Phthalic acid esters are also known as the endocrine disrupting phthalates owing to their adverse effect on various organs of the endocrine system. PAEs the emerging environmental pollutants that are released into the environment from a wide variety of sources during production, use and disposal. Water contamination due to PAEs is therefore a serious concern.
Hence, this study focused on the biodegradation of different PAEs at various concentration combination using different bioengineered system, viz. continuous stirred tank bioreactor (CSTB), two-phase partitioning bioreactor (TPPB) and continuous with biomass recycle system followed by microfiltration under aerobic condition.
In a preliminary experiment using three bacteria (Rhodococcus opacus, Cellulosimicrobium funkei and Ochrobactrum sp.), C. funkei was identified to be the best for the biodegradation of DMP and DEP as a single substrate. However, using batch shake flask, low degradation efficiency was obtained at high initial concentration of the phthalates. Hence, a continuous stirred tank bioreactor (CSTB) was examined for enhancing the biodegradation of DMP and DEP by C. funkei, and complete degradation was achieved even up to 3000 and 2000 mg/L initial concentrations of DMP and DEP, respectively. High degradation efficiency using the CSTB was attributed to the controlled conditions of aeration, agitation and pH in the bioreactor.
Stoichiometric and mass balance analyses carried out in this batch bioreactor study clearly established the effectiveness of the biodegradation process using the CSTB. Degradation of a mixture of DMP and DEP was also found to be enhanced using the CSTB.
As compared to batch operation, fed-batch operation further revealed the high efficiency of degradation of the DMP and DEP mixture, even at their high initial concentrations. Under the continuous mode of operation using the CSTB, a high degradation rate (178.37 mg/Lh) was achieved even at high total inlet loading rate (ILR) of 218.75 mg/Lh. The degradation rate under the continuous mode of operation was further enhanced (218.68 mg/Lh) by recycling
the biomass into the bioreactor by microfiltration using an indigenous tubular ceramic membrane. High GI and low brine shrimp mortality values were observed from the phthalate degraded samples, demonstrating the potential of the CSTB integrated with microfiltration for treating PAEs containing wastewater. Thus, C. funkei demonstrated the efficient degradation of DMP and DEP as single and dual substrates using the CSTB under different operation modes, in particular continuous with 100% biomass recycle.
In addition to C. funkei, another novel bacterium, Gordonia sp., showed complete degradation of a mixture of six phthalates including BBP, DEHP and DnOP as high molecular weight phthalate in the CSTB system. Complete degradation (100%) of phthalates, even at very high total ILR (61.67 mg/Lh) was achieved using CSTB under continuous with biomass recycle mode suggesting that integrated biodegradation-microfiltration approach was best suitable for efficient degradation of phthalates for treating PAEs containing wastewater. In addition, toxicity analysis of the degraded phthalates revealed very high GI and low mortality of brine shrimps, further confirming the potential of the bioengineered system for treating such wastewater. Hence, the bioengineered system consisting of CSTB with the degrading bacterium demonstrated successful biodegradation of different phthalates in wastewater under different operation modes: batch, fed-batch, continuous and continuous with biomass recycle.
The major mechanism of PAEs biodegradation by Gordonia sp. and C. funkei was shown to involve the hydrolysis and/or esterification of alkyl side chain of phthalates. Intermediates formed during the biodegradation of PAEs further confirmed phthalic acid as the central metabolite. The mixture study reveals that an increase in the concentration of low molecular weight (LMW) phthalates (DMP, DEP and DBP) resulted in a high degradation of high molecular weight (HMW) phthalates (BBP, DEHP and DnOP), whereas LMW phthalate degradation efficiency values reduced with an increase in HMW phthalate concentrations.
Due to the low molecular weight and a short side chain of LMW phthalate, its specific degradation rate was more than that of the HMW phthalates.
A novel bioengineered system, a two-phase partitioning bioreactor (TPPB) system showed very high biodegradation efficiency of phthalates by C. funkei even at very high initial concentration of DMP and DEP, and within a short duration. In this TPPB system, the non- aqueous phase liquid (NAPL) silicone oil used was found to be biocompatible, non- bioavailable and suitable for DEP delivery to the degrading bacterium. The mechanism of phthalate degradation in the TPPB system involved the slow release of the EDPs from the non- aqueous phase (silicone oil) to the aqueous phase as per real-time demand of C. funkei, which also aided in overcoming the substrate inhibition effect of the EDPs. Besides, a very high volumetric oxygen mass transfer coefficient due to the added silicone oil in the TPPB was observed for enhancing the EDPs biodegradation in this bioengineered system. Very high biodegradation efficiency values of the compounds by C. funkei were reported using the TPPB system. A high GI value of 86.17% of chickpea seeds soaked in phthalate degraded sample taken from the TPPB system along with a low mortality value of 13.33% of brine shrimps, demonstrated the utility of the TPPB-based bioengineered system in toxicity removal and treatment of phthalates from wastewater.
Scope for future work
Following are some scope for future research work based on the findings of this thesis:
1) Biodegradation of a mixture of low and high molecular weight PAEs by Gordonia sp.
in the TPPB system.
2) PAEs biodegradation using co-culture of C. funkei and Gordonia sp.: process intensification and optimization
3) Cost analysis of different treatment systems used for biodegradation of PAEs.
4) Genetic engineering strategies to overcome substrate inhibition at high PAEs concentration.