Degradation of lignin and other 14C-labelled compounds in compost and soil with an emphasis on white-rot fungi

Puu, ruohokasvit ja näistä tehdyt tuotteet kuten mekaanisesta massasta valmistettu sanomalehtipaperi sisältävät ligniiniä, joka hajoaa yleensä hyvin hitaasti luonnossa. Valkolahosienet hajottavat ligniiniä tehokkaimmin, ja koska niiden tuottamat entsyymit hajottavat myös muita vaikeasti hajoavia yhdisteitä, voidaan valkolahosienten avulla mahdollisesti puhdistaa saastuneita maita. Tässä työssä haluttiin selvittää, säilyttävätkö valkolahosienet (Abortiporus biennis, Bjerkandera adusta, Dichomitus squalens, Phanerochaete chrysosporium, Phanerochaete sordida, Phlebia radiata, Pleurotus ostreatus, Trametes hirsuta ja Trametes versicolor) aktiivisuutensa ja kasvavatko ne maassa. Aktiivisuutta mitattiin seuraamalla sienten synteettisen ligniinin (14C-DHP) hajotuskykyä. T. versicolor (silkkivyökääpä) osoittautui tehokkaimmaksi ligniinin hajottajaksi ja sen pentakloorifenolin (PCP) hajotuskykyä tutkittiin erillisessä kokeessa. Entiset tai pitkään käytössä olleet saha-alueet ovat yhä saastuneet puun käsittelyaineista peräisin olevilla kloorifenoleilla. Biohajoavien muovien kehitystyö sekä kompostoinnin yleistyminen jätteiden käsittelymenetelmänä ovat luoneet tarpeen materiaalien biohajoavuuden määrittämiseen. Euroopan standardisoimisjärjestön (CEN) kontrolloidussa kompostitestissä biohajoavuus määritetään materiaalin hajoamisen aikana muodostuvan hiilidioksidin perusteella. Hiilidioksidin tuotto mitataan sekä näytettä sisältävästä kompostista että kompostista ilman näytettä, ja tällöin oletetaan, että kompostin orgaaninen aines molemmissa komposteissa (tausta) tuottaa yhtä paljon hiilidioksidia. Testin puutteeksi saattaa osoittautua kompostissa tai maassa esiintyvä "priming effect". Tällä tarkoitetaan materiaalin lisäämisen jälkeen esiintyvää epänormaalin suurita tai pientä hiilidioksidin muodostusta, minkä seurauksena testin tulosksena saatava biohajoavuus on virheellinen. Ligniinin hajotessa muodostuu enemmän humusta kuin hiilidioksidia, koska ligniini on humuksen tärkein lähtöaine. Näin ollen ligniiniä sisältävät paperituotteet saattavat testin mukaan vaikuttaa biologisesti hajoamattomilta. Valkolahosienet hajottivat 4-23% ligniinistä hiilidioksidiksi ja T. versicolor 29% PCP:sta. Kompostissa ligniini hajosi hiilidioksidiksi 58°C:ssa huomattavasti vähemmän (8%) kuin lämpötiloissa 35°C ja 50°C (23-24%). Kompostin todennäköisesti tärkeimpien ligniinin hajottajien, termofiilisten sienten, tyypillinen optimilämpötila on 45°C, eivätkä ne ole enää aktiivisia 58°C:ssa. Sekä maassa että kompostissa ligniini sitoutui kuitenkin suurimmaksi osaksi humukseen. Valkolahosienet hajottivat sekä humukseen sitoutunutta ligniiniä että PCP:ia, mutta kompostin sekapopulaatio ei tähän pystynyt, ja ligniiniä sitoutui humukseen yhä enemmän kompostoinnin aikana. T. versicolor hajotti PCP:ia tehokkaasti, eikä se tuottanut myrkyllisiä kloorianisoleja, joita jotkut valkolahosienet saattavat muodostaa kloorifenoleista. Priming effect ilmiötä tutkittiin eri ikäisissä ja kypsyydeltään erilaisissa komposteissa. Kompostit erosvat toisistaan myös hajoamattoman jätteen määrän ja mikrobipopulaation suhteen. Negatiivinen priming effect havaittiin kaikissa epästabiileissa komposteissa (ikä enintään 6 kk), ja sen lisäksi yhdessä näistä komposteista positiivinen priming effect kokeen lopussa. Stabiileissa komposteissa (ikä vähintään 6 kk) ilmiötä ei sen sijaan havaittu. Epästabiileissa komposteissa biohajoavuudelle saadut tulokset eivät siis ole luotettavia. Työn tulosten perusteella valkolahosienet, ja erityisesti T. versicolor, ovat lupaavia saastuneen maan puhdistukseen, joskin sienirihmaston mahdollisuudet säilyä aktiivisena maan alkuperäisen mikrobipopulaation kanssa täytyy vielä selvittää. Kompostin sekapopulaatio, joka ei sisällä valkolahosieniä, hajotti ligniiniä yllättävän tehokkaasti termofiilisille sienille sopivissa lämpötiloissa, vaikka ligniini sitoutuikin pääasiallisesti humukseen. Kompostin kypsyys osoittautui tärkeäksi tekijäksi kontrolloidun kompostitestin onnistumisen kannalta. Priming effect ilmiön välttämiseksi on varmistettava, että testissä käytetty komposti on riittävän kypsä. Kompostien mikrobipopulaation koostumusta kompostoinnin eri vaiheissa tulisi tarkemmin selvittää, koska stabiilien ja epästabiilien kompostien ero aiheutui todennäköisesti populaatioiden rakenteessa vallitsevista eroista. Näin myös priming effect ilmiön syyt voitaisiin selittää paremmin.

