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**Metagenomic sequencing for improved farming techniques with ethical considerations:**

**Abstract:**

(Figure 1) [] Global population increase, sustainable development and modern technologies have the potential to be a hinderence in the next 40-50 years, given that farming and environmental pressures placed on soil environments and the populations that inhabit them will be placed under more and more stress and strain. Without utilising modern technologies there cannot be any solution to such issues as soil depletion, eutrophication or loss of biodiversity due to the nature of globalisation and ever increasing demand of modern technologies that have the ability of making our lives simpler.

By using an integrated metagenomics (Figure 1) techniques to sequence soils and, by using other technologies such as web based management tools and statistics farmers are learning to better manage their land, which may be the solution to the many food issues, soil health issues and other sustainability problems. However, ethically there is a need to ensure such technologies are utilised with the full knowledge that there may also be consequences not yet known. This should not however detract from the use of such technologies but only serve as a method for consideration, so not to jeopardise their potential benefits.

//Key Words: Environment, Sustainability, Metagenomics, Soil, Technology, Ethics, Food//

**Objectives (Broader Master’s research, ISO 8801 and future ISO 8802 unit objectives):**

Consider a predicted doubling of a wealthier global population over the next six decades, from 7 billion people to 14 billion people (Pimentel 2011) (Figure 2). (Figure 2) [] This increase is predicted to rapidly drive the demand consequently for securing food. It expected also to increase the demand for land by 109 hectares of which will need to be converted from natural systems to agricultural farming systems and will require an increase in phosphorus and nitrogen and potassium fertilisers in the range of 2.4 – 2.7 times that already being used globally (Walsh 2009; Iqbal et al. 2010; Suter et al. 2010). Farming systems are related to environmental factors such as geography, climate, soil type, water availability and management. Farms however also require large amounts of fertiliser, pesticide and fuel, all of which are known to effect the surrounding environment and put increasing pressure on humanity. (Figure 3) [] The Haber-Bosch process and advances in mechanised farming techniques (Figure 2) have lead to a drastic increase in the world’s population. This has major social, economic and geographic implications in relation to sustainability of soil environments, including soils associated within the farming industry (Hong el al. 2010; Townsend and Howarth 2010). Microbial community dynamics relate to physical, chemical and biological properties when evaluating soil environments (Abbott and Murphy 2003; Cotching & Kidd 2010). Soil physical, chemical and biological properties are thus important because they are directly related to the surrounding environment and enable determination of human impacts (Marzaioli 2010). []
 * 1) To relate the diversity of microbial species found in dairy soil rhizospheres to specific measured soil physical, chemical and biological properties of those soils via statistical analysis, (as part of a Master’s Degree). Hence, to examine some nitrogen-induced changes associated with microbial diversity and abundance in nitrogen cycling processes (ammonia volatisation, nitrogen mineralisation, nitrogen fixation, nitrification, and de-nitrification) in Australian dairy farms.
 * 2) To investigate the possibility of using targeted gene specific metagenomic sequencing (via primer selection) to sequence the microbes extracted from dairy farm soil DNA, and to link these to efficient nutrient use, feed conservation efficiency, through the use of a glasshouse experiment.
 * 3) To investigate the outlook and potential concerns of farmers or general public in relation to genetically modified organisms, food stuffs and the impacts these may have in relation to the advancement of faming in Australia and globally and to provide an investigation into the ethics of such practices from a scientific point of view, especially in relation to metagenomics technology.
 * Background: **

