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MEASUREMENTS OF SOIL MICROBIAL BIOMASS CARBON, NITROGEN AND NRN Oguz Can Turgay 1, Akira Haraguchi 2 1 Department of Environmental Science, Faculty of Science, 2 Niigata University, Igarashi, 2-8050, Niigata, 950-2181, JAPAN ABSTRACT The measurement of soil microbial biomass (SMB) is one of the most reliable procedures commonly used for a better comprehension of the nutrient cycle in soil. We used several SMB indices to monitor microbial pool in a wider scale in soil. The objectives of this study were to (i) quantify biomass C, N, (BC, BN,) and also Ninhydrin Reactive Nitrogen (BNRN), which is a fraction consisted of mainly NH4+ N and amino groups (amino acids, proteins and peptides etc.) included by microorganisms and (ii) to test the reliability of these parameters by comparing with soil properties in an andosol soil under different managements. The soil samples were collected from three agricultural plots, under different management in Muramatsu Experimental Station and from neighboring forest site (pine) in Niigata Prefecture, Japan. Following soil fumigation with ethanol free chloroform, a group of samples was extracted with 0.5 M K2SO4 and extractable NRN, C, total N, NH4+ N were determined in fumigated and unfumigated K2SO4 extracts. All SMB values were calculated according to the chloroform-fumigation extraction (CFE) procedure. Biomass parameters showed no remarkable difference between manure and slurry-amended plots. However, marked differences in all soil biomass values were observed among adjacent agricultural and forest ecosystems, in the same climatic environment related with the management. The results, overall, showed that SMB measurements were effective on the determination of microbial pool and were well accorded with soil characteristics. INTRODUCTION Soil organic matter is known to represent the primary origin of energy for microorganisms and can be divided into several fractions that vary in turnover time from hours to thousands of years. The active fraction of organic matter consisted of amino acids, groups of proteins and carbohydrates, represents small, but dynamic portion of the huge and slowly changing background of stabile organic matter. This labile pool is readily available for microbial use and is mostly stored by soil microorganisms. A portion of such kind of organic substances can be quantified as an indicator of actual amount of microbial populations. Thus, there has been increasing interest to definite measurements of the soil microbial biomass and several methods have been attempted to improve more accurate and useful procedure for microbial biomass measurements over the last two decades. Among these methods, Chloroform Fumigation Extraction (CFE) method (Vance et al, 1987) has been more commonly used due to its simplicity and applicability for wide group of soils. This technique is theoretically based on the quantitative extraction of a particular compound, found in all components of the microbial community but in no other constituents of soil. Furthermore, various organic forms such as soluble free sugars, carbohydrates and proteins can be measured in the same extracts (DeLuca and Keeney, 1993b; DeLuca, 1998; Joergensen et al, 1996). However, there are not so many works regarding the determination of particular organic forms in SMB. Ninhydrin, which is a reagent forming a purple complex with varying molecules comprising ?-amino nitrogen and with ammonium and other compounds with free ?-amino groups such as amino acids, peptides and proteins (Moore and Stein1948) has been lately used as a simple an reliable parameter in microbial biomass determinations (Joergensen and Brookes 1990). In the present study, we assayed ninhydrin reactive compounds to evaluate SMB pool in a wider scale. Our aims were to quantify microbial biomass and also NRN under different soil and test the reliability of these parameters by comparing each other and soil parameters. MATERIALS and METHODS The arable soils (0-10 cm) were sampled from 3 soil plots of Muramatsu Experimental Station, which is located in central Niigata (1390 11 E, 370 41 N), and from a forest site, near the station in May 1999. The soil type was Andosol which has highly good soil structure, porosity, water holding capacity, permeability, and which is rich in allophanes and humus containing humified black colored humic substances. Recent crop pattern was watermelon in two of the arable plots, which had been amended with slurry and manure previously. In these plots, soil sampling was made after harvest, before following cultivation. The third plot was a 10-years-old fruit garden, planted with apricot trees. Forest sample was collected after removal of the litter layer from the same depth with arable plots. Each site was sampled 10 times with 50x50 mm soil cores. The samples then bulked and large pieces of plant materials were picked up before