Molecular Biomonitoring during Rhizoremediation of Oil-Contaminated Soil

Rhizoremediation is the use of microbial populations present in the rhizosphere of plants for environmental cleanup. The idea of this work was that bacteria living in the rhizosphere of a nitrogen-fixing leguminous plant, goat's rue (Galega orientalis), could take part in the degradation of harmful monoaromatic hydrocarbons, such as benzene, toluene and xylene (BTEX), from oil-contaminated soils. In addition to chemical (e.g. pollutant concentration) and physical (e.g. soil structure) information, the knowledge of biological aspects (e.g. bacteria and their catabolic genes) is essential when developing the rhizoremediation into controlled and effective bioremediation practice. Therefore, the need for reliable biomonitoring methods is obvious. The main aims of this thesis were to evaluate the symbiotic G. orientalis - Rhizobium galegae system for rhizoremediation of oil-contaminated soils, to develop molecular methods for biomonitoring, and to apply these methods for studying the microbiology of rhizoremediation. In vitro, Galega plants and rhizobia remained viable in m-toluate concentrations up to 3000 mg/l. Plant growth and nodulation were inhibited in 500 mg/l m-toluate, but were restored when plants were transferred to clean medium. In the greenhouse, Galega showed good growth, nodulation and nitrogen fixation, and developed a strong rhizosphere in soils contaminated with oil or spiked with 2000 mg/l m-toluate. The high aromatic tolerance of R. galegae and the viability of Galega plants in oil-polluted soils proved this legume system to be a promising method for the rhizoremediation of oil-contaminated soils. Molecular biomonitoring methods were designed and/or developed further for bacteria and their degradation genes. A combination of genomic fingerprinting ((GTG)5-PCR), taxonomic ribotyping of 16S rRNA genes and partial 16S rRNA gene sequencing were chosen for molecular grouping of culturable, heterogeneous rhizosphere bacteria. PCR primers specific for the xylE gene were designed for TOL plasmid detection. Amplified enzyme-coding DNA restriction analysis (AEDRA) with AluI was used to profile both TOL plasmids (xylE primers) and, in general, aromatics-degrading plasmids (C230 primers). The sensitivity of the direct monitoring of TOL plasmids in soil was enhanced by nested C23O-xylE-PCR. Rhizosphere bacteria were isolated from the greenhouse and field lysimeter experiments. High genetic diversity was observed among the 50 isolated, m-toluate tolerating rhizosphere bacteria in the form of five major lineages of the Bacteria domain. Gram-positive Rhodococcus, Bacillus and Arthrobacter and gram-negative Pseudomonas were the most abundant genera. The inoculum Pseudomonas putida PaW85/pWW0 was not found in the rhizosphere samples. Even if there were no ecological niches available for the bioaugmentation bacterium itself, its conjugative catabolic plasmid might have had some additional value for other bacterial species and thus, for rhizoremediation. Only 10 to 20% of the isolated, m-toluate tolerating bacterial strains were also able to degrade m-toluate. TOL plasmids were a major group of catabolic plasmids among these bacteria. The ability to degrade m-toluate by using enzymes encoded by a TOL plasmid was detected only in species of the genus Pseudomonas, and the best m-toluate degraders were these Pseudomonas species. Strain-specific differences in degradation abilities were found for P.oryzihabitans and P. migulae: some of these strains harbored a TOL plasmid - a new finding observed in this work, indicating putative horizontal plasmid transfer in the rhizosphere. One P. oryzihabitans strain harbored the pWW0 plasmid that had probably conjugated from the bioaugmentation Pseudomonas. Some P. migulae and P. oryzihabitans strains seemed to harbor both the pWW0- and the pDK1-type TOL plasmid. Alternatively, they might have harbored a TOL plasmid with both the pWW0- and the pDK1-type xylE gene. The breakdown of m-toluate by gram-negative bacteria was not restricted to the TOL pathway. Also some gram-positive Rhodococcus erythropolis and Arthrobacter aurescens strains were able to degrade m-toluate in the absence of a TOL plasmid. Three aspects of the rhizosphere effect of G. orientalis were manifested in oil-contaminated soil in the field: 1) G. orientalis and Pseudomonas bioaugmentation increased the amount of rhizosphere bacteria. G. orientalis especially together with Pseudomonas bioaugmentation increased the numbers of m-toluate utilizing and catechol positive bacteria indicating an increase in degradation potential. 2) Also the bacterial diversity, when measured as the amount of ribotypes, was increased in the Galega rhizosphere with or without Pseudomonas bioaugmentation. However, the diversity of m-toluate utilizing bacteria did not significantly increase. At the community level, by using the 16S rRNA gene PCR-DGGE method, the highest diversity of species was also observed in vegetated soils compared with non-vegetated soils. Diversified communities may best guarantee the overall success in rhizoremediation by offering various genetic machineries for catabolic processes. 3) At the end of the experiment, no TOL plasmid could be detected by direct DNA analysis in soil treated with both G. orientalis and Pseudomonas. The detection limit for TOL plasmids was encountered indicating decreased amount of degradation plasmids and thus, the success of rhizoremediation. The use of G. orientalis for rhizoremediation is unique. In this thesis new information was obtained about the rhizosphere effect of Galega orientalis in BTEX contaminated soils. The molecular biomonitoring methods can be applied for several purposes within environmental biotechnology, such as for evaluating the intrinsic biodegradation potential, monitoring the enhanced bioremediation, and estimating the success of bioremediation. Environmental protection by using nature's own resources and thus, acting according to the principle of sustainable development, would be both economically and environmentally beneficial for society. Keywords: molecular biomonitoring, genetic fingerprinting, soil bacteria, bacterial diversity, TOL plasmid, catabolic genes, horizontal gene transfer, rhizoremediation, rhizosphere effect, Galega orientalis, aerobic biodegradation, petroleum hydrocarbons, BTEX