Nitrogen is involved in the transcription and expression of genes in the polymerase chain reaction (PCR), forms the building of nucleic acids found in DNA molecules, most organic proteins, amino acids, polypeptides and enzymes, hence every known organism relies on nitrogen to some extent for its survival (Griffiths et al. 2008). Farmers use a range of inorganic fertilisers (anhydrous ammonium nitrate, NPK, saltpeter and urea) to selectively increase and control rates of production of plant species to increase crop yields, however nitrogen use by living plants and animals is up to 70% inefficient and anthropogenic alterations of nitrogen cycling of terrestrial and aquatic environments have lead to the following, globally,
 * An approximate doubling of the rate of nitrogen input into the terrestrial nitrogen cycle, with these rates still increasing;
 * Increased concentrations of the potent greenhouse gas N2O globally, and increased concentrations of other oxides of nitrogen that drive the formation of photochemical smog over large regions of Earth;
 * Caused losses of soil nutrients, such as calcium and potassium that are essential for the long-term maintenance of soil fertility;
 * Contributed substantially to the acidification of soils, streams, and lakes;
 * Greatly increased the transfer of nitrogen through rivers to estuaries and coastal oceans through leaching.

It’s also suggested with certainty that human alterations have: (Vitousek 1997; Gourley et al. 2007; Prosser et al. 2009; Galloway 2010; Liu et al. 2010)
 * Increased the quantity of organic carbon stored within terrestrial ecosystems;
 * Accelerated losses of biological diversity, especially plants adapted to efficient use of nitrogen, and losses of the animals and microorganisms that depend on them; and
 * Caused changes in the composition and functioning of estuarine and near shore ecosystems, and contributed to long-term declines in coastal marine fisheries.

For these reasons management of soil environments with respect to environmental ecosystems is important because it can be related to understanding sustainability of certain farming practices (Abbott & Murphy 2003; Walsh 2009; Iqbal et al. 2010; Suter et al. 2010). Measuring microbial diversity should include multiple methods integrating ‘holistic measures’ at the total community level and ‘partial approaches’ to target structural or functional subsets.

‘Metagenomic techniques’ (the field that analyses genetic materials recovered directly from environmental samples) (Figure 4) has shown that links can be made between land use type and specific, categorised physiological and functional observations of microbial communities and applied at the genetic level. This research will attempt to enable models of relationships between community diversity, abundance and function over particular ecological scales and land use types to be constructed through a process of metagenomic sequencing, interpretation and implementation (Prosser et al. 2009). (Figure 4) []

In relation to microbial community dynamics and land use type, diversity needs to be considered as distribution of an amount as information, which is directly applicable to the total genetic diversity or complexity in the community and this approach applied to the science (O’Malley and Dupre 2010). In this case, ‘soils associated with microbial communities in agricultural soil’ and the relationships this has with management on the broader farming scale needs assessment. Technologies aimed at aiding humanity such as those where functions of gene specific sequencing could be used in vertical glasshouse design. The two pictures below represent the possible progression, from DNA sequencing to glasshouse design for deserts. Such technology is possible (Courtesy Google Images).

As suggested earlier management of soil ecosystems can be modeled to basic ecosystem functions (Prosser et al. 2009). The nitrogen cycle thus is of vital importance when considering the management of farm systems in relation to sustainability principles.
 * Ecosystem Processes: **

Natural undisturbed ecosystems are systems where the difference between exchange of matter between compartments is entirely non-anthropogenic; for example in relation to nitrogen the overall net effect on nitrogen cycling and the net effect on the physical, chemical and biological properties to the surroundings is efficient and does not change (much) with respect to time (Krebs 2009). Many of the original plant species living in these ecosystems are adapted to, and function optimally in soils and solutions with low levels of available nitrogen representing efficient nitrogen usage (Vitousek et al. 1997). An example of a natural undisturbed ecosystem in Western Australia would be unlogged jarrah forests that have been fenced off to keep exotic species out.
 * Natural Ecosystems: **

Over the last few decades nitrogen inputs globally have been increasing and these rates are largely due to anthropogenic sources. In the associated dynamics of agricultural ecosystems these are vastly different to that of natural undisturbed ecosystems, due to induced alterations to the ecosystem required in changing from a natural state to one able, through management to support primary production such as crops or livestock. It is widely accepted that grasslands and managed pastures into the surrounding environment; that the amounts can be modeled to land use (Papale & Valentini, 2010).
 * Agricultural Ecosystems: **

The website [|http://www.soilquality.org.au] is a database, which includes web based calculators and fact sheets, management charts, and practical tools dedicated to providing information to farmers and the general public that may help them in managing land or for research based purposes. In this case the website will be used specifically in relation to the dairy industry. Another source of information regarding soils and soil health is the website [|http://www.soilhealth.org] which has been set up by Winthrop Professor Lynette Abbott and has information on it in relation mycorrhizas (fungi) and information to possibly the first soil scientist Charles Darwin and his observations of earth worms living in pots outside his verandah.

