partitioning bioreactor 25 26 27 13 28 29 14 Abstract 30 31 32 15 The potential of algal-bacterial symbiosis for off-gas abatement was investigated for the first 33 16 time by comparatively evaluating the performance of a bacterial (CB) and an algal-bacterial 34 35 17 (PB) airlift bioreactors during the treatment of a 6 g m-3 toluene laden air emission. The 36 37 18 influence of biomass concentration and of the addition of a non-aqueous phase was also 38 19 investigated. A poor and fluctuating performance was recorded during the initial stages of the 39 40 20 experiment, which was attributed to the low biomass concentration present in both reactors and 41 42 21 to the accumulation of toxic metabolites. In this sense, an increase in the dilution rate from 0.23 43 22 to 0.45 d-1 and in biomass concentration from ~1 to ~5 g L-1 resulted in elimination capacities 44 45 23 (ECs) of 300 g m-3 h-1 (corresponding to removal efficiencies ~ 90 %). Microalgae activity 46 47 24 allowed for a reduction in the emitted CO2 and an increase in dissolved O2 concentration in the 48 -1 25 PB. However, excess biomass growth over 11 g L hindered light penetration and severely 49 50 26 decreased photosynthetic activity. The addition of silicone oil at 20 % (on a volume basis) 51 -1 52 27 stabilized system performance, leading to dissolved O2 concentrations of 7 mg L and steady 53 ECs of 320 g m-3 h-1 in the PB. The ECs here recorded were considerably higher than those 54 28 55 29 previously reported in toluene-degrading bioreactors. Finally, microbial population analysis by 56 57 30 DGGE-sequencing demonstrated the differential specialization of the microbial community in 58 59 31 both reactors, likely resulting in different toluene degradation pathways and metabolite 60 32 production. 61 62 1 63 64 65 33 1. Introduction 1 2 34 Due to its valuable psychoactive properties, the amount of toluene used in the chemical 3 4 5 35 industry has noticeably increased over the last decades, leading to a concomitant 6 7 36 increase in the atmospheric emissions of this aromatic pollutant (WHO 2000, EURAR- 8 9 10 37 T 2003). Toluene causes toxic effects to both human health and the environment, and is 11 12 38 considered a priority pollutant by the US-Environmental Protection Agency. Physical- 13 14 39 chemical technologies for toluene removal, such as adsorption in activated carbon, 15 16 17 40 thermal incineration or UV oxidation, generate hazardous by-products and/or wastes 18 19 41 and entail high investment and operating costs. In this context, bioremediation based on 20 21 22 42 bacterial or fungal activity has arisen as a cost-effective and environmentally 23 24 43 sustainable alternative to conventional physical-chemical methods (Harding et al. 2003). 25 26 27 44 In particular, pneumatically agitated suspended-growth systems such as airlift 28 29 30 45 bioreactors have demonstrated a cost-effective toluene abatement performance while 31 32 46 avoiding typical operational problems encountered in packed-bed bioreactors: excessive 33 34 35 47 biomass growth, flooding, channeling, pressure drop build-up or formation of anaerobic 36 37 48 or dry zones (Vergara-Fdez et al. 2008). Nevertheless, airlift bioreactors for the 38 39 49 treatment of toluene still face severe limitations such as (i) a limited pollutant mass 40 41 42 50 transfer from the gas to the liquid phase due to its high Henry law, which results in a 43 44 51 low pollutant bioavailability, (ii) microbial inhibition at high toluene inlet 45 46 47 52 concentrations, and (iii) O2 limitation when treating high toluene loads. An enhanced 48 49 53 oxygen supply to the microbial community in the reactor by increasing the turbulence of 50 51 52 54 the culture broth might be problematic since intensive mechanical aeration in 53 54 55 bioreactors is highly costly and might cause volatilization and re-dispersion of the 55 56 56 toluene present in the aqueous phase (Muñoz et al. 2005). 