Microbial Degradation Of Sparingly Soluble Diethyl Phthalate (DEP) And Dibutyl Phthalate (DBP) By Pure Microbes Using Microcalorimetry

PAEs are synthesized in massive amounts to produce various plastics and have become ubiquitous/widespread in environments and have been found in the pedosphere (soils, landfills and leachates), plants, hydrosphere (i.e. natural waters, fresh and ocean waters, groundwaters), the atmosphere, and human and other living organisms' fatty tissues (compounds are lipophilic/lyphophilic; i.e. fat-soluble) as a result of the production, usage, and disposal of plastics. Refractory water-soluble organic compounds perform a vital function in the etiology of certain chronic diseases in humans, reduce the viability of many plant and animal species, and pose a threat to one or more populations in natural ecosystems. Such worrying findings have aroused interest in the biodegradation of these compounds in soils and waters. Biodegradation of these compounds is a critical process affecting the environmental fate of PAEs. The sole aim of this study was to evaluate the microbial degradability of two potentially toxic PAEs (i.e., DEP and DBP) by two bacteria, (Escherichia coli and Staphylococcus aureus); and a fungus (Aspergillus niger) using microcalorimetry by monitoring their growth on media amended with/without DEP or DBP. To achieve this aim, the specific objectives were carried out and the results are presented: 1. Microcalorimetric methodThe k, I and IC50 values were determined from the TAMⅢMicrocalorimeter with correlation coefficients (r) greater than 0.99. Inhibitory ratio (Ⅰ) is important index to evaluate toxicological effect of PAEs and was each calculated for each microbe in relation to these compounds. The k values, decreasing with increase in the DEP or DBP doses, show that these compounds had potential antimicrobial activity even though they were almost completely consumed in these experiments. The IC50 values (E. coli,197.37μg mL-1; S. aureus,154.43μg mL-1; and A. niger,127.84μg mL-1) indicate that A. niger and E. coli are the most sensitive and most tolerant microbes to DEP, respectively; and those (E. coli,154.43μg mL-1; S. aureus,137.95μg mL-1; and A. niger,155.68μg mL-1) indicate that A. niger and S. aureus are the most tolerant and most sensitive microbes to DBP, respectively.It was observed that the growth rate constants decrease with the increasing concentrations of PAEs. When the concentrations of PAEs reached 300μg mL-1, the PAEs exerted complete inhibitory effects on the growth of the microbes, in this case no thermal effects can be observed in the power-time curve during the experimental time.The DEP degradation efficiencies (δ) for E. coli, S. aureus, and A. niger are 51.9-93.5,91.9-99.8 and 59.8-99.9%, respectively; deducing that S. aureus (meanδ=96.18%) is the most efficient DEP degrader and E. coli (meanδ=83.83 %) the least and those for DBP for E. coli, S. aureus, and A. niger are 63.8-99.1, 13.7-98.5 and 92.3-100.0%, respectively; inferring that A. niger (meanδ=97.27 %) is the most efficient DBP degrader and S. aureus (meanδ=75.95%) the least.It is also interesting to note that the heights of the second peaks on the P-T curves increased greater than the first at low and medium DEP or DBP doses and little or diminishing peaks on curves for higher DEP or DBP doses (200 and 300μg mL-1). Therefore, it may be concluded that the first way of metabolism has been influenced greater than the second way and that in the second way of metabolism, these facultative microbes must have consumed the DEP or DBP and subsequently its related degradation intermediates (monoethyl phthalate/monobutyl phthalate, MEP/MBP; phthalic acid, PA; and protocatechuic acid) that may have accumulated during the primary degradation of DEP or DBP, as sources of carbon and energy needed for their continued growth for a definite time. Biodegradation of PAEs (DEP or DBP), respectively, follows the degradative pathway proposed somewhere.The rate constants (k) were found to be independent of initial concentrations, and the mean first-order equations for E. coli+DEP, S. aureus+DEP, A. niger+ DEP, E. coli+DBP, S. aureus+DBP, and A. niger+DBP could be expressed as: In C=-0.00134t+A, In C=-0.00160t+A, In C=-0.00175t+A, In C=-0.00156t+ A, In C=-0.00086t+A, and In C=-0.00200t+A, respectively and their corresponding mean biodegradation half-lives were t1/2(DEP/E. coli=8.67 h,t1/2(DEP/S. aureus)=7.54 h, t1/2(DEP/A. niger)=10.92 h, t1/2(DBP/E. coli)=9.66 h,t1/2(DBP/S. aureus)=18.81 h, t1/2(DBP/A. niger)=9.60 h, respectively. The first-order rate coefficient (k) decreased gradually, suggesting that the higher initial concentrations (200 and 300μg mL-1) of DEP and DBP hindered the biodegradation.The biodegradability of the PAEs by these microbes appeared to be related to the length of the alkyl side chains. Relatively, the shorter-chain DEP was degraded at higher rate than the longer-chain DBP. It was observed that as the octanol/water partition coefficient (kow) increased and the water solubility decreased, the biodegradability of PAEs by these microbes declined. The order of the biodegradability of the two PAEs by these microbes was DEP (S. aureus)>DBP (A. niger) >DEP(A. niger)>DEP(E. coli)>DBP(E. coli)>DBP(S. aureus).Generally, A. niger, the fungus was found to be an efficient degrader of these two PAEs.2. Spectrophotometric analyses.Microbial Cell Surface Hydrophobicity and autoaggregation.There were significant differences in CSH (E. coli,64.90%; S. aureus,78.25%; and A. niger,51.30%) and the autoaggregation abilities (E. coli,63.00%; S. aureus, 78.25%; and A. niger,56.25%) amongst the microbes without DEP or