Because soil microbial communities play important roles in soil processes, the dynamic relationships between them or other physical, chemical and biological properties can be used to estimate soil microbial diversity and assess the management options for soils (Supaphol et al. 2011) (Figure 5). Processes occurring in soils depend largely on the land use type, due to the relationships between land use type and microbial species diversity, abundance and function for both natural and ed ecosystems (Marzaioli et al. 2010). (Figure 5) []
 * Microbial Processes in Soil: **

Land management practices as suggested by Abbott and Murphy (2003) have a considerable impact on the size and dynamics (physical, chemical and biological) of microbial populations, this view is shared by Botton et al. (2006) who suggests that this may be due to changes that alter the food web of the ecosystem whereby the resilience of the microbial community changes as a result of land use practices being applied to the surrounding ecology. This could explain why microbial communities regulate enzyme activity and preserve energy and nutrients in their biomass as suggested in Vitousek et al. (1997).

Without doubt soil fertility and the relationship it has with climate particularly rainfall and temperature is a significant factor in the nitrogen cycle. A study by Barton et al. (2010) showed that gaseous flux rates of nitrogen emissions from soils after rainfall events increased shortly after each event. “Respiration of soil microbes is related to temperature and emissions of NO2 (g) and NO3 (g)… cooler temperatures may promote dormancy of certain types of microbes in soils.”

Microbial soil interactions affecting nitrogen cycling and soil processes from Young et al. (2008) include; (i) Characteristics of the soil structure, (ii) Moisture characteristics, (iii) 3D structure–water interactions, (iv) Water-film thickness, (v) Surface area, (vi) Spatial and temporal distribution of microbes, (vii) Fungi in soil, (viii) Visualisation and quantification of fungal hyphae in soil, (ix) Relevance of spatial temporal dynamics to ecosystem function, (x) Habitat–biofilm interactions, (xi) Bacterial movement, (xii) The biofilm environment, (xiii) Celluose in soil, (xiv) Surfactants in soil, (xv) Habitat–microbe interactions, (xvi) Regulatory feedbacks in soil–microbe interactions and (xvii) The soil–microbe complex as a complex adaptive system.

In any soil study, the questions that are posed ultimately determine the sampling design that is appropriate (Smalla and van Elsas 2010). Evidence linking molecular techniques to farm management can be found in physiological and functional observations of microbial communities at the genetic level that may enable the determination of relationships between community structures and functions and even single microorganism ecological functions as achieved in Lauber et al. (2008) and Prosser et al. (2009). These studies essentially integrate land use type with a range of physical, chemical and biological soil dynamics to provide insights into the soil microbial diversity, abundance and function of the particular agricultural areas.
 * Metagenomics: **

Metagenomics is a relatively new (circa 1990’s) series of methods for the analysis of samples containing the elements of life (essentially C, N, O and H). Within soil science, metagenomics has become to be known as the analysis of genetic materials directly from environmental samples. The methods are now the most widely used and are cost effective and readily available for assessing soil microbial relationships at diverse scalar environments.

Studies have been done on the continental (Leininger et al. 2006), oceanic (Tringe et al. 2005),terrestrial(Quince et al. 2008) scales, and on managed agricultural pastures (Buckley and Schmidt 2001; Acosta-Mart´nez et al. 2010), and most specifically in analysing the effect of varying land use on dairy soils relating to microbial community diversity and abundance (Peacock et al. 2001). Where relevant metagenomic techniques fit into the broader scheme of the science and the methods available for this research is ultimately determined by the desired outcome of the research as mentioned earlier.