How weed control and fertilisation influence tree physiological processes and growth at early establishment in an exotic F1 hybrid pine plantation of subtropical Australia

Purpose This study investigated how nitrogen (N) nutrition and key physiological processes varied under changed water and nitrogen competition resulting from different weed control and fertilisation treatments in a 2-year-old F1 hybrid (Pinus elliottii Engelm var. elliottii × P. caribaea var. hondurensis Barr. ex Golf.) plantation on a grey podzolic soil type, in Southeast Queensland. Materials and methods The study integrated a range of measures including growth variables (diameter at ground level (DGL), diameter at breast height (DBH) and height (H)), foliar variables (including foliar N concentration, foliar δ13C and δ15N) and physiological variables (including photosynthesis (An), stomatal conductance (gs), transpiration (E), intrinsic water use efficiency (WUEi) (A/gs) and xylem pressure potential (ΨXPP)) to better understand the mechanisms influencing growth under different weed control and fertilisation treatments. Five levels of weed control were applied: standard (routine), luxury, intermediate, mechanical and nil weed control, all with routine fertilisation plus an additional treatment, routine weed control and luxury fertilisation. Relative weed cover was assessed at 0.8, 1.1 and 1.6 years after plantation establishment to monitor the effectiveness of weed control treatments. Soil investigation included soil ammonium (NH4 +-N), nitrate (NO3 −-N), potentially mineralizable N (PMN), gravimetric soil moisture content (MC), hot water extractable organic carbon (HWETC), hot water extractable total N (HWETN), total C, total N, stable C isotope composition (δ13C), stable N isotope composition (δ15N), total P and extractable K. Results and discussion There were significant relationships between foliar N concentrations and relative weed cover and between tree growth and foliar N concentration or foliar δ15N, but initial site preparation practices also increased soil N transformations in the planting rows reducing the observable effects of weed control on foliar δ15N. A positive relationship between foliar N concentration and foliar δ13C or photosynthesis indicated that increased N availability to trees positively influenced non-stomatal limitations to photosynthesis. However, trees with increased foliar N concentrations and photosynthesis were negatively related to xylem pressure potential in the afternoons which enhanced stomatal limitations to photosynthesis and WUEi. Conclusions Luxury and intermediate weed control and luxury fertilisation positively influenced growth at early establishment by reducing the competition for water and N resources. This influenced fundamental key physiological processes such as the relationships between foliar N concentration, A n, E, gs and ΨXPP. Results also confirmed that time from cultivation is an important factor influencing the effectiveness of using foliar δ15N as an indicator of soil N transformations.