DPB treatment.S. aureus showed high while E. coli and A. niger showed moderate hydrophobicity and autoaggregation abilities in the absence of DEP or DBP treatment. The fungus (A. niger) displayed lower CSH and autoaggregation than that of the bacterial microbes (E. coli and S. aureus), illustrating that remarkable differences might exist in the bacterial and fungal surface composition and structure (i.e., strain-specific characteristics). When DEP or DBP (50μg mL-1) was added to the culture media, there were decreases in both CSH [(E. coli,43.85%; S. aureus, 59.40%; and A. niger,31.70%) for DEP; (E. coli,57.70%; S. aureus,65.95%; and A. niger,43.10%) for DBP] and autoaggregation ability [(E. coli,35.00%; S. aureus, 58.00%; and A. niger,23.00%) for DEP; (E. coli,43.25%; S. aureus,65.00%; and A. niger,36.25%) for DBP] for the microorganisms. When compared with the blank (0.0μg mL-1), the two moderately hydrophobic cells recorded considerable reductions in autoaggregation ability (for DEP:E. coli, 44.44%; S. aureus,25.88%; and A. niger,59.11%; and for DBP:E. coli,31.35%; S. aureus,16.93%; and A. niger,35.56%) than the strongly hydrophobic cell. Similar reduction in hydrophobicity for all microbes (for DEP:E. coli,32.90%; S. aureus,24.09%; and A. niger,19.64%; and for DBP:E. coli,11.09%; S. aureus,15.72%; and A. niger, 15.98%) was also observed. Regression analysis shows a significant positive linear correlation between hydrophobicity of microbial cultures and autoaggregation abilities of the microbes with r=0.9584 for DEP and r=0.9968, which is consistent with other literatures.Biomass and TurbidityThe relationship between biomass and turbidity was observed to be linear. The changes in turbidity and biomass (log phase), as a result of PAE addition, with incubation time were found to be exponential [(hence, obeying the thermokinetic equation] and dose-dependent; and depict the phases of growth.Although 300μg mL-1 each of these compounds can also be degraded completely, the times needed for degradation were longer than the ones needed for lower initial concentrations of DEP or DBP for S. aureus, A. niger and E. coli, respectively. It's noteworthy that all three microbes completely degraded both PAEs within the same time period (i.e. about 84 to 100 h).Research data also demonstrated that the biomass concentration increased with increasing initial. DEP or DBP concentration. In addition, it was found that the microorganism continued to grow for a certain time after DEP or DBP was consumed, which indicated that intermediates accumulated during the primary degradation of DEP or DBP could subsequently be used for microbial growth. DEP and DBP biodegradations follow the general pathway.3. Chromatographic analysesResults from the GC (Hewlett-Packard Model 5890; J.W., USA) analyses showed that DEP and DBP were rapidly and completely degraded within 84 h (i.e., 3.5 days) and 120 h (i.e.,5 days) maximum, respectively. However, the degradation rates of DBP were slow. These degradation trends were also observed in the microcalorimetric assays and the spectrophotometric methods. The biodegradation rates of these two PAEs appeared to be related to the length of the alkyl side chains, influencing the length of the alkyl group of the di-ester, and hence the biodegradabilityThe shorter-chain DEP was degraded at a higher rate than the longer-chain DBP. The three cultures were able to degrade actively up to 300μg mL-1 dose of DEP and DBP, however, at slower rates. The degradations of these two tested phthalates conform to the first-order reaction model.The order of the biodegradability of the two PAEs by these microbes was observed to be in this order:DEP(S. aureus)>DBP(A. niger)>DEP(A. niger)>DEP(E. coli)> DBP(E. coli)>DBP (S. aureus).This trend was also observed in the microcalorimetric experiment, and also confirmed such observation made by other researchers. Generally, A. niger, the fungus was found to be the most efficient degrader of these two PAEs.The metabolites of PAE biodegradation were identified by the Thin-layer chromatography (TLC). Based on the Rf values, the intermediates released into the medium were identified to be monoethyl phthalate (MEP), monobutyl phthalate (MBP) and phthalic acid (PA). Protocatechuate, a key intermediate during aerobic degradation of DBP via MBP and PA by numerous microorganisms, was not detected in this study. These results suggested that MEP (from DEP degradation) MBP (from DBP degradation) and PA were the ultimate products of DEP and DBP hydrolysis.The degradation pathways exploration of phthalates possibly will help to understand their mineralization process and the toxicological behavior of their metabolites. This study is vital to tackle possible biodegradation concerns and to significantly contribute in the proper modelling of the in situ bioremediation processes (processes involving the use of microorganisms to degrade environmental pollution; the extent of bioremediation is critically dependent upon the presence of degrading microorganisms, the environmental conditions, the types of contaminants and the bioavailability of the contaminants to biodegradative microorganisms) for future field applications which could deal with the discharge of these compounds into the environment.In conclusion, microcalorimetric investigations on the biodegradation and associated stimulatory effects of the PAE compounds on microorganisms are possible and promising. It is believed that microcalorimetry is a useful and accurate method for studying the complete biodegradation mechanism of microorganisms in the presence of the PAEs and can offer vital information for researches in the field of microbiology.