The metagenomic methods available at UWA are a follow on from that of the work of Supaphol et al. (2011) and represent a new range of options available at UWA. These methods are still in development to date but as of this publication, the procedures include; (i) DNA extraction from microbes contained in soil, (ii) DNA amplification using targeted gene specific primers, (iii) Metagenomic sequencing of amplified targeted gene sets via Ion Torrent (Figure 6) technology based on the principle that protons released during DNA polymerization can detect nucleotide incorporation, (iv) Decoding sequenced DNA amplicons via uploading onto a cloud computer online database located at [|http://www.amazon.com], and (v) Cross reference use of The Kyoto Encyclopedia of Genes and Genomes (KEGG) and BLAST® (Basic Local Alignment Search Tool) located within NCBI (The National Center for Biotechnology Information). (Figure 6) []

The NCBI database allows sequenced (AT and GC) base pairs to be BLAST(ed) for taxanomic approximations of species identities (given as maximum identity in %) from within the entire nucleotide sample database (Zvelebil and Baum 2008). This is all done on the targeted genes through an Amazon run cloud computer application.

The website based local alignment search assessment tool (BLAST) is located at;

(Figure 7) [] Development of predictive frameworks is crucial for managing soil biology and the essential functions and services. Key areas include; (i) causal links between soil biology and structure, physicochemical factors and ecological processes (e.g. nutrient cycling, soil carbon sequestration) that contribute to plant community development and function, (ii) how soil communities respond to and impact on plant succession and weed species, (iii) the role of plant–soil feedbacks in determining the evolutionary dynamics of soil mutualists and pathogens and (iv) impacts of anthropogenic disturbance on soil diversity and function.
 * Microbial Gene Function Relationships to Diversity (Figure 7) **:  [[image:http://rd.springer.com/chapter/10.1007%2F978-90-481-3333-8_10/lookinside/000.png]]

Practical issues concerning soil health provide example of where eco-genomic and metagenomic approaches can open opportunities to ask entirely new questions (Thrall et al. 2011).

Microbes in communities’ genes are compact and rich, coding for function as a fraction of the genome, the distribution of the bacterial genome is related to size and distribution. Bacteria with small genome sizes tend to have narrow ecological niches, whereas bacteria with larger genomes (with more diverse functions) are typically more ecologically diverse and metabolically active (Cole et al. 2010).

Characterised genes known to be associated with nitrogen pathways and cycling in the nitrogen cycling include those associated with nitrogen fixation (nifH) which encodes for an iron protein (Poly et al. 2001; Yan 2010), nitrifiers (including amoA) (Leininger et al. 2006), the denitrifiers nirK and nirS (attributed to the conversion of NO2 à NO3) (Enwall et al. 2010) and the nosZ gene (attributed to the conversion of NO à N2O) (Mele and Crowley 2008).

The main effects that nitrogen has on microbial communities and dynamics, relates directly to the transcription of genes that are associated with a particular part(s) of the nitrogen cycle e.g. N fixation, nitrification, ammonification and de-nitrification. This is due to the regulatory networks that respond to the availability of fixed nitrogen being highly controlled at the transcriptional level.

Yan et al. (2010) suggests that there are possibly undiscovered genes involved directly in the nitrogen fixation process. In summary measures of N cycling genes could be used as an additional indicator of soil health to assess potential ecosystem functions to improve sustainability and efficiency (Cavagnaro et al.2008). Metagenomics can demonstrate relationships between distributions of microorganisms at a range of scales, as mentioned earlier. However there are limitations. Important aspects of diversity and abundance at the ecosystem level are the range of processes and the complexity of interactions, which can vary greatly due to many factors. Some critical aspects of modern metagenomic techniques and analysis include but are not necessarily limited to the following;
 * What are Limitations to Methods of Metagenomic Analyses? **

