The application of new microbial isolates for environmental biotechnology

Taha Elfouly, M 2015, The application of new microbial isolates for environmental biotechnology, Doctor of Philosophy (PhD), Applied Sciences, RMIT University.

Document type: Thesis
Collection: Theses

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Title The application of new microbial isolates for environmental biotechnology
Author(s) Taha Elfouly, M
Year 2015
Abstract The shortage and overconsumption of our (finite) energy resources (i) as well as increasingly contaminated water supplies arising from industrial wastewaters (ii) represent significant global threats. Environmental biotechnology potentially offers biological solutions to these two fundamental problems. This project aims to use environmental biotechnology to overcome the technical barriers involved in both the generation of alternative bioenergy and the biodegradation of textile industrial effluent in wastewaters. The research begins with the isolation, purification and characterisation of lignocelluolose degrading microbial isolates (bacteria and fungi) and the subsequent assessment of the environmental biotechnological potential of these isolates in relation to the two areas. A rapid approach to the screening, isolation, purification and identification of the lignocellulolytic derivative potential of microbial isolates was developed based on the activity against a variety of ball-milled straws (wheat, rice sugarcane and pea straw) using a Biolog (MT2) microplate-based assay as an alternative and effective method compared to the existing traditional methods that are costly, time consuming and largely not environmentally friendly. Sixty bacterial and fifty fungal isolates were successfully isolated mainly from compost and soil located from an old straw pile. Thirty bacterial isolates that showed high lignocellulolytic activity were screened (for three lignocellulolytic activities; cellulase, xylanase, strawase) using both conventional methodologies (crude enzyme production; submerged mode) and by the developed Biolog (MT2) microplate-based assay. Biolog (MT2) microplate arrays resulted in significant positive correlations (R2 values up to 0.86) between Biolog and the traditional enzyme methodologies with respect to bacterial isolates and their lignocellulosic activities. The results confirmed the suitability of Biolog microplate-based assays as an alternative screening method for lignocellulolytic bacteria. Differences between the degradability of different straws material were found to be consistent with differences in the surface structure of these different straws observed by ESEM suggesting that ball-milled sugarcane and rice straws were degraded more readily than other straws.

In the second part of this study, the biotechnological potential of these lignocellulose degrading isolates were further examined using a novel approach involving co-culture of microbial isolates using four fungal and five bacterial isolates (selected out of 18 fungal and 30 bacterial isolates) for the construction of dual and triple defined microbial combinations. Co-culturing was used to investigate and understand the synergistic effects on saccharification of four different ball-milled straw substrates: wheat, rice, sugarcane and pea and comparison with the individual isolates. Some fungal and bacterial combinations such as Neosartorya fischeri–Myceliophthora thermophila and Aeromonas hydrophila–Pseudomonas poae enhanced higher saccharification (3- and 6.6 fold respectively) compared with their monocultures indicating the beneficial and compatible synergetic effects of mixed cultures. Ball-milled rice and sugarcane straws which had similar Fourier transform-infrared spectroscopy (FT-IR) profiles were more degradable, inducing more hydrolytic enzyme production than wheat and pea straws. The result highlights the potential of microbial co-culturing to improve saccharification of lignocellulosic substrates.

In the third part of this study the ability of fungal isolates were further examined, in this case to overcome the technical barriers involved in the production of biofuels from microalgae (3rd generation). The high-energy input required for harvesting of microalgal biomass (using existing harvesting techniques) can account for up to 50% of the total cost of biofuels production. Co-cultivation of fungal (oleaginous) and microalgal cells is receiving increasing attention because of the high efficiency of bio-flocculation of microalgal cells (up to 90%) with no requirement for added chemicals and low energy inputs. Screening of the new fungal isolates for their ability to bioflocculate 11 different microalgae representing freshwater, marine, small (5 µm), large (over 300 µm), heterotrophic, photoautotrophic, motile and non-motile strains (most of these strains are commercially used for biofuels production) led to the identification of one fungal isolate, the filamentous fungus A. fumigatus as being capable of efficiently harvesting 10 of the 11 microalgal strains. The harvested fungal-microalgal pellets showed additive and synergistic effects, increasing biomass production and lipid yields. Further, the ability of A. fumigatus/Thraustochytrid (Af/Thr) and A. fumigatus/T. chuii (Af/Tc) pellets to further grow in (25% and 10%) swine wastewater as an alternative and sustainable source of nutrient supply was investigated. The incubation of both fungal-microalgal pellets for 48 h in 10% wastewater resulted in the removal of 96% of NH4+ and 84% of PO4-3 in the wastewater together with a lipid yield and biomass increase of 1.4-fold for both pellets. The results confirmed the suitability and the commercial applicability of bioflocculation process using A. fumigatus for harvesting marine and freshwater microalgal strains effectively.

The final part of this study investigated the ability of another new isolate (thermophilic fungi) to treat industrial wastewater arising from the textile industry. Microorganisms can adsorb dyes via metabolism dependent or independent mechanisms, although fungal biomass is more frequently used for bioadsorption. Dye effluents are generally discharged at relatively high temperature between 50°C and 60°C. Here, the dye decolourizing abilities of a thermophilic fungal isolate, Thermomucor indicae-seudaticae at different temperatures (30, 45 and 55°C) and dye concentrations (100, 500 and 1000 mg l-1) were investigated in an azo–anthraquinone dye mixture (Azure B, Congo Red, Trypan Blue and Remazol Brilliant Blue R) over 6 days. Assays with living and inactivated T. indicae-seudaticae, Aspergillus fumigatus and the combined culture indicated that inactivated fungi were substantially better at dye decolourization. Inactivated T. indicae-seudaticae was a faster and more effective dye decolourizer in the temperature range, 30–55°C at 100, 500 and 1000 mg l-1 concentrations over 12 h than either A. fumigatus or the combined culture. At 1000 mg l-1 and 55°C, Thermomucor adsorbed up to 1.7-fold (74.93% decolourization) more than Aspergillus (44.67%) over 12 h. The results highlight the potential of using thermophilic fungus; T. indicae-seudaticae (for the first time) as a significant biological-agent for dye adsorption significantly at elevated temperatures and concentrations using activated and inactivated biomass.

In summary, this study has demonstrated the importance of microbial isolations, based on novel isolation and screening techniques to quickly assess their lignocellulolytic activity. Assessment of the environmental biotechnological potential of these microbial isolates (used individually or in co-cultures) has confirmed the potential of environmental biotechnology to overcome technical barriers involved in the generation of alternative bioenergy as well as the biodegradation of wastewaters. The biological approaches of microbial strains isolated in this study represent cost effective, green technology that can be applied economically at large scale.
Degree Doctor of Philosophy (PhD)
Institution RMIT University
School, Department or Centre Applied Sciences
Subjects Environmental Biotechnology not elsewhere classified
Keyword(s) Microbial isolates (bacteria and fungi)
Second generation of biofuels
Cellulosic bioethanol
Third generation of biofuels
Fungal-algal bioflocculation (Harvesting microalgal biomass)
Wastewater treatment
Industrial wastewater treatment (dye decolourisation)
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Created: Tue, 29 Sep 2015, 11:20:17 EST by Keely Chapman
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