Production and characterization of poly-(3-hydroxybutyrate) from recombinant Escherichia coli grown on cheap renewable carbon substrates

Although the biopolymer poly-(3-hydroxybutyrate), P[3HB], presents physicochemical properties that make it an alternative material to conventional plastics, its biotechnological production is quite expensive. As carbon substrates contribute greatly to P[3HB] production cost, the utilization of a cheaper carbon substrate and less demanding micro-organisms should decrease its cost. In the present study a 23 factorial experimental design was applied, aiming to evaluate the effects of using hydrolysed corn starch (HCS) and soybean oil (SBO) as carbon substrates, and cheese whey (CW) supplementation in the mineral medium (MM) on the responses, cell dried weigh (DCW), percentage P[3HB] and mass P[3HB] by recombinant Escherichia coli strains JM101 and DH10B, containing the P[3HB] synthase genes from Cupriavidus necator (ex-Ralstonia eutropha). The analysis of effects indicated that the substrates and the supplement and their interactions had positive effect on CDW. Statistically generated equations showed that, at the highest concentrations of HCS, SO and CW, theoretically it should be possible to produce about 2 g L(1) DCW, accumulating 50% P[3HB], in both strains. To complement this study, the strain that presented the best results was cultivated in MM added to HCS, SBO and CW ( in best composition observed) and complex medium (CM) to compare the obtained P[3HB] in terms of physicochemical parameters. The obtained results showed that the P[3HB] production in MM (1.29 g L(-1)) was approximately 20% lower than in CM (1.63 g L(-1)); however, this difference can be compensated by the lower cost of the MM achieved by the use of cheap renewable carbon sources. Moreover, using differential scanning calorimetry and thermogravimetry analyses, it was observed that the polymer produced in MM was the one which presented physicochemical properties (Tg and Tf) that were more similar to those found in the literature for P[3HB].

Laccase bio-grafting a versatile modification tool from green chemistry technologies

Today more than 99% of plastics are petroleum-based because of availability and cost of the raw material. The durability of these disposed plastics contributes to the environmental problems as waste and their persistence in the environment causes deleterious effects on the ecosystem. Environmental pollution awareness and the demand for green technology have drawn considerable attention of both academia and industry into biodegradable polymers. In this regard green chemistry technology has the potential to provide solution to this problematic issue. Laccase bio-grafting has recently been the focus of green chemistry technologies due to the growing environmental concerns, legal restrictions and increasing availability of scientific knowledge. In the last several years, research covering various applications of laccases has been increased rapidly particularly in the field of grafting. In principle, laccase-assisted graft co-polymerization may impart a variety of new functionalities to a polymer. The modified polymers through grafting have a bright future and their development is practically boundless. In present work, novel biodegradable graft copolymers combining the advantages of bacterial cellulose backbone and PHB side chains will be prepared by introducing enzymatic grafting technique. The present research will be a first step in the biopolymer modification. To date no report has been found in literature explaining the enzymatic grafting of PHAs. The technique would also provide an efficient modulation approach to improve the biodegradability and biocompatibility of the graft copolymer. The newly grafted copolymers will exhibit unique functionalities with wider range of potential applications mainly in tissue engineering, biosensors, pharmaceutical industry (drug delivery systems) and bio-plastics.

Expression of hepatitis B surface antigen gene (HBsAg) in Laminaria japonica (Laminariales, Phaeophyta)

A transformation model for Laminaria japonica was established from 1993 to 1998, on the basis of which the transgenic kelp with heterologous gene encoding hepatitis B surface antigen (HBsAg) was obtained by using the micro-particle bombardment transformation method. Results of quantitative ELISA showed that HBsAg in transgenic kelp was 0.529 mug/mg soluble proteins on average and the highest value was 2.497 mug/mg, implying that recombinant HBsAg had natural epitope. Further support for the integration of HBsAg gene into kelp genome was obtained by PCR-Southern and total DNA hybridization. Prospect of kelp bio-reactor producing high value materials such as edible HBV vaccine was discussed as well.