It is thus of vital importance that the correct procedure be selected and the research directed specifically at the objectives of the research such that the most suitable option be selected. (Figure 8)[] There has always been a concern that by modifying the natural environment that there might be negative impacts in relation to the food chain (Figure 8). Such food stuffs are known as genetically modified food stuffs. Issues in the environmental assessment of GM crops are putative invasiveness, vertical or horizontal gene flow, other ecological impacts, effects on biodiversity and the impact of presence of GM material in other products, at the broad scale. There are also highly interdisciplinary and complex issues directly related to the abovementioned issues (Curtis et al. 2004).
 * A broad range of techniques is critical for comprehensive and realistic understanding of biological communities (Liu et al. 2010),
 * Approximately 60% of genes in all micro-organisms are ubiquitous and have different functions in similar species and organisms, such that the presence of DNA sequences may not be sufficient to link genes to specific function(s) Guazzaroni et al. (2010),
 * Low abundance (<0.1%) of genes can be linked to important pathway functions. Therefore, relative abundances of certain groups of microorganisms are not necessarily linked to the importance of that group in a community, particularly in relation to nitrogen pathways and cycling Guazzaroni et al. (2010),
 * Some studies suggest that specific changes in edaphic properties, not necessarily land-use type itself, may best predict shifts in microbial community (Lauber et al. 2008),
 * Single celled organisms are taxonomically diverse and estimations of species or phyla numbers are difficult to determine due to their ability to use free extracellular DNA to evolve and adapt via a complex series of mechanisms in horizontal gene transfer (Fierer et al. 2007; Koonin and Wolf 2008; Xu 2010),
 * Metagenomics based on single or communities of organisms alone may not provide a quantitative understanding of microbial communities (Blow 2008).
 * The 16 S rDNA gene is, only 0.05% of the bacterial genome in length on average, and all but in a few cases (e.g. the nitrifying bacteria) is of limited value in predicting physiology or environmental interaction of organisms (O’Donnell et al. 2003).
 * Discussion on Ethical Considerations of Employing Eugenics via Technology in Farming: ** [[image:http://www.marymeetsdolly.com/blog/uploads/mban1450l.jpg]]

A crucial component for a proper assessment is defining the appropriate baseline for comparison and decision (Conner et al. 2003). Some issues associated with the use of GM ingredients in food products from the consumer point of view are largely negative in many of the developed countries in the European Union as well as Japan. Consumer skepticism in these countries is usually attributed to the unknown environmental and health consequences of genetically modified crops. Such consequences include, but are not limited to, unanticipated allergic responses, the spread of pest resistance or herbicide tolerance to wild plants, and inadvertent toxicity to wildlife (Curtis et al. 2004).

Increasingly, the use of GM crops will require research agronomists, ecologists, farmers and policy makers alike to take more of a systems perspective which considers the broader evolutionary consequences of the traits in question. If we choose to design plants containing genes that may enable better nutrient-use efficiency, an understanding of function in an agricultural production context must first be gained. This is because induced genetic changes may also alter multiple management systems that in turn could alter species competitive interactions. For example, there is potential for both positive and negative ecological and evolutionary feedbacks between novel crops, herbivores and weeds, and soil microbial communities (Thrall et al. 2011). Having said this, “explicit recognition of the immense value of information from such initiatives could facilitate a quantum leap in our understanding of the ecological and evolutionary responses of agro-ecosystems” (Thomas et al. 2012).

The real question in considering the human induced evolution of organisms through technologies is how much different are these processes of forced evolution differ to that of natural selection (Figure 9)? Essentially when humans take the genes from something and place them in something else we are speeding up the polymerase chain reaction, the process that enables DNA to self replicate. The genes that make up the DNA molecule within an organism can be transferred through both vertical and horizontal gene transfer. What if any are the actual consequences of this? Does human genetic manipulation of DNA differ to that of gene transfer in the environment? []

One way to look at it is to ask whether humans exist solely independently of the surrounding environment, or whether humans exist solely dependently of the surrounding environment. We are at the top of the food chain, but does this exclude us from our interaction with the smallest organisms? Either way the technology at our fingertips is evolving too, along with our DNA and the entire global combined DNA also. Is this an independent global process? I would think yes, even if humans were not on earth, unless there was an Armageddon DNA would still continue to exist and evolve without us. Hence ethically although genetic manipulation of microbes and plants and animals may be seen as negative by many people, it does not exclude us from what is happening in nature independently of time.