Environmentally benign Fe chelates in plant nutrition

The low solubility of iron (Fe) depresses plant growth in calcareous soils. In order to improve Fe availability, calcareous soils are treated with synthetic ligands, such as ethylenediaminetetraacetic acid (EDTA) and ethylenediimi-nobis(2-hydroxyphenyl)acetic acid (EDDHA). However, high expenses may hinder their use (EDDHA), and the recalcitrance of EDTA against biodegra-dation may increase the potential of cadmium (Cd) and lead (Pb) leaching. This study evaluated the ability of biodegradable ligands, i.e. different stereo-isomers of ethylenediaminedisuccinic acid (EDDS), to provide Fe for lettuce (Lactuca sativa L.) and ryegrass (Lolium perenne cv. Prego), their effects on uptake of other elements and solubility in soils and their subsequent effects on the activity of oxygen-scavenging enzymes in lettuce. Both EDTA and EDDHA were used as reference ligands. In unlimed and limed quartz sand both FeEDDS(S,S) and a mixture of stereo-isomers of FeEDDS (25% [S,S]-EDDS, 25% [R,R]-EDDS and 50% [S,R]/[R,S]-EDDS), FeEDDS(mix), were as efficient as FeEDTA and FeEDDHA in providing lettuce with Fe. However, in calcareous soils only FeEDDS(mix) was comparable to FeEDDHA when Fe was applied twice a week to mimic drip irrigation. The Fe deficiency increased the manganese (Mn) concentration in lettuce in both acidic and alkaline growth media, whereas Fe chelates depressed it. The same was observed with zinc (Zn) and copper (Cu) in acidic growth media. EDDHA probably affected the hormonal status of lettuce as well and thus depressed the uptake of Zn and Mn even more. The nutrient concentrations of ryegrass were only slightly affected by the Fe availability. After Fe chelate splitting in calcareous soils, EDDS and EDTA increased the solubility of Zn and Cu most, but only the Zn concentration was increased in lettuce. The availability of Fe increased the activity of oxygen-scavenging enzymes (ascorbate peroxidase, guaiacol peroxidase, catalase). The activity of Cu/ZnSOD (Cu/Zn superoxide dismutase) and MnSOD in lettuce leaves followed the concentrations of Zn and Mn. In acidic quartz sand low avail-ability of Fe increased the cobalt (Co) and nickel (Ni) concentrations in let-tuce, but Fe chelates decreased them. EDTA increased the solubility of Cd and Pb in calcareous soils, but not their uptake. The biodegradation of EDDS was not affected by the complexed element, and [S,S]-EDDS was biodegraded within 28 days in calcareous soils. EDDS(mix) was more recalcitrant, and after 56 days of incubation water-soluble elements (Fe, Mn, Zn, Cu, Co, Ni, Cd and Pb) corresponded to 10% of the added EDDS(mix) concentration.