Enzymatic grafting: A novel approach to develop multifunctional materials of interest

Today more than 99% of plastics are petroleum-based because of the availability and cost of the raw material. The durability of disposed plastics contributes to the environmental problems as waste and their persistence in the environment causes deleterious effects on the ecosystem. Environmental pollution awareness and the demand for green technology have drawn considerable attention of both academia and industry into biodegradable polymers. In this regard green chemistry technology has the potential to provide solution to this issue. Enzymatic grafting has recently been the focus of green chemistry technologies due to the growing environmental concerns, legal restrictions, and increasing availability of scientific knowledge. Over the last several years, research covering various applications of robust enzymes like laccases and lipases has been increased rapidly, particularly in the field of polymer science, to graft multi-functional materials of interest. In principle, enzyme-assisted grafting may modify/impart a variety of functionalities to the grafted composites which individual materials fail to demonstrate on their own. The modified polymers through grafting have a bright future and their development is practically boundless. In the present study series of graft composites with poly(3-hydroxybutyrate) (P(3HB) as side chain and cellulose as a backbone polymer were successfully synthesised by introducing enzymatic grafting technique where laccase and lipase were used as model catalysts [1-3]. Subsequently, the resulting composites were removed from the casting surface under ambient environment and characterised by Fourier-transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and X-ray diffraction (XRD) in detail. Moreover, the thermo-mechanical behaviours of the grafted composites were investigated by differential scanning calorimetry (DSC) and dynamic mechanical analyser (DMA) measurements, respectively. In addition, hydrophobic and hydrophilic characteristics of the grafted polymers were studied through drop contour analysis using water contact angle (WCA). In comparison to the individual counterparts improvement was observed in the thermo- mechanical properties of the composites to varied extent. The tensile strength, elongation at break, and Young’s modulus values of the composites reached their highest levels in comparison to the films prepared with pure P(3HB) only which was too fragile to measure any of the above said characteristics. Interestingly, untreated P(3HB) was hydrophobic in nature and after lipase treatment P(3HB) and P(3HB)-EC-based graft composite attained higher level of hydrophilicity. This is a desired characteristic that enhances the biocompatibility of the materials for proper cell adhesion and proliferation therefore suggesting potential candidates for tissue engineering/bio-medical type applications [3]. The present research will be a first step in the biopolymer modification. To date no report has been found in literature explaining the laccase/lipase assisted grafting of P(3HB) [1-3]. The newly grafted composites exhibit unique functionalities with wider range of potential applications in bio-plastics, pharmaceutical, and cosmetics industries, tissue engineering, and biosensors. [1] H.M.N. Iqbal, G. Kyazze, T. Tron and T. Keshavarz, Cellulose 21, 3613-3621 (2014). [2] H.M.N. Iqbal, G. Kyazze, T. Tron and T. Keshavarz, Carbohydrate Polymers 113, 131-137 (2014). [3] H.M.N. Iqbal, G. Kyazze, T. Tron and T. Keshavarz, Polymer Chemistry In-Press, DOI: 10.1039/C4PY0 0857J (2014).

Production and Characterization of Lignin Peroxidases from Mangrove Ascomycetes

In this thesis, the production and characterization of ligninolytic enzymes using the fungi isolated from mangrove area are studied. The objective of the present work are isolation and screening of dye decolorizing micro-organisms from mangrove area, screening of the selected microorganisms for the production of lignin degrading enzymes, identification of the potent micro-organisms, characterization of the crude enzyme, lignin peroxidase, of the selected fungi—Aspergillus sp. SIP 11 and Penicillium sp. SIP 10 etc. This included the determination of the optimum pH, temperature, veratryl alcohol and H2O2 concentration. Besides the stability of crude LiP at different pHs and temperatures were studied. The immense applications, particularly in bioremediation, to which the lignin degrading micro-organisms could be used make this study important, the ascomycetes and deuteromycetes fungi, especially form the marine environment were studied with respect to their ligninolytic enzyme system making this study an initial step in unraveling the vast hidden potential of these microbes in bioremediation, the marine microbes are halophilic in nature which make them better suited to cope with the high salinity of industrial effluents thereby giving them added advantage in the filed of bioremediation. The thesis deals with the isolation and screening of lignin degrading enzyme-producing microbes from mangrove area. The identification of the most potent fungal isolates and characterization of LiP from these are also done.