Regardless of such debate it is important that metagenomics be taught to students and young scientists in the same way that other techniques and approaches have been in the past because science has been at the forefront of improving human existence ever since the great alchemists and scholars over the centuries delved into areas in the belief that they were doing so for the betterment of humanity.

// “Every revolutionary idea seems to evoke three stages of reaction. They may be summed up by the phrases: 1- It's completely impossible. 2- It's possible, but it's not worth doing. 3- I said it was a good idea all along.” A.C. Clarke //

The work presented here is a continuation and supplementary to that which I am doing for my Master’s Degree. It is integration and collaboration, whereby I complete the following two experiments proposed below. Instead of answering the hypotheses and working toward objectives of the Master’s project, I will fulfill the objectives and hypotheses set out in this Report and continue on into the ISO 8802 unit, in relation to the experiments described below.
 * Experimental Design / Pre-schedule: **

Two glasshouse experiments that use a range of treatments: plant species, nitrogen and phosphorus, sampled at different times based on previous literature reviews and project work,
 * Glasshouse Rhizosphere Experiment: **

Experimental Design (Exp 1): 3 N x 3 plant x 2 cycles x 3 reps
 * N: (i) 0, (ii) 0.5 kg N per ha per day, (iii) 2.5 kg N per ha per day,
 * Plant: (i) ryegrass, (ii) clover, (iii) ryegrass + clover.

Mycorrhizal development (2 months), plant growth and soil microbial diversity assessment (soil DNA extraction, Ion Torrent sequencing of N cycling genes via targeted gene specific primers*)

Experimental Design (Exp 2): 3 N x 3 P 1 plant (clover) x 2 cycles x 3 reps, Cycle 1 - Mycorrhiza development (at 2 months), plant growth, plant N and P to be assessed
 * N: (i) 0, (ii) 0.5 kg N per ha per day, (iii) 2.5 kg N per ha per day
 * P: (i) 0, (ii) 1X, (iii) 2X (based on farmer use) and soil testing

Cycle 2 - Mycorrhizal development assessed (at 4 months) plant growth, plant N and P to be assessed

* The methods for the Ion Torrent Sequencer are still yet to be ascertained through collaboration with other working groups within the University structure.

Microbial diversity and abundance assessments will be measured against the physicochemical parameters, and canonical correspondence analysis charts (CCA) or multivariate statistics constructed showing diversity of microbial communities represented in soils via proximity to vectors.

Environmental variables are presented as vectors on the two CCA charts. The charts show the relationships between farm paddock treatments and microbial organisms to the vectors. From the two graphs relationships between land use and specific organisms can be found and related back to management of the farm land use. For example if a paddock on farm A has more nitrogen fixers then it could be suggested to grow plant types that do not fix their own nitrogen well as a management option. These graph have been completed as part of a Master’s Preliminary Course in 2010/11.


 * Global Ethical Management Committee Estabilshment: **

To investigate the creation of a global ethical management committee in relation to using technologies for the benefit of humanity by looking at if there are any such groups already established especially in relation to metagenomics and other sequencing technologies. [] To bring community groups together to discuss their opinions in relation to forming such a committee and to collectively forge a new way of thinking about the use of technology in relation to farming, soils, water and air. This could be related to any form of technology but the overarching theme would be to discuss amoungst the group any technology and its benefits, its shortfalls, pit traps etc. and to then some look at the legislation and to see if there would be a way to allow the committee to approve under sectional guidelines within patent law, or broader environmental law. Thus new policies can be made. Essentially we are not trying to achieve what has been happening in the above cartoon. []
 * Aim: **

I will attempt to do some of this during the 8802 unit, should I chose to enroll.

