Eric M. Adetutu

In situ microbial treatment of landfill leachate using aerated lagoons

The aim of this study was to assess the efficiency of leachate treatment by microbial oxidation in four connected on-site aerated lagoons at a landfill site. The landfill site was found to be in an ageing methanogenic state, producing leachate with relatively low COD (mean value 1740 mg l(-1)) and relatively high ammonium concentrations (mean value 1241 mg l(-1)). Removal of COD averaged 75%, with retention times varying from 11 to 254 days. Overall 80% of the N load was removed within the plant, some by volatilisation of ammonium. Microbial community profiling of the water from each lagoon showed a divergent community profile, presumably a reflection of the nutrient status in each lagoon. In municipal solid waste landfills under similar conditions, leachate treatment through a facultative aerobic system in which sequential aerobic and anaerobic microbial oxidations occurred can readily be achieved using a simple two-lagoon system, suggesting this technology can be economic to install and simple to run.

Re-use of remediated soils for the bioremediation of waste oil sludge

We investigated the possibility of re-using remediated soils for new bioremediation projects by spiking these soils with waste oil sludge in laboratory based microcosms. The level of Total Petroleum Hydrocarbon (TPH) reduction was high (>80%) in naturally attenuated microcosms and was not significantly improved by biostimulation, bioaugmentation and the combined treatment of bioaugmentation and biostimulation by week 12. This indicated that the observed TPH reduction might have been related to the soils inherent hydrocarbon-degrading potential. Microbial community analysis (16S rDNA and ITS-based Denaturing Gradient Gel Electrophoresis fingerprints) confirmed the dominance of hydrocarbon degrading genera such as Alcanivorax and Scedosporium. Cluster and Shannon diversity analysis revealed similar but stable bacterial and fungal communities in naturally attenuated and amended microcosms indicating that rapid reduction in TPH may not always be accompanied by changes in soil microbial communities. This study has therefore shown that soils previously used for bioremediation can have an improved hydrocarbon degrading potential which was successfully re-harnessed for new projects. This ability to re-harness this potential is attractive because it substantially reduces operational costs as no additional bioremediation treatments are needed. It can also extend a landfills lifespan as soils can be re-used again before landfill disposal.

Assessment of five bioaccessibility assays for predicting the efficacy of petroleum hydrocarbon biodegradation in aged contaminated soils.

In this study, the bioaccessibility of petroleum hydrocarbons in aged contaminated soils (1.6-67gkg(-1)) was assessed using four non-exhaustive extraction techniques (100% 1-butanol, 100% 1-propanol, 50% 1-propanol in water and hydroxypropyl-β-cyclodextrin) and the persulfate oxidation method. Using linear regression analysis, residual hydrocarbon concentrations following bioaccessibility assessment were compared to residual hydrocarbon concentrations following biodegradation in laboratory-scale microcosms in order to determine whether bioaccessibility assays can predict the endpoint of hydrocarbon biodegradation. The relationship between residual hydrocarbon concentrations following microcosm biodegradation and bioaccessibility assessment was linear (r(2)=0.71-0.97) indicating that bioaccessibility assays have the potential to predict the extent of hydrocarbon biodegradation. However, the slope of best fit varied depending on the hydrocarbon fractional range assessed. For the C(10)-C(14) hydrocarbon fraction, the slope of best fit ranged from 0.12 to 0.27 indicating that the non-exhaustive or persulfate oxidation methods removed 3.5-8 times more hydrocarbons than biodegradation. Conversely, for the higher molecular weight hydrocarbon fractions (C(29)-C(36) and C(37)-C(40)), biodegradation removed up to 3.3 times more hydrocarbons compared to bioaccessibility assays with the resulting slope of best fit ranging from 1.0-1.9 to 2.0-3.3 respectively. For mid-range hydrocarbons (C(15)-C(28)), a slope of approximately one was obtained indicating that C(15)-C(28) hydrocarbon removal by these bioaccessibility assays may approximate the extent of biodegradation. While this study demonstrates the potential of predicting biodegradation endpoints using bioaccessibility assays, limitations of the study include a small data set and that all soils were collected from a single site, presumably resulting from a single contamination source. Further evaluation and validation is required using soils from a range of hydrocarbon contamination sources in order to develop robust assays for predicting bioremediation endpoints in the field.