Diversidade e evolução na simbiose entre bactérias e formigas Attini

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Biorefineries – creating economic opportunities from biofuels and bio-products

Biorefineries, producing fuels, green chemicals and bio-products, offer great potential for improving the profitability and sustainability of tropical agricultural industries. Biomass from tropical crops like sugarcane, sweet sorghum, palm and cassava offer great potential because of the high biomass growth potential under favourable climatic conditions. Biorefineries aim to convert waste residues through biochemical and enzymatic processes to low cost fermentable sugars which are a platform for value-adding. Through subsequent fermentation utilising microbial biotechnologies or chemical synthesis, the sugars can be converted to fuels including ethanol and butanol, oils, organic acids such as lactic and levulinic acid and polymer precursors. Other biorefinery products can include food and animal feeds, plastics, fibre products and resins. Pretreatment technologies are a key to unlocking this potential and new technologies are emerging. This paper will address the opportunities available for tropical biorefineries to contribute to the future profitability of tropical agricultural industries. The importance of pretreatment technologies will be discussed.

Manganese: Minerals, Microbes, and the Evolution of Oxygenic Photosynthesis

Oxygenic photosynthesis fundamentally transformed our planet by releasing molecular oxygen and altering major biogeochemical cycles, and this exceptional metabolism relies on a redox-active cubane cluster of four manganese atoms. Not only is manganese essential for producing oxygen, but manganese is also only oxidized by oxygen and oxygen-derived species. Thus the history of manganese oxidation provides a valuable perspective on our planet’s environmental past, the ancient availability of oxygen, and the evolution of oxygenic photosynthesis. Broadly, the general trends of the geologic record of manganese deposition is a chronicle of ancient manganese oxidation: manganese is introduced into the fluid Earth as Mn(II) and it will remain only a trace component in sedimentary rocks until it is oxidized, forming Mn(III,IV) insoluble precipitates that are concentrated in the rock record. Because these manganese oxides are highly favorable electron acceptors, they often undergo reduction in sediments through anaerobic respiration and abiotic reaction pathways.

The following dissertation presents five chapters investigating manganese cycling both by examining ancient examples of manganese enrichments in the geologic record and exploring the mineralogical products of various pathways of manganese oxide reduction that may occur in sediments. The first chapter explores the mineralogical record of manganese and reports abundant manganese reduction recorded in six representative manganese-enriched sedimentary sequences. This is followed by a second chapter that further analyzes the earliest significant manganese deposit 2.4 billon years ago, and determines that it predated the origin of oxygenic photosynthesis and thus is supporting evidence for manganese-oxidizing photosynthesis as an evolutionary precursor prior to oxygenic photosynthesis. The lack of oxygen during this early manganese deposition was partially established using oxygen-sensitive detrital grains, and so a third chapter delves into what these grains mean for oxygen constraints using a mathematical model. The fourth chapter returns to processes affecting manganese post-deposition, and explores the relationships between manganese mineral products and (bio)geochemical reduction processes to understand how various manganese minerals can reveal ancient environmental conditions and biological metabolisms. Finally, a fifth chapter considers whether manganese can be mobilized and enriched in sedimentary rocks and determines that manganese was concentrated secondarily in a 2.5 billion-year-old example from South Africa. Overall, this thesis demonstrates how microbial processes, namely photosynthesis and metal oxide-reducing metabolisms, are linked to and recorded in the rich complexity of the manganese mineralogical record.