pretreatments. All samples were passed through a sieve (2 mm), adjusted to a WHC of 40%, pre-incubated at 25 0C a week and stored at 4 0C before analysis. Soil pH was measured in H2O and KCl with a soil to solution ratio of 1:2.5. Soil organic C and N were determined C-N Analyzer (Sumigraph NC-90A). All SMB parameters were measured by the fumigation-extraction procedure according to Vance et al. (1987) and the extracts were stored at -20 0C prior to analysis. Ninhydrin Reactive Nitrogen determination in soil extracts was carried out as indicated by Joergensen and Brookes (1990). Following addition of ninhydrin solution, reaction was measured colorimetrically at 570 nm. Extractable total N was tested by total persulfate oxidation procedure based on the oxidation of total N to NO3-N in an alkali at elevated temperature by using persulfate as described by Cabrera and Beare (1993); Total N, which had been oxidized to NO3-N, was reduced to NO2- N within copperized cadmium reduction unit. NO2- N was then measured according to modified Gries Ilosvay method (Keeney and Nelson,1982). Extractable NH4 in soil extracts was determined colorimetrically, similar to the original indophenol blue procedure (Alef and Nannipieri, 1995). Extractable C in soil extracts was measured with automated carbon analyzer (SHIMADZU, TOC 5000 model), accelerating and simplifying the organic C determination by using combustion, oxidation and infrared ray absorption processes (Wu et al, 1990; Shibara and Inubushi 1995). Soil microbial biomass calculations : Biomass C (BC) was calculated as indicated by (Wu et al, 1990), BC = EC: kEC where EC: (extractable C in fumigated soil extracts) - (extractable C in non-fumigated soil extracts) and kEC: 0.45 (extractable part of microbial C after fumigation) Biomass-N was calculated according to Jenkinson (1988). (BN) = EN: kEN, where EN: (total N, determined in fumigated extracts) - (total N, determined in non-fumigated soil extracts) and kEN: 0.45 (extractable part of microbial N after fumigation) Biomass ninhydrin reactive N, (BNRN) and extractable NH4+-N (ENH4) were calculated based on the same principle of the FE method BNRN = (ninhydrin-N in extracts of fumigated soils) - (ninhydrin-N in extracts of non-fumigated soils) and ENH4 = (NH4+-N in extracts of fumigated soils) - (NH4+-N in extracts of non-fumigated soils) (Joergensen and Brooke, 1990). a-amino N, which represents amino acids, proteins and peptides included by microorganisms, was calculated from the difference (BNRN and ENH4) as indicated by Joergensen and Brooke (1990). With the exception of NRN values obtained from five replicate measurements, the results presented are arithmetic means of three replicate measurements and expressed on an oven dry basis (24h at 105 °C). Statistical analyses (simple linear regression and Pearson covariance test) were calculated by MINITAB.  RESULTS AND DISCUSSION The soil properties (Table 1) and extractable fractions of the soil biomass measured by different techniques are shown for each site (Table 2). The forest soil showed the highest values for all extractable biomass materials, 836 mg C, 82.9 mg N and 138.2 mg NRN kg-1 soil, followed by fruit garden soil, 474 mg C, 45.6 mg N and 117.8 mg NRN kg-1 soil respectively. Slurry and manure amended plot appeared to have same amount of extractable N values, 14.8 mg N, 22 mg NRN kg-1 soil as a mean of both plots. However, extractable C in manure amended plot was slightly higher than that in slurry amended plot. The results indicated that extractable SMB components (EC, EN, BNRN) were significantly correlated with soil organic C, total N and also with each other (Table 4). Soil pH and C:N ratios did not show reasonable correlation with biomass parameters. C:N ratios were not significantly different between cultivated soils (15.3 as an average of cultivated soils and 18.0 for forest soil) which suggests that C and N are in an identical balance in cultivated soils. However, it is clear that three systems are fundamentally different in management. Therefore, C:N ratios does not reflect the status of internal C and N cycle on microbial biomass efficiently.  Comparison of microbial biomass under different managements : A particular proportion of the soil organic matter input is readily utilized by the organisms, such that the biomass C generally comprises only 1-5% of the soil organic carbon in soil (Jenkinson and Ladd, 1981; Sparling, 1985; Smith and Paul, 1990). The results found in our study suggest that microbial C was approximately 1% of soil organic matter in cropped plots, which have been managed under intensive crop rotation (corn, potato, been, leek, sunflower, and also watermelon) since 1987, comparatively lower than fruit garden (1.51%) and forest soil (1.66%) (Table 3). Probably, this is because of the cultivation, causing significant disturbance to soil organisms. Active biomass pool in cropped plots apparently regulated by