Microbial community and ecotoxicity analysis of bioremediated, weathered hydrocarbon-contaminated soil

Bioremediated soils are usually disposed of after meeting legislated guidelines defined by chemical and ecotoxicity tests. In many countries including Australia, ecotoxicity tests are not yet mandatory safety requirements. This study investigated the biotreatment of weathered hydrocarbon-contaminated soils in 12-week laboratory-based microcosms. Monitored natural attenuation resulted in ~43% reduction of total petroleum hydrocarbon contaminant to 5503mg/kg (C16-C35), making the soil suitable for disposal as waste under current guidelines (pesticide and metal contents within safe limits).16S rDNA(universalandAlkB) andITS-based DGGE fingerprintsshowed stableand adaptedmicrobial communities throughout the experimental period. However, ecotoxicology assays showed 100% mortality of earthworms (Eisena fetida) in potting soils containing � 50% (� 2751mg/kg, legally safe in situ concentrations) contaminated soil over 14 days. Up to 70% reduction in radish (Raphanus sativus) seed germination was observed in potting soils containing � 10% contaminated soil (� 550mg/kg, legally safe ex situ concentrations for soil disposal into residential areas). The results indicate the toxicity of these soils to soil biota despite meeting legislated Australian safe levels and guidelines for disposal or use in residential areas.

Plant residues — A low cost, effective bioremediation treatment for petrogenic hydrocarbon-contaminated soil

Petrogenic hydrocarbons represent the most commonly reported environmental contaminant in industrialised countries. In terms of remediating petrogenic contaminated hydrocarbons, finding sustainable non-invasive technologies represents an important goal. In this study, the effect of 4 types of plant residues on the bioremediation of aliphatic hydrocarbons was investigated in a 90 day greenhouse experiment. The results showed that contaminated soil amended with different plant residues led to statistically significant increases in the utilisation rate of Total Petroleum Hydrocarbon (TPH) relative to control values. The maximum TPH reduction (up to 83% or 6800 mg kg(-1)) occurred in soil mixed with pea straw, compared to a TPH reduction of 57% (4633 mg kg(-1)) in control soil. A positive correlation (0.75) between TPH reduction rate and the population of hydrocarbon-utilising microorganisms was observed; a weaker correlation (0.68) was seen between TPH degradation and bacterial population, confirming that adding plant materials significantly enhanced both hydrocarbonoclastic and general microbial soil activities. Microbial community analysis using Denaturing Gradient Gel Electrophoresis (DGGE) showed that amending the contaminated soil with plant residues (e.g., pea straw) caused changes in the soil microbial structure, as observed using the Shannon diversity index; the diversity index increased in amended treatments, suggesting that microorganisms present on the dead biomass may become important members of the microbial community. In terms of specific hydrocarbonoclastic activity, the number of alkB gene copies in the soil microbial community increased about 300-fold when plant residues were added to contaminated soil. This study has shown that plant residues stimulate TPH degradation in contaminated soil through stimulation and perhaps addition to the pool of hydrocarbon-utilising microorganisms, resulting in a changed microbial structure and increased alkB gene copy numbers. These results suggest that pea straw in particular represents a low cost, effective treatment to enhance the remediation of aliphatic hydrocarbons in contaminated soils.

Commercial feasibility of lignocellulose biodegradation: possibilities and challenges

The main source of energy supply worldwide is generated from fossil fuels, which undoubtedly are finite and non-environmental friendly resources. Bioethanol generated from edible resources also has economic and environmental concerns. Despite the immense attention to find an alternative (inedible) source of energy in the last two decades, the total commercial production of 1st generation biofuels is limited and equivalent only to approximately 3% of the total road transport fuel consumption. Lignocellulosic waste represents the most abundant biomass on earth and could be a suitable candidate for producing valuable products including biofuels. However, cellulosic bioethanol has not been produced on a large scale due to the technical barriers involved that make the commercial production of cellulosic bioethanol not economically feasible. This review examines some of the current barriers to commercialization of the process.

Impact of bacterial and fungal processes on 14C-hexadecane mineralisation in weathered hydrocarbon contaminated soil