Molecular interactions of arbuscular mycorrhizal fungi with mycotoxin-producing fungi and their role in plant defense responses

Les trichothécènes de Fusarium appartiennent au groupe des sesquiterpènes qui sont des inhibiteurs la synthèse des protéines des eucaryotes. Les trichothécènes causent d’une part de sérieux problèmes de santé aux humains et aux animaux qui ont consommé des aliments infectés par le champignon et de l’autre part, elles sont des facteurs importants de la virulence chez plantes. Dans cette étude, nous avons isolé et caractérisé seize isolats de Fusarium de la pomme de terre infectée naturellement dans un champs. Les tests de pathogénicité ont été réalisés pour évaluer la virulence des isolats sur la pomme de terre ainsi que leur capacité à produire des trichothécènes. Nous avons choisi F. sambucinum souche T5 comme un modèle pour cette étude parce qu’il était le plus agressif sur la pomme de terre en serre en induisant un flétrissement rapide, un jaunissement suivi de la mort des plantes. Cette souche produit le 4,15-diacétoxyscirpénol (4,15-DAS) lorsqu’elle est cultivée en milieu liquide. Nous avons amplifié et caractérisé cinq gènes de biosynthèse trichothécènes (TRI5, TRI4, TRI3, TRI11, et TRI101) impliqués dans la production du 4,15-DAS. La comparaison des séquences avec les bases de données a montré 98% et 97% d'identité de séquence avec les gènes de la biosynthèse des trichothécènes chez F. sporotrichioides et Gibberella zeae, respectivement. Nous avons confrenté F. sambucinum avec le champignon mycorhizien à arbuscule Glomus irregulare en culture in vitro. Les racines de carotte et F. sambucinum seul, ont été utilisés comme témoins. Nous avons observé que la croissance de F. sambucinum a été significativement réduite avec la présence de G. irregulare par rapport aux témoins. Nous avons remarqué que l'inhibition de la croissance F. sambucinum a été associée avec des changements morphologiques, qui ont été observés lorsque les hyphes de G. irregulare ont atteint le mycélium de F. sambucinum. Ceci suggère que G. irregulare pourrait produire des composés qui inhibent la croissance de F. sambucinum. Nous avons étudié les patrons d’expression des gènes de biosynthèse de trichothécènes de F. sambucinum en présence ou non de G. irregulare, en utilisant le PCR en temps-réel. Nous avons observé que TRI5 et TRI6 étaient sur-exprimés, tandis que TRI4, TRI13 et TRI101 étaient en sous-exprimés en présence de G. irregulare. Des analyses par chromatographie en phase-gazeuse (GC-MS) montrent clairement que la présence de G. irregulare réduit significativement la production des trichothécènes par F. sambucinum. Le dosage du 4,15-DAS a été réduit à 39 μg/ml milieu GYEP par G. irregulare, comparativement à 144 μg/ml milieu GYEP quand F. sambucinum est cultivé sans G. irregulare. Nous avons testé la capacité de G. irregulare à induire la défense des plants de pomme de terre contre l'infection de F. sambucinum. Des essais en chambre de croissance montrent que G. irregulare réduit significativement l’incidence de la maladie causée par F. sambucinum. Nous avons aussi observé que G. irregulare augmente la biomasse des racines, des feuilles et des tubercules. En utilisant le PCR en temps-réel, nous avons étudié les niveaux d’expression des gènes impliqué dans la défense des plants de pommes de terre tels que : chitinase class II (ChtA3), 1,3-β-glucanase (Glub), peroxidase (CEVI16), osmotin-like protéin (OSM-8e) et pathogenèses-related protein (PR-1). Nous avons observé que G. irregulare a induit une sur-expression de tous ces gènes dans les racines après 72 heures de l'infection avec F. sambucinum. Nous avons également trové que la baisse provoquée par F. sambucinum des gènes Glub et CEVI16 dans les feuilles pourrait etre bloquée par le traitement AMF. Ceci montre que l’inoculation avec G. irregulare constitut un bio-inducteur systémique même dans les parties non infectées par F. sambucinum. En conclusion, cette étude apporte de nouvelles connaissances importantes sur les interactions entre les plants et les microbes, d’une part sur les effets directs des champignons mycorhiziens sur l’inhibition de la croissance et la diminution de la production des mycotoxines chez Fusarium et d’autre part, l’atténuation de la sévérité de la maladie dans des plantes par stimulation leur défense. Les données présentées ouvrent de nouvelles perspectives de bio-contrôle contre les pathogènes mycotoxinogènes des plantes.

