Effect of Co‐culture and Nutrients Supplementation on Bioremediation of Crude Petroleum Sludge

Ex‐situ bioremediation of real‐field crude petroleum sludge was evaluated to elucidate the role of co‐culture (bioaugmentation) and external nutrients supplementation (biostimulation) under anaerobic microenvironment. Maximum removal of total petroleum hydrocarbons (TPH) was observed by integrating biostimulation with bioaugmentation (R5, 44.01%) followed by bioaugmentation alone (R4, 34.47%), co‐substrate supplemented operations [R6, 23.36%; R3, 16.5%; R2, 9.88%] and control (R1, 4.36%). Aromatics fraction showed higher degradation in all the conditions studied. Fate of six selected polycyclic aromatic hydrocarbons (PAHs) was evaluated during bioremediation. Among these, four ring PAHs compounds showed good degradation by integration of biostimulation with bioaugmentation (R5) while bioaugmentation alone (R4) documented good degradation of three ring PAHs. Lower ring PAHs compounds showed good degradation with the application of biostimulation (R6). Fluorescent in situ hybridization (FISH) detected the presence of known PAHs degrading microorganisms viz., Bacillus, Pseudomonas, Acido bacteria, Sulphur reducing bacteria Firmicutes, etc. Application of biostimulation and bioaugmentation strategies alone or in combinations documented noticeable influence on the degradation of petroleum sludge.     

Evidence of subsurface anaerobic biodegradation of hydrocarbons and potential secondary methanogenesis in terrestrial mud volcanoes

The assessment of gas origin in mud volcanoes and related petroleum systems must consider post-genetic processes which may alter the original molecular and isotopic composition of reservoir gas. Beyond eventual molecular and isotopic fractionation due to gas migration and microbial oxidation, investigated in previous studies, we now demonstrate that mud volcanoes can show signals of anaerobic biodegradation of natural gas and oil in the subsurface. A large set of gas geochemical data from more than 150 terrestrial mud volcanoes worldwide has been examined. Due to the very low amount of C₂₊ in mud volcanoes, isotopic ratios of ethane, propane and butane (generally the best tracers of anaerobic biodegradation) are only available in a few cases. However, it is observed that ¹³C-enriched propane is always associated with positive δ¹³C_CO2 values, which are known indicators of secondary methanogenesis following anaerobic biodegradation of petroleum. Data from carbon isotopic ratio of CO₂ are available for 134 onshore mud volcanoes from 9 countries (Azerbaijan, Georgia, Ukraine, Russia, Turkmenistan, Trinidad, Italy, Japan and Taiwan). Exactly 50% of mud volcanoes, all releasing thermogenic or mixed methane, show at least one sample with δ¹³C_CO2 > +5‰ (PDB). Thermogenic CH₄ associated with positive carbon isotopic ratio of CO₂ generally maintains its δ¹³C-enriched signature, which is therefore not perturbed by the lighter secondary microbial gas. There is, however, high variability in the δ¹³C_CO2 values within the same mud volcanoes, so that positive δ¹³C_CO2 values can be found in some vents and not in others, or not continuously in the same vent. This can be due to high sensitivity of δ¹³C_CO2 to gas–water–rock interactions or to the presence of differently biodegraded seepage systems in the same mud volcano. However, finding a positive δ¹³C_CO2 value should be considered highly indicative of anaerobic biodegradation and further analyses should be made, especially if mud volcanoes are to be used as pathfinders of the conditions indicative of subsurface hydrocarbon accumulations in unexplored areas.     

Effects of Fenton Pre‐Treatment on Waste Activated Sludge Properties

Fenton process was investigated for the purpose of biological sludge disintegration. The Box–Wilson experimental design was employed to evaluate the effects of major process variables (Fe(II) and H₂O₂ concentrations) on both disintegration and dewatering performance of sludge. Results showed that 4 g Fe(II)/kg total solids (TSs) and 60 g H₂O₂/kg TS are efficient for floc disintegration. Fenton pre‐treatment enhanced the biodegradability of sludge. For 4 g Fe(II)/kg TS and 60 g H₂O₂/kg TS, 19.4% higher methane production was achieved compared to raw sludge in biochemical methane potential assay. Fenton pre‐treatment resulted in the release of organic sludge components into the liquid phase. For 4 g Fe(II)/kg TS and 60 g H₂O₂/kg TS, dissolved organic carbon and total nitrogen in sludge's supernatant increased by 75.74 and 60.60%, respectively. Fenton pre‐treatment enhanced the filterability of sludge and it can be applied for conditioning purpose before mechanical dewatering units.     

Enzymatic mobilisation of trace metals from estuarine sediment

Proteinase K, a non-specific protease that is representative of enzymes encountered in the digestive environment of deposit-feeding organisms, has been employed to determine the nature and extent of enzymatic mobilisation of trace metals (Al, Fe, Mn, As, Cu, Pb and Zn) from contaminated estuarine sediment. The extent of metal release by the enzyme was critically dependent on whether and how the sample had been dried and, for all metals except Mn, mobilisation was greatest when the sample had not been dried. The fraction of metal available to the enzyme, f_e, defined as the amount of metal released from undried sediment after 4 h incubation relative to the total metal concentration, was less than 1% in all cases. Significantly, however, there was a strong correlation between f_e and published factors defining the enrichment of metals in the gut fluids of the deposit-feeding lugworm, Arenicola marina. This suggests that, at least qualitatively, the mechanics of metal mobilisation in situ are replicated by the commercial enzyme. Thermally denaturing the enzyme resulted in mobilisation that was greater than (As and Cu) or similar to (all other metals) release engendered by the active enzyme, suggesting that the principal mechanism of metal mobilisation is not related to proteolysis but more likely involves complexation or exchange with component amino acids of the enzyme.     

Degree of Sulfate‐Reducing Activities on COD Removal in Various Reactor Configurations in Anaerobic Glucose and Acetate‐fed Reactors

Sulfate‐reduction data from various anaerobic reactor configurations, e. g., upflow anaerobic sludge blanket reactor (UASBR), completely stirred tank reactor (CSTR), and batch reactor (BR) with synthetic wastewaters, having glucose and acetate as the substrates and different levels of sulfate, were evaluated to determine the level of sulfate‐reducing activity by sulfate‐reducing bacteria coupled to organic matter removal. Anaerobic reactors were observed for the degree of competition between sulfate‐reducing sulfidogens and methane producing bacteria during the degradation of glucose and acetate. Low sulfate‐reducing activity was obtained with a maximum of 20% of organic matter degradation with glucose‐fed upflow anaerobic sludge bed reactors (UASBRs), while a minimum of 2% was observed with acetate‐fed batch reactors. The highest sulfate removal performance (72–89%) was obtained from glucose fed‐UASB reactors, with the best results observed with increasing COD/SO₄ ratios. UASB reactors produced the highest level of sulfidogenic activity, with the highest sulfate removal and without a performance loss. Hence, this was shown to be the optimum reactor configuration. Dissolved sulfide produced as a result of sulfate reduction reached 325 mg/L and 390 mg/L in CST and UASB reactors, respectively, and these levels were tolerated. The sulfate removal rate was higher at lower COD/SO₄ ratios, but the degree of sulfate removal improved with increasing COD/SO₄ ratios.