LITHUANIAN UNIVERSITY OF AGRICULTURE LITHUANIAN INSTITUTE OF AGRICULTURE Loreta Piaulokait ė Motuzien ė
THE SPREADING AND DYNAMICS OF MICROORGANISMS, WITCH PARTICIPATE IN NITROGEN TRANSFORMATION, IN DIFFERENT BY pH AND FERTILIZATION ALBELUVISOL
Summary of doctoral dissertation Biomedical sciences, agronomy (06 B)
Dissertation was worked out during 2001-2005 at the Lithuanian Institute of Agriculture, Vezaiciai Branch. This work was supported by the State Science and Studies Foundation of the Republic of Lithuania. Scientific supervisors: Prof. Dr. habil. Edmundas Lapinskas (Lithuanian Institute of Agriculture, biomedical sciences, agronomy-06 B) Scientific consultant: Doc. Dr. Dalia Ambrazaitien ė (Klaipeda University, biomedical sciences, biology-01 B) The dissertation will be defended at the Council of Agronomy Sciences at the Lithuanian University of Agriculture: Chairman: Prof. Dr. habil. Pavelas Duchovskis (Lithuanian Institute of Horticulture, biomedical sciences, agronomy-06 B) Members: Prof. Dr. habil. Algirdas Juozas Motuzas (Lithuanian University of Agriculture, biomedical sciences, agronomy-06 B) Prof. Dr. habil. Algirdas Sliesaravicius (Lithuanian University of Agriculture, biomedical sciences, agronomy-06 B) Dr. habil. Liudmila Tripolskaja (Lithuanian Institute of Agriculture, biomedical sciences, agronomy-06 B) Dr. K ę stutis Armolaitis (Lithuanian Institute of Forestry, biomedical sciences, ecology and environmental sciences -03 B) Opponents: Prof. Dr. habil. Albinas Lugauskas (Institute of