Co-production of bio-oil and propylene through the hydrothermal liquefaction of polyhydroxybutyrate producing cyanobacteria

A polyhydroxybutyrate (PHB) producing cyanobacteria was converted through hydrothermal liquefaction (HTL) into propylene and a bio-oil suitable for advanced biofuel production. HTL of model compounds demonstrated that in contrast to proteins and carbohydrates, no synergistic effects were detected when converting PHB in the presence of algae. Subsequently, Synechocystis cf. salina, which had accumulated 7.5wt% PHB was converted via HTL (15% dry weight loading, 340°C). The reaction gave an overall propylene yield of 2.6%, higher than that obtained from the model compounds, in addition to a bio-oil with a low nitrogen content of 4.6%. No propylene was recovered from the alternative non-PHB producing cyanobacterial strains screened, suggesting that PHB is the source of propylene. PHB producing microorganisms could therefore be used as a feedstock for a biorefinery to produce polypropylene and advanced biofuels, with the level of propylene being proportional to the accumulated amount of PHB.

Co-production of bio-oil and propylene through the hydrothermal liquefaction of polyhydroxybutyrate producing cyanobacteria

A polyhydroxybutyrate (PHB) producing cyanobacteria was converted through hydrothermal liquefaction (HTL) into propylene and a bio-oil suitable for advanced biofuel production. HTL of model compounds demonstrated that in contrast to proteins and carbohydrates, no synergistic effects were detected when converting PHB in the presence of algae. Subsequently, Synechocystis cf. salina, which had accumulated 7.5wt% PHB was converted via HTL (15% dry weight loading, 340°C). The reaction gave an overall propylene yield of 2.6%, higher than that obtained from the model compounds, in addition to a bio-oil with a low nitrogen content of 4.6%. No propylene was recovered from the alternative non-PHB producing cyanobacterial strains screened, suggesting that PHB is the source of propylene. PHB producing microorganisms could therefore be used as a feedstock for a biorefinery to produce polypropylene and advanced biofuels, with the level of propylene being proportional to the accumulated amount of PHB.

Optimisation of bio-oil extraction process from Beauty Leaf (Calophyllum inophyllum) oil seed as a second generation biodiesel source

The Beauty Leaf tree (Calophyllum inophyllum) is a potential source of non-edible vegetable oil for producing future generation biodiesel because of its ability to grow in a wide range of climate conditions, easy cultivation, high fruit production rate, and the high oil content in the seed. This plant naturally occurs in the coastal areas of Queensland and the Northern Territory in Australia, and is also widespread in south-east Asia, India and Sri Lanka. Although Beauty Leaf is traditionally used as a source of timber and orientation plant, its potential as a source of second generation biodiesel is yet to be exploited. In this study, the extraction process from the Beauty Leaf oil seed has been optimised in terms of seed preparation, moisture content and oil extraction methods. The two methods that have been considered to extract oil from the seed kernel are mechanical oil extraction using an electric powered screw press, and chemical oil extraction using n-hexane as an oil solvent. The study found that seed preparation has a significant impact on oil yields, especially in the screw press extraction method. Kernels prepared to 15% moisture content provided the highest oil yields for both extraction methods. Mechanical extraction using the screw press can produce oil from correctly prepared product at a low cost, however overall this method is ineffective with relatively low oil yields. Chemical extraction was found to be a very effective method for oil extraction for its consistence performance and high oil yield, but cost of production was relatively higher due to the high cost of solvent. However, a solvent recycle system can be implemented to reduce the production cost of Beauty Leaf biodiesel. The findings of this study are expected to serve as the basis from which industrial scale biodiesel production from Beauty Leaf can be made.