With the advent of many modern farm techniques such as large scale mechanised cropping machines, bio-informatics approaches, metagenomics and bio-economics these have a profound impacts in relation to microbial community assessments. Especially in relation to how these complex and diverse systems evolve and can be managed. Based on the information within this report it is expected that there may be relationships directly related to species diversity, abundance and composition at the genetic level, which will be modeled statistically.
 * Conclusion: **

Given that farms must operate within new emerging environmental legislation, all the surrounding environmental dynamics and ecological processes must now be considered within the farm management context if the ultimate goal is sustainability. This is because some of the main drivers of farming success are probably related to emerging niches including; microbial evolution in natural ecosystems, change of microbial diversity, change of abundance and change of function over time.

Having said this careful consideration must be placed on the ethical decision making processes in relation to ecological and evolutionary responses of agro-ecosystems to genetic modification that much of this research relates to. Special consideration needs to be placed on the overall amount and distribution of information in a gene pool of a community, directly applicable to total genetic diversity or complexity in the while community, because the sum of all genetic information is freely available to any other organism via gene transfer (Fierer et al. 2007; Koonin and Wolf 2008; Xu 2010). This has significant implication in relation to genetically modified food and the use of sequencing technology too.

These technologies have the potential to transform the farming industry though improved efficiency of farming practices, because metagenomics technology allows for specialised treatments of individual soil types, paddocks and farms due to the diverse nature of the soils within each land use type/farm. However to what extent should this technology go, and what are the major ethical questions that need to be asked?

Do all surrounding environmental processes affecting microbial communities and individuals generate enough information for comparisons and determinations between diversity, abundance and function to be made (quantitatively) in relation to farming management decisions? Or are the sample populations of microbes in environmental samples constantly adapting and evolving, such that farming management decisions based on such observations continue to elude scientific efforts? A significant ‘gap’ is in that there are so many limitations to the technology as mentioned earlier. Further hindrance with respect to estimation of function of microbes in soil exists due to pluralisms in the ncbi database with possibly multiple copies of the same species existing in the library making estimations of function difficult. Why so? Possibly the most difficult aspect of this research however is in displaying the information discovered in the research to farmers such that they can then make the most of the technological information which is being presented.
 * Questions that will be asked in 8802 after more research: **

Where are we going as a global civilisation in the environment as it is today? Will a world where modern technology and advancements in robotics and genetics as futurised in Clark’s 3001 become true? Modern technologies particularly robots have to be considered as major uses in a world where population increase and demand for resources put more pressure on humanity. Holistically if one were to consider Asimov’s 3 laws of robotics; (i) A robot may not injure a human being or, through inaction, allow a human being to come to harm, (ii) A robot must obey the orders given to it by human beings, except where such orders would conflict with the First Law and (iii) A robot must protect its own existence as long as such protection does not conflict with the First or Second Laws, in relation to metagenomics and gene sequencing (all robotic functions) described above the outcomes can only be described as beneficial to humanity. http://svati.wikispaces.com/
 * The future: **


 * Hypotheses: **
 * 1) Diversity and relative abundance of microbial organisms in soil is related to land management practices associated with management of; (i) soil type, (ii) fertiliser application, (iii) pasture species selection and (iv) other environmental dynamics (e.g. temperature and rainfall).
 * 2) Detection of nitrogen cycling genes within soil microbes varies with N application, plant community composition, soil physicochemical properties and the particular metagenomic methods employed for analysis. This technology in turn has a potential benefit to enable enhanced food production with lesser environmental impacts. However there are possible ethical decisions that need to be made in relation to the technology.
 * 3) The future of agriculture will be dependent on efficiency that maximises sustainable use of fertilisers and minimises environmental impacts these have on the surrounding environment through the use of modern technologies including metagenomics and robotics (mechanised farming, fully automated feeding and milking systems).

Abbott LK, Murphy DV (2003) What is soil biological fertility? In ‘Soil Biological Fertility – A Key to Sustainable Land Use in Agriculture’. (Eds. LK Abbott, DV Murphy) pp. 1–16. (Kluwer Academic Publishers: The Netherlands)
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