Rural Income Generation and Diversification : A Case Study in Eastern Zambia

Rural income diversification has been found to be rather the norm than the exception in developing countries. Smallholder households tend to diversify their income sources because of the need to manage risks, secure a smooth flow of income, allocate surplus labour, respond to various kinds of market failures, and apply coping strategies. The Agricultural Household Model provides a theoretical rationale for income diversification in that rural households aim at maximising their utility. There are several elements involved, such as agricultural production for their own consumption and markets, leisure activities and income from non-farm sources. The aim of the present study is to enhance understanding of the processes of rural income generation and diversification in eastern Zambia. Specifically, it explores the relationship between household characteristics, asset endowments and income-generation patterns. According to the sustainable- rural-livelihoods framework, the assets a household possesses shape its capacity to seize new economic opportunities. The study is based on two surveys conducted among rural smallholder households in four districts of Eastern Province in Zambia in 1985/86 and 2003. Sixty-seven of the interviewed households were present in both surveys and this panel allows comparison between the two points of time. The initial descriptive analysis is complemented with an econometric analysis of the relationships between household assets and income sources. The results show that, on average, 30 per cent of the households income originated from sources outside their own agriculture. There was a slight increase in the proportion of non-farm income from 1985/86 to 2003, but total income clearly declined mainly on account of diminishing crop income. The land area the household was able to cultivate, which is often dependent on the available labour, was the most significant factor affecting both the household-income level and the diversification patterns. Diversification was, in most cases, a coping strategy rather than a voluntary choice. Measured as income/capita/day, all households were below the poverty line in 2003. The agricultural reforms in Zambia, combined with other trends such as changes in rainfall pattern, the worsening livestock situation and the incidence of human disease, had a negative impact on agricultural productivity and income between 1985/86 and 2003. Sources of non-farm income were closely linked to agriculture either upstream or downstream and the income they generated was not enough to compensate for the decline of agricultural income. Household assets and characteristics had a smaller impact on diversification patterns than expected, which could reflect the lack of opportunities in the remote rural environment.

Assessing the sustainability of wheat-based cropping systems using simulation modelling: sustainability

Concepts of agricultural sustainability and possible roles of simulation modelling for characterising sustainability were explored by conducting, and reflecting on, a sustainability assessment of rain-fed wheat-based systems in the Middle East and North Africa region. We designed a goal-oriented, model-based framework using the cropping systems model Agricultural Production Systems sIMulator (APSIM). For the assessment, valid (rather than true or false) sustainability goals and indicators were identified for the target system. System-specific vagueness was depicted in sustainability polygons-a system property derived from highly quantitative data-and denoted using descriptive quantifiers. Diagnostic evaluations of alternative tillage practices demonstrated the utility of the framework to quantify key bio-physical and chemical constraints to sustainability. Here, we argue that sustainability is a vague, emergent system property of often wicked complexity that arises out of more fundamental elements and processes. A 'wicked concept of sustainability' acknowledges the breadth of the human experience of sustainability, which cannot be internalised in a model. To achieve socially desirable sustainability goals, our model-based approach can inform reflective evaluation processes that connect with the needs and values of agricultural decision-makers. Hence, it can help to frame meaningful discussions, from which actions might emerge.

Sustainable grazing management for temporal and spatial variability in north Australian rangelands – a synthesis of the latest evidence and recommendations

Rainfall variability is a major challenge to sustainable grazing management in northern Australia, with management often complicated further by large, spatially-heterogeneous paddocks. This paper presents the latest grazing research and associated bio-economic modelling from northern Australia and assesses the extent to which current recommendations to manage for these issues are supported. Overall, stocking around the safe long-term carrying capacity will maintain land condition and maximise long-term profitability. However, stocking rates should be varied in a risk-averse manner as pasture availability varies between years. Periodic wet-season spelling is also essential to maintain pasture condition and allow recovery of overgrazed areas. Uneven grazing distributions can be partially managed through fencing, providing additional water-points and in some cases patch-burning, although the economics of infrastructure development are extremely context-dependent. Overall, complex multi-paddock grazing systems do not appear justified in northern Australia. Provided the key management principles outlined above are applied in an active, adaptive manner, acceptable economic and environmental outcomes will be achieved irrespective of the grazing system applied.

Can sustainable cotton production systems be developed for tropical northern Australia?

This article reviews research coordinated by the Australian Cotton Cooperative Research Centre (CRC) that investigated production issues for irrigated cotton at five targeted sites in tropical northern Australia, north of 21°S from Broome in Western Australia to the Burdekin in Queensland. The biotic and abiotic issues for cotton production were investigated with the aim of defining the potential limitations and, where appropriate, building a sustainable technical foundation for a future industry if it were to follow. Key lessons from the Cotton CRC research effort were: (1) limitations thought to be associated with cotton production in northern Australia can be overcome by developing a deep understanding of biotic and environmental constraints, then tailoring and validating production practices; and (2) transplanting of southern farming practices without consideration of local pest, soil and climatic factors is unlikely to succeed. Two grower guides were published which synthesised the research for new growers into a rational blueprint for sustainable cotton production in each region. In addition to crop production and environmental impact issues, the project identified the following as key elements needed to establish new cropping regions in tropical Australia: rigorous quantification of suitable land and sustainable water yields; support from governments; a long-term funding model for locally based research; the inclusion of traditional owners; and development of human capacity.