Prospects for the development of sugarcane biorefineries

Sugarcane biorefineries co-producing fuels, green chemicals and bio-products offer great potential for improving the profitability and sustainability of sugarcane industries around the world. Sugarcane bagasse is widely regarded as one of the best biomass feedstocks for early adoption and commercialisation of biorefining technologies because of the large scale of the resource and its availability at sugar factories. Biomass biorefineries aim to convert bagasse through biochemical and thermochemical processes to produce low cost fermentable sugars which are a platform for value-adding. Through subsequent fermentation technologies or chemical synthesis, the sugars can be converted to fuels including ethanol and butanol, oils, organic acids such as succinic and levulinic and polymer precursors. Other biorefinery products can include food and animal feeds, plastics, fibre products and resins. Recent advances in biorefinery production technologies are being demonstrated in a unique research facility at the Queensland University of Technology’s Mackay Renewable Biocommodities Pilot Plant in Mackay, Australia. This pilot scale production facility located at Mackay Sugar Ltd’s Racecourse Mill is demonstrating the production of a range of fuels and other products from sugarcane bagasse. This paper will address the opportunities available for sugarcane biorefineries to contribute to future profitability and sustainability of the sugarcane industry.

Bio-hydrogen production from food waste through anaerobic fermentation

In order to protect our planet and ourselves from the adverse effects of excessive CO2 emissions and to prevent an imminent non-renewable fossil fuel shortage and energy crisis, there is a need to transform our current ‘fossil fuel dependent’ energy systems to new, clean, renewable energy sources. The world has recognized hydrogen as an energy carrier that complies with all the environmental quality and energy security, demands. This research aimed at producing hydrogen through anaerobic fermentation, using food waste as the substrate. Four food waste substrates were used: Rice, fish, vegetable and their mixture. Bio-hydrogen production was performed in lab scale reactors, using 250 mL serum bottles. The food waste was first mixed with the anaerobic sewage sludge and incubated at 37°C for 31 days (acclimatization). The anaerobic sewage sludge was then heat treated at 80°C for 15 min. The experiment was conducted at an initial pH of 5.5 and temperatures of 27, 35 and 55°C. The maximum cumulative hydrogen produced by rice, fish, vegetable and mixed food waste substrates were highest at 37°C (Rice =26.97±0.76 mL, fish = 89.70±1.25 mL, vegetable = 42.00±1.76 mL, mixed = 108.90±1.42 mL). A comparative study of acclimatized (the different food waste substrates were mixed with anaerobic sewage sludge and incubated at 37°C for 31days) and non-acclimatized food waste substrate (food waste that was not incubated with anaerobic sewage sludge) showed that acclimatized food waste substrate enhanced bio-hydrogen production by 90 - 100%.

The economic value of new tropical biorefinery industries in Australia

Biorefineries, co-producing fuels, green chemicals and bio-products, offer great potential for enhancing agricultural value, and developing new industries in the bioeconomy. Biomass biorefineries aim to convert agricultural crops and wastes through biochemical and enzymatic processes to low cost fermentable sugars and other products which are platforms for value-adding. Through subsequent fermentation or chemical synthesis, the bio-based platforms can be converted to fuels including ethanol and butanol, oils, organic acids such as lactic and levulinic acid and polymer precursors. Other biorefinery products can include food and animal feeds, plastics, fibre products and resins. In 2014, QUT commissioned a study from Deloitte Access Economics and Correlli Consulting to assess the potential future economic value of tropical biorefineries to Queensland. This paper will report on the outcomes of this study and address the opportunities available for tropical biorefineries to contribute to the future profitability and sustainability of tropical agricultural industries in Queensland and more broadly across northern Australia.