Lignocellulose degradation and humus modification by the fungus Paecilomyces inflatus

Composting is the biological conversion of solid organic waste into usable end products such as fertilizers, substrates for mushroom production and biogas. Although composts are highly variable in their bulk composition, composting material is generally based on lignocellulose compounds derived from agricultural, forestry, fruit and vegetable processing, household and municipal wastes. Lignocellulose is very recalcitrant; however it is rich and abundant source of carbon and energy. Therefore lignocellulose degradation is essential for maintaining the global carbon cycle. In compost, the active component involved in the biodegradation and conversion processes is the resident microbial population, among which microfungi play a very important role. In composting pile the warm, humid, and aerobic environment provides the optimal conditions for their development. Microfungi use many carbon sources, including lignocellulosic polymers and can survive in extreme conditions. Typically microfungi are responsible for compost maturation. In order to improve the composting process, more information is needed about the microbial degradation process. Better knowledge on the lignocellulose degradation by microfungi could be used to optimize the composting process. Thus, this thesis focused on lignocellulose and humic compounds degradation by a microfungus Paecilomyces inflatus, which belongs to a flora of common microbial compost, soil and decaying plant remains. It is a very common species in Europe, North America and Asia. The lignocellulose and humic compounds degradation was studied using several methods including measurements of carbon release from 14C-labelled compounds, such as synthetic lignin (dehydrogenative polymer, DHP) and humic acids, as well as by determination of fibre composition using chemical detergents and sulphuric acid. Spectrophotometric enzyme assays were conducted to detect extracellular lignocellulose-degrading hydrolytic and oxidative enzymes. Paecilomyces inflatus secreted clearly extracellular laccase to the culture media. Laccase was involved in the degradation process of lignin and humic acids. In compost P. inflatus mineralised 6-10% of 14C-labelled DHP into carbon dioxide. About 15% of labelled DHP was converted into water-soluble compounds. Also humic acids were partly mineralised and converted into water-soluble material, such as low-molecular mass fulvic acid-like compounds. Although laccase activity in aromatics-rich compost media clearly is connected with the degradation process of lignin and lignin-like compounds, it may preferentially effect the polymerisation and/or detoxification of such aromatic compounds. P. inflatus can degrade lignin and carbohydrates also while growing in straw and in wood. The cellulolytic enzyme system includes endoglucanase and β-glucosidase. In P. inflatus the secretion of these enzymes was stimulated by low-molecular-weight aromatics, such as soil humic acid and veratric acid. When strains of P. inflatus from different ecophysiological origins were compared, indications were found that specific adaptation strategies needed for lignocellulosics degradation may operate in P. inflatus. The degradative features of these microfungi are on relevance for lignocellulose decomposition in nature, especially in soil and compost environments, where basidiomycetes are not established. The results of this study may help to understand, control and better design the process of plant polymer conversion in compost environment, with a special emphasis on the role of ubiquitous microfungi.

What determines soil organic carbon stocks in the grazing lands of north-eastern Australia?

This study aimed to unravel the effects of climate, topography, soil, and grazing management on soil organic carbon (SOC) stocks in the grazing lands of north-eastern Australia. We sampled for SOC stocks at 98 sites from 18 grazing properties across Queensland, Australia. These samples covered four nominal grazing management classes (Continuous, Rotational, Cell, and Exclosure), eight broad soil types, and a strong tropical to subtropical climatic gradient. Temperature and vapour-pressure deficit explained >80% of the variability of SOC stocks at cumulative equivalent mineral masses nominally representing 0-0.1 and 0-0.3m depths. Once detrended of climatic effects, SOC stocks were strongly influenced by total standing dry matter, soil type, and the dominant grass species. At 0-0.3m depth only, there was a weak negative association between stocking rate and climate-detrended SOC stocks, and Cell grazing was associated with smaller SOC stocks than Continuous grazing and Exclosure. In future, collection of quantitative information on stocking intensity, frequency, and duration may help to improve understanding of the effect of grazing management on SOC stocks. Further exploration of the links between grazing management and above- and below-ground biomass, perhaps inferred through remote sensing and/or simulation modelling, may assist large-area mapping of SOC stocks in northern Australia. © CSIRO 2013.