In this study, the impact of bacterial and fungal processes on (14)C-hexadecane mineralisation was investigated in weathered hydrocarbon contaminated soil. The extent of (14)C-hexadecane mineralisation varied depending on the bioremediation strategy employed. Under enhanced natural attenuation conditions, (14)C-hexadecane mineralisation after 98 days was 8.5 ± 3.7% compared to <1.2% without nitrogen and phosphorus additions. (14)C-hexadecane mineralisation was further enhanced through Tween 80 amendments (28.9 ± 2.4%) which also promoted the growth of a Phanerochaete chyrsosporium fungal mat. Although fungal growth in weathered hydrocarbon contaminated soil could be promoted through supplementing additional carbon sources (Tween 80, sawdust, compost, pea straw), fungal (14)C-hexadecane mineralisation was negligible when sodium azide was added to soil microcosms to inhibit bacterial activity. In contrast, when fungal activity was inhibited through nystatin additions, (14)C-hexadecane mineralisation ranged from 6.5 ± 0.2 to 35.8 ± 3.8% after 98 days depending on the supplied amendment. Bacteria inhibition with sodium azide resulted in a reduction in bacterial diversity (33-37%) compared to microcosms supplemented with nystatin or microcosms without inhibitory supplements. However, alkB bacterial groups were undetected in sodium azide supplemented microcosms, highlighting the important role of this bacterial group in (14)C-hexadecane mineralisation.

Phylogenetic diversity of fungal communities in areas accessible and not accessible to tourists in Naracoorte Caves.

The fungal diversity in areas accessible and not accessible to tourists at UNESCO World Heritage-listed Naracoorte Caves was investigated with culture-dependent and culture-independent techniques for assistance in cave management protocol development. The caves were selected based on tourist numbers and configurations: Stick Tomato (open, high numbers), Alexandra (lockable openings, high numbers) and Strawhaven (control; no access). Culture-based survey revealed Ascomycota dominance irrespective of sampling area with Microascales (Trichurus sp.) being most frequently isolated. Some Hypocreales-like sequences belonging to Fusarium sp., Trichoderma sp. and Neonectria sp. (Stick Tomato) were cultured only from areas not accessible to tourists. These orders also were detected by DGGE assay irrespective of sampling area. The predominance of Ascomycota (especially Microascales) suggested their important ecological roles in these caves. Culture-independent analysis showed higher Shannon fungal diversity values (from ITS-based DGGE profiles) in tourist-accessible areas of these caves than in inaccessible areas with the fungal community banding patterns being substantially different in Stick Tomato Cave. Further investigations are needed to determine the cause of the differences in the fungal communities of Stick Tomato Cave, although cave-related factors such as use, configuration and sediment heterogeneity might have contributed to these differences.

Enhanced Biological Straw Saccharification Through Coculturing of Lignocellulose-Degrading Microorganisms

Lignocellulosic waste (LCW) is an abundant, low-cost, and inedible substrate for the induction of lignocellulolytic enzymes for cellulosic bioethanol production using an efficient, environmentally friendly, and economical biological approach. In this study, 30 different lignocellulose-degrading bacterial and 18 fungal isolates were quantitatively screened individually for the saccharification of four different ball-milled straw substrates: wheat, rice, sugarcane, and pea straw. Rice and sugarcane straws which had similar Fourier transform-infrared spectroscopy profiles were more degradable, and resulted in more hydrolytic enzyme production than wheat and pea straws. Crude enzyme produced on native straws performed better than those on artificial substrates (such as cellulose and xylan). Four fungal and five bacterial isolates were selected (based on their high strawase activities) for constructing dual and triple microbial combinations to investigate microbial synergistic effects on saccharification. Combinations such as FUNG16-FUNG17 (Neosartorya fischeri–Myceliophthora thermophila) and RMIT10-RMIT11 (Aeromonas hydrophila–Pseudomonas poae) enhanced saccharification (3- and 6.6-folds, respectively) compared with their monocultures indicating the beneficial effects of synergism between those isolates. Dual isolate combinations were more efficient at straw saccharification than triple combinations in both bacterial and fungal assays. Overall, co-culturing can result in significant increases in saccharification which may offer significant commercial potential for the use of microbial consortia.

Bio-harvesting and pyrolysis of the microalgae Botryococcus braunii.

The microalgae Botryococcus braunii is widely recognized as a potentially important biofuel-feedstock whose commercial exploitation is limited by difficulties with its cultivation and harvesting. In this study, two B. braunii strains, Kossou-4 and Overjuyo-3 were successfully cultured at a 500 l-scale for 60-days. Harvesting by bio-flocculation with Aspergillus fumigatus at an optimum ratio of 1:40 of fungus to microalgal culture resulted in up to 98% recovery of biomass in the two strains. Ultimate analysis (C, N, H, S, ash, high heating value) and pyrolysis (analytical and preparative pyrolysis and GC-MS assays) showed that co-harvesting with fungi did not cause any impairment of the feedstock value of the microalgal biomass. This work represents the first report on the successful culturing and harvesting of these strains at a 500 l-scale using bio-flocculation. The use of A. fumigatus represents an efficient and economical method for the harvest of B. braunii for biofuel production.