agricultural practices, which alter nutrient input and output frequently. Comparing with these plots, fruit garden and forest soils have much larger biomass C, N and NRN contents due to the facts that the main source of organic input to the biomass in natural ecosystems is from plant material, consisting of roots, leaf, stem litter and actual biomass pool does not alter apart from the fluctuational effects of seasonal changes. Fruit garden has been apricot-planted plot for more than 10 years, means much less cultivation disturbing natural biomass and providing a relatively stabile nutrient flow which is similar to that in forest ecosystem. In grassland and arable soils, the microbial biomass contains an average 3.1% of total N, ranging from 0.5% to 6.6% (Joergensen and Mueller, 1996).  We observed identical inclination in biomass N as a function of total soil N in all soil sites (0.94% for intensively cultivated soils, 2.07% for fruit garden soil and 2.98% for forest soil). BNRN values and their ratios in total N, ranging between 0.64% and 2.23% (Table 3), were also in agreement with biomass C % and biomass N%. Ninhydrin reactive compounds represent certain cytoplasmic products of microorganisms which are mainly amino acids and NH4+ N. Besides, it reacts with free ?-amino groups consisting of amino acids, peptides and proteins (Moore and Stein, 1948). The substrates such as amino acids and carbohydrates, which form a temporary pool reflecting microbial activity and the delicate balance between degradation and synthesis of organic materials, have been used as a measure of organic matter quality (Stevenson 1982; Arshad et al, 1990).We, therefore, measured microbial NH4+ N separately to calculate the fraction of ?-amino N (see biomass calculations ), and obtain a sensitive parameter. NH4+ N contents of non-fumigated soils, which were taken as soil NH4+ N, ranged from 17.7 to 28.1 mg kg-1 soil and did not show good correlation with soil and biomass parameters. In contrast to soil NH4+ N, the fraction of ?-amino N widely ranged from 18.8 to 106.5 mg kg-1 (Table 2) and was significantly correlated with organic C, total N and also biomass parameters (Table 4) which suggests that organic N is mostly accumulated by microbial biomass as proteins, peptides, amino acids etc. and microorganisms are highly competitive for N in the ecosystems where an available C source is not restricted (Jackson et al 1989; Schimel et al 1989).  CONCLUSION It is well understood that cultivation accelerates decomposition of organic matter and results in lower levels of microbial biomass. However, there is no much information regarding organic matter quality and if it is influenced by cultivation. Specific or total determinations of amino acids and carbohydrates are highly reasonable alternatives to evaluate organic matter quality and provide a comparison of microbial N content but represents a limited fraction of total biomass N. Thus, we tested biomass C, and N in addition to NRN and ?-amino N measurements to evaluate the actual size of the microbial pool under different managements. We concluded that NRN was significantly correlated with biomass C, and N and can be used as a reliable biomass parameter monitoring amino acid pool within a wide extent in soil. Acknowledgments We would like to thank Mr. Akihiro Furubayashi, Faculty of Agriculture, Niigata University, and also Seiichi Nohara, NIES, Tsukuba for their technical assistance. REFERENCES Alef, K. and Nannipieri, P. 1995. Methods in Applied Soil Microbiology and Biochemistry. Soil nitrogen. Academic Press Limited. 24-28 Oval Road, London NW1. 7DX. pp 79-87 Arshad, M.A., Schnitzer, M., Angers, D.A. and Ripmeester, J.A., 1990. Effects of till vs no-till on the quality of soil organic matter. Soil Biol. Biochem 22, 595-599 Cabrera, M.R., and Beare, M.H. 1993. Alkaline persulphate oxidation for determining total nitrogen in microbial biomass extracts. Soil Sci. Soc. Am. J. 57, 1007-1012 DeLuca, T.H., and Keeney, D.R., 1993b. Soluble anthrone reactive carbon in soils; effect of carbon and nitrogen amendments. Soil Sci. Soc. Am. J. 57, 1296-1300 DeLuca, T.H., 1998. Relationship of 0.5 M K2SO4 extractable anthrone reactive carbon to indices of microbial activity in forest soils. Soil Biol Biochem. 30, 1293-1299 Jackson, L.E., Schimel, J.P. and Firestone, M.K. 1989. Short-term partitioning of ammonium and nitrate between plants and microbes in an annual grassland. Soil Biol. Biochem. 21, 409-415 Jenkinson, D.S. and Ladd, J.M. 1981. Microbial biomass in soil: measurement and turnover. In: Soil Biochemistry, vol 5 Paul E.A., Ladd J.N (eds). Dekker, New York, pp. 415-471 Jenkinson, D.S. 1988. The determination of microbial biomass C and nitrogen in soil. In: Advances in nitrogen cycling in agricultural ecosystems. Wilson JR (ed) CAB International, Wallingford, 368-386 Joergensen, R.G. and Brookes, P.C. 1990. 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