Soil nitrogen—crop response calibration relationships and criteria for winter cereal crops grown in Australia

More than 1200 wheat and 120 barley experiments conducted in Australia to examine yield responses to applied nitrogen (N) fertiliser are contained in a national database of field crops nutrient research (BFDC National Database). The yield responses are accompanied by various pre-plant soil test data to quantify plant-available N and other indicators of soil fertility status or mineralisable N. A web application (BFDC Interrogator), developed to access the database, enables construction of calibrations between relative crop yield ((Y0/Ymax) × 100) and N soil test value. In this paper we report the critical soil test values for 90% RY (CV90) and the associated critical ranges (CR90, defined as the 70% confidence interval around that CV90) derived from analysis of various subsets of these winter cereal experiments. Experimental programs were conducted throughout Australia’s main grain-production regions in different eras, starting from the 1960s in Queensland through to Victoria during 2000s. Improved management practices adopted during the period were reflected in increasing potential yields with research era, increasing from an average Ymax of 2.2 t/ha in Queensland in the 1960s and 1970s, to 3.4 t/ha in South Australia (SA) in the 1980s, to 4.3 t/ha in New South Wales (NSW) in the 1990s, and 4.2 t/ha in Victoria in the 2000s. Various sampling depths (0.1–1.2 m) and methods of quantifying available N (nitrate-N or mineral-N) from pre-planting soil samples were used and provided useful guides to the need for supplementary N. The most regionally consistent relationships were established using nitrate-N (kg/ha) in the top 0.6 m of the soil profile, with regional and seasonal variation in CV90 largely accounted for through impacts on experimental Ymax. The CV90 for nitrate-N within the top 0.6 m of the soil profile for wheat crops increased from 36 to 110 kg nitrate-N/ha as Ymax increased over the range 1 to >5 t/ha. Apparent variation in CV90 with seasonal moisture availability was entirely consistent with impacts on experimental Ymax. Further analyses of wheat trials with available grain protein (~45% of all experiments) established that grain yield and not grain N content was the major driver of crop N demand and CV90. Subsets of data explored the impact of crop management practices such as crop rotation or fallow length on both pre-planting profile mineral-N and CV90. Analyses showed that while management practices influenced profile mineral-N at planting and the likelihood and size of yield response to applied N fertiliser, they had no significant impact on CV90. A level of risk is involved with the use of pre-plant testing to determine the need for supplementary N application in all Australian dryland systems. In southern and western regions, where crop performance is based almost entirely on in-crop rainfall, this risk is offset by the management opportunity to split N applications during crop growth in response to changing crop yield potential. In northern cropping systems, where stored soil moisture at sowing is indicative of minimum yield potential, erratic winter rainfall increases uncertainty about actual yield potential as well as reducing the opportunity for effective in-season applications.

Soil phosphorus–crop response calibration relationships and criteria for winter cereal crops grown in Australia

Soil testing is the most widely used tool to predict the need for fertiliser phosphorus (P) application to crops. This study examined factors affecting critical soil P concentrations and confidence intervals for wheat and barley grown in Australian soils by interrogating validated data from 1777 wheat and 150 barley field treatment series now held in the BFDC National Database. To narrow confidence intervals associated with estimated critical P concentrations, filters for yield, crop stress, or low pH were applied. Once treatment series with low yield (<1 t/ha), severe crop stress, or pHCaCl2 <4.3 were screened out, critical concentrations were relatively insensitive to wheat yield (>1 t/ha). There was a clear increase in critical P concentration from early trials when full tillage was common compared with those conducted in 1995–2011, which corresponds to a period of rapid shift towards adoption of minimum tillage. For wheat, critical Colwell-P concentrations associated with 90 or 95% of maximum yield varied among Australian Soil Classification (ASC) Orders and Sub-orders: Calcarosol, Chromosol, Kandosol, Sodosol, Tenosol and Vertosol. Soil type, based on ASC Orders and Sub-orders, produced critical Colwell-P concentrations at 90% of maximum relative yield from 15 mg/kg (Grey Vertosol) to 47 mg/kg (Supracalcic Calcarosols), with other soils having values in the range 19–27 mg/kg. Distinctive differences in critical P concentrations were evident among Sub-orders of Calcarosols, Chromosols, Sodosols, Tenosols, and Vertosols, possibly due to differences in soil properties related to P sorption. However, insufficient data were available to develop a relationship between P buffering index (PBI) and critical P concentration. In general, there was no evidence that critical concentrations for barley would be different from those for wheat on the same soils. Significant knowledge gaps to fill to improve the relevance and reliability of soil P testing for winter cereals were: lack of data for oats; the paucity of treatment series reflecting current cropping practices, especially minimum tillage; and inadequate metadata on soil texture, pH, growing season rainfall, gravel content, and PBI. The critical concentrations determined illustrate the importance of recent experimental data and of soil type, but also provide examples of interrogation pathways into the BFDC National Database to extract locally relevant critical P concentrations for guiding P fertiliser decision-making in wheat and barley.