57 58 59 60 61 62 2 63 64 65 57 Photosynthetic oxygenation in algal-bacterial photobioreactors constitutes an efficient 1 2 58 alternative to conventional aeration methods. In this process, the oxygen 3 4 5 59 photosynthetically produced by microalgae in the presence of light and CO2 is used by 6 7 60 heterotrophic bacteria to in situ oxidize the organic pollutant, producing in return the 8 9 10 61 CO2 needed for microalgae photosynthesis. Unlike bacterial processes where the 11 12 62 mineralization of organic pollutants releases CO2 and H2O as main end-products, 13 14 63 microalgal processes are able to fix and recover carbon and other nutrients as valuable 15 16 17 64 biomass besides furnishing O2 to the heterotrophic community. Moreover, some 18 19 65 microalgae are capable of biotransforming xenobiotic organic contaminants (Semple et 20 21 22 66 al. 1999; Muñoz and Guieysse 2006). Therefore, microalgae may boost the 23 24 67 biodegradation of toluene either by directly biotransforming the pollutant or by 25 26 27 68 enhancing the degradation potential of the microbial community present in the 28 29 69 bioreactor. Besides, additional O2 supply by photosynthetic activity might prevent 30 31 70 oxygen limitation and accelerate the bacterial degradation of toluene (Semple et al. 32 33 34 71 1999). Up to date, most studies on toluene biodegradation were based on the activity of 35 36 72 bacteria and fungi. On the contrary, microalgae-supported biodegradation of aromatic 37 38 39 73 contaminants has been scarcely investigated, and the catabolic pathways of 40 41 74 biodegradation of aromatic compounds in microalgae are still largely unknown. Thus, 42 43 44 75 despite the potential benefits of microalgae-based off-gas treatment, there is no single 45 46 76 study comparatively evaluating the performance of algal-bacterial and bacterial 47 48 49 77 bioreactors for the treatment of volatile organic compounds (VOCs). 50 51 78 On the other hand, the limited mass transfer of toluene to the liquid phase might be 52 53 54 79 overcome by the addition to the bioreactor of a non-aqueous phase (NAP) with high 55 56 80 affinity for this pollutant, resulting in a so-called two-liquid phase partitioning 57 58 59 81 bioreactor (TLPPB). TLPPBs have been successfully applied to treat both high and low 60 61 62 3 63 64 65 82 VOC-laden gas emissions, improving the performance of biological processes (Muñoz 1 2 83 et al. 2012, Lebrero et al. 2013). The NAP not only supports an enhanced mass transfer 3 4 5 84 of the target pollutant and O2 from the gas phase to the microorganisms, but also acts as 6 7 85 a buffer against surges in pollutant or metabolite concentrations that might be 8 9 10 86 potentially toxic to the microbial community (Lebrero et al. 2015). 11 12 87 This work was devised to comparatively evaluate a bacterial and an algal-bacterial 13 14 15 88 airlift bioreactors treating toluene at high loading rates. The influence of biomass 16 17 89 concentration and of the addition of a non-aqueous phase in order to overcome mass 18 19 20 90 transfer limitations and the inhibition resulting from high toluene loads were also 21 22 91 investigated. Moreover, the differential specialization of the microbial communities in 23 24 25 92 both bioreactors was also assessed by molecular techniques. 26 27 28 93 29 30 31 94 2. Materials and Methods 32 33 34 95 2.1 Microorganisms and culture conditions 35 36 37 96 Both bioreactors were inoculated with a mixture of Chlorella sorokiniana (0.5 L), 38 39 97 activated sludge from Valladolid wastewater treatment plant (0.1 L) and a toluene 40 41 42 98 acclimated Pseudomonas putida DSM 6899 culture (0.35 L) to an initial total suspended 43 44 99 solids concentration (TSS) of 0.79 g L-1. The mineral salt medium (MSM) used 45 46 -1 47 100 throughout the entire experiment was composed of (g L ): KNO3 1.25, CaCl2·H2O 48 49 101 0.1105, H3BO3 0.1142, FeSO4·7H2O 0.0498, ZnSO4·7H2O 0.0882, MnCl2·4H2O 50 51 52 102 0.0144, MoO3 0.0071, CuCl2·2H2O 0.0157, CoCl2·2H2O 0.0049, EDTA (C10H16N2O8) 53 54 103 0.5, KH2PO4 0.6247 and K2HPO4 1.3251. 55 56 57 104 58 59 60 105 2.2 Experimental set-up 61 62 4 63 64 65 106 The experimental plant consisted of two identical internal-loop airlift glass bioreactors 1 2 107 with a total volume of 2.5 L and a working volume of 2.2 L, operated in parallel in an 3 4 5 108 air-conditioned room at a constant temperature of 25 ºC (Figure 1). The inner tube had a 6 7 109 diameter of 0.055 m and a height of 0.295 m, while the external cylinder diameter and 8 9 10 110 height were 0.09 m and 0.42 m, respectively.