Degradation of monogalactosyl diglyceride and diagalactosyl diglyceride by sheep pancreatic enzymes

1. Saline extract of sheep pancreas acetone-dried powder was shown to catalyse acyl ester hydrolysis of spinach leaf galactosyl diglycerides and also galactosylglucosyl diglyceride of Lactobacillus casei. 2. Sodium deoxycholate stimulated the enzyme activity. Ca2+ had no effect on the hydrolysis of monogalactosyl diglyceride, but it enhanced that of digalactosyl diglyceride. When added together, there was considerably less activity with both the substrates. 3. Optimal hydrolysis was observed at pH7.2. 4. The initial point of hydrolysis was at position-1, leading to the formation of monogalactosyl monoglyceride and digalactosyl monoglyceride. Further hydrolysis to the corresponding galactosylglycerols and later to galactose and glycerol was also observed, indicating the presence of a- and b-galactosidases in the enzyme preparation. 5. Formation of monogalactosyl diglyceride from digalactosyl diglyceride by the action of a-galactosidase was noted. 6. Monogalactosyl diglyceride was also hydrolysed by b-galactosidase to a limited extent, giving rise to diacylglycerol and galactose. 7. Attempts at purification of monogalactosyl diglyceride acyl hydrolase by using protamine sulphate treatment, Sephadex G-100 filtration and DEAE-cellulose chromatography gave a partially purified enzyme which showed 9- and 81-fold higher specific activity towards monogalactosyl diglyceride and digalactosyl diglyceride respectively. This still showed acyl ester hydrolysis activity towards methyl oleate, phosphatidylcholine and triacylglycerol. 8. When sheep, rat and guinea-pig tissues were compared, guinea-pig tissues showed the highest activity towards both monogalactosyl diglyceride and digalactosyl diglyceride. In all the species pancreas showed higher activity than intestine.

A comparison of stocking methods for beef production in northern Australia: pasture and soil surface condition responses

Historical stocking methods of continuous, season-long grazing of pastures with little account of growing conditions have caused some degradation within grazed landscapes in northern Australia. Alternative stocking methods have been implemented to address this degradation and raise the productivity and profitability of the principal livestock, cattle. Because information comparing stocking methods is limited, an evaluation was undertaken to quantify the effects of stocking methods on pastures, soils and grazing capacity. The approach was to monitor existing stocking methods on nine commercial beef properties in north and south Queensland. Environments included native and exotic pastures and eucalypt (lighter soil) and brigalow (heavier soil) land types. Breeding and growing cattle were grazed under each method. The owners/managers, formally trained in pasture and grazing management, made all management decisions affecting the study sites. Three stocking methods were compared: continuous (with rest), extensive rotation and intensive rotation (commonly referred to as 'cell grazing'). There were two or three stocking methods examined on each property: in total 21 methods (seven continuous, six extensive rotations and eight intensive rotations) were monitored over 74 paddocks, between 2006 and 2009. Pasture and soil surface measurements were made in the autumns of 2006, 2007 and 2009, while the paddock grazing was analysed from property records for the period from 2006 to 2009. The first 2 years had drought conditions (rainfall average 3.4 decile) but were followed by 2 years of above-average rainfall. There were no consistent differences between stocking methods across all sites over the 4 years for herbage mass, plant species composition, total and litter cover, or landscape function analysis (LFA) indices. There were large responses to rainfall in the last 2 years with mean herbage mass in the autumn increasing from 1970 kg DM ha(-1) in 2006-07 to 3830 kg DM ha(-1) in 2009. Over the same period, ground and litter cover and LFA indices increased. Across all sites and 4 years, mean grazing capacity was similar for the three stocking methods. There were, however, significant differences in grazing capacity between stocking methods at four sites but these differences were not consistent between stocking methods or sites. Both the continuous and intensive rotation methods supported the highest average annual grazing capacity at different sites. The results suggest that cattle producers can obtain similar ecological responses and carry similar numbers of livestock under any of the three stocking methods.

Integrating organic matter into banana plantation in North Queensland: the effects on soil properties

The major banana production areas in Australia are particularly sensitive to environments due to their close proximity to areas of World Heritage rainforest and the Great Barrier Reef catchment. Management of soil quality, nutrients and pesticides are vital to maintaining the integrity of these sensitive areas. Studies on cropping systems have suggested that integrating organic matter into ground cover management would improve the quality of soil under banana cultivation. In this study, an alternative management practice for bananas, which addresses the management of organic matter and fertiliser application, was assessed and compared to the conventional practice currently employed in the banana industry. Several chemical, physical and biological soil parameters were measured including: pH, electrical conductivity, water stable aggregates, bulk density, water filled pore space, porosity, water content, fluorescein diacetate hydrolyis (FDA) and beta-glucosidase activity. The alternative management practice did not have a significant impact of the production and growth of bananas but overall improved the quality of the soil. Although some differences were observed, the chemical and physical soil characteristics did not differ dramatically between the two management systems. The addition of organic matter resulted in the soil under alternative practice having higher FDA and beta-glucosidase levels, indicating higher microbial activity. The integration of organic matter into the management of bananas resulted in positive benefits on soil properties under bananas, however, methods of maintaining organic matter in the soil need to be further researched.

Temporal and spatial patterns of soil water extraction and drought resistance among genotypes of a perennial C-4 grass

The objective of this study was to investigate patterns of soil water extraction and drought resistance among genotypes of bermudagrass (Cynodon spp.) a perennial C-4 grass. Four wild Australian ecotypes (1-1, 25a1, 40-1, and 81-1) and four cultivars (CT2, Grand Prix, Legend, and Wintergreen) were examined in field experiments with rainfall excluded to monitor soil water extraction at 30-190 cm depths. In the study we defined drought resistance as the ability to maintain green canopy cover under drought. The most drought resistant genotypes (40-1 and 25a1) maintained more green cover (55-85% vs 5-10%) during water deficit and extracted more soil water (120-160 mm vs 77-107 mm) than drought sensitive genotypes, especially at depths from 50 to 110 cm, though all genotypes extracted water to 190 cm. The maintenance of green cover and higher soil water extraction were associated with higher stomatal conductance, photosynthetic rate and relative water content. For all genotypes, the pattern of water use as a percentage of total water use was similar across depth and time We propose the observed genetic variation was related to different root characteristics (root length density, hydraulic conductivity, root activity) although shoot sensitivity to drying soil cannot be ruled out.

Can soil nematode community structure be used to indicate soil carbon dynamics in horticultural systems?

There is a need to develop indicators that relate the dynamics of soil organic carbon (SOC) with changes in land management of horticultural production systems. Soil nematode communities have been shown to be sensitive to land management changes, but often do not include plant-parasites in the calculation of soil nematode community indices. The concept of nematode functional guilds was used to estimate the proportion of carbon entering the soil ecosystem through different channels, such as through decomposition of organic material, the detrital channel, through the roots of plants, the root channel or recycled through the activity of predators, a predation channel. Calculations of the indices were developed and validated using case studies in the north Queensland banana industry. Firstly, a survey of organic and conventional banana farms found a greater proportion of C entering the soil ecosystem through the detrital channel and a reduced proportion of C originating through the root channel at the organic sites relative to conventional sites. Secondly, a field experiment comparing compost amendments, found application of fresh compost significantly increased the proportion of C entering the soil ecosystem through the detrital channel and decreased proportion of C originating from the root channel. Thirdly, a field experiment comparing 'conventional' banana production to an 'alternative' system which incorporated organic matter, found the proportion of C entering the soil ecosystem through the root channel was significantly greater in the conventional systems relative to the alternative system. This research demonstrates that nematode indices can be used to assess horticultural systems, by indicating the origins of SOC.

The effect of pasture utilization rate on stocks of soil organic carbon and total nitrogen in a semi-arid tropical grassland

The influence of grazing management on total soil organic carbon (SOC) and soil total nitrogen (TN) in tropical grasslands is an issue of considerable ecological and economic interest. Here we have used linear mixed models to investigate the effect of grazing management on stocks of SOC and TN in the top 0.5 m of the soil profile. The study site was a long-term pasture utilization experiment, 26 years after the experiment was established for sheep grazing on native Mitchell grass (Astrebla spp.) pasture in northern Australia. The pasture utilization rates were between 0% (exclosure) and 80%, assessed visually. We found that a significant amount of TN had been lost from the top 0.1 m of the soil profile as a result of grazing, with 80% pasture utilization resulting in a loss of 84 kg ha−1 over the 26-year period. There was no significant effect of pasture utilization rate on TN when greater soil depths were considered. There was no significant effect of pasture utilization rate on stocks of SOC and soil particulate organic carbon (POC), or the C:N ratio at any depth; however, visual trends in the data suggested some agreement with the literature, whereby increased grazing pressure appeared to: (i) decrease SOC and POC stocks; and, (ii) increase the C:N ratio. Overall, the statistical power of the study was limited, and future research would benefit from a more comprehensive sampling scheme. Previous studies at the site have found that a pasture utilization rate of 30% is sustainable for grazing production on Mitchell grass; however, given our results, we conclude that N inputs (possibly through management of native N2-fixing pasture legumes) should be made for long-term maintenance of soil health, and pasture productivity, within this ecosystem.

Molecular Modelling of Estrogen-Producing Enzymes CYP450 Aromatase and 17beta-Hydroxysteroid Dehydrogenase Type 1

Breast cancer is the most common cancer in women in the western countries. Approximately two-thirds of breast cancer tumours are hormone dependent, requiring estrogens to grow. Estrogens are formed in the human body via a multistep route starting from cholesterol. The final steps in the biosynthesis include the CYP450 aromatase enzyme, converting the male hormones androgens (preferred substrate androstenedione ASD) into estrogens(estrone E1), and the 17beta-HSD1 enzyme, converting the biologically less active E1 into the active hormone 17beta-hydroxyestradiol E2. E2 is bound to the nuclear estrogen receptors causing a cascade of biochemical reactions leading to cell proliferation in normal tissue, and to tumour growth in cancer tissue. Aromatase and 17beta-HSD1 are expressed in or near the breast tumour, locally providing the tissue with estrogens. One approach in treating hormone dependent breast tumours is to block the local estrogen production by inhibiting these two enzymes. Aromatase inhibitors are already on the market in treating breast cancer, despite the lack of an experimentally solved structure. The structure of 17beta-HSD1, on the other hand, has been solved, but no commercial drugs have emerged from the drug discovery projects reported in the literature. Computer-assisted molecular modelling is an invaluable tool in modern drug design projects. Modelling techniques can be used to generate a model of the target protein and to design novel inhibitors for them even if the target protein structure is unknown. Molecular modelling has applications in predicting the activities of theoretical inhibitors and in finding possible active inhibitors from a compound database. Inhibitor binding at atomic level can also be studied with molecular modelling. To clarify the interactions between the aromatase enzyme and its substrate and inhibitors, we generated a homology model based on a mammalian CYP450 enzyme, rabbit progesterone 21-hydroxylase CYP2C5. The model was carefully validated using molecular dynamics simulations (MDS) with and without the natural substrate ASD. Binding orientation of the inhibitors was based on the hypothesis that the inhibitors coordinate to the heme iron, and were studied using MDS. The inhibitors were dietary phytoestrogens, which have been shown to reduce the risk for breast cancer. To further validate the model, the interactions of a commercial breast cancer drug were studied with MDS and ligand–protein docking. In the case of 17beta-HSD1, a 3D QSAR model was generated on the basis of MDS of an enzyme complex with active inhibitor and ligand–protein docking, employing a compound library synthesised in our laboratory. Furthermore, four pharmacophore hypotheses with and without a bound substrate or an inhibitor were developed and used in screening a commercial database of drug-like compounds. The homology model of aromatase showed stable behaviour in MDS and was capable of explaining most of the results from mutagenesis studies. We were able to identify the active site residues contributing to the inhibitor binding, and explain differences in coordination geometry corresponding to the inhibitory activity. Interactions between the inhibitors and aromatase were in agreement with the mutagenesis studies reported for aromatase. Simulations of 17beta-HSD1 with inhibitors revealed an inhibitor binding mode with hydrogen bond interactions previously not reported, and a hydrophobic pocket capable of accommodating a bulky side chain. Pharmacophore hypothesis generation, followed by virtual screening, was able to identify several compounds that can be used in lead compound generation. The visualisation of the interaction fields from the QSAR model and the pharmacophores provided us with novel ideas for inhibitor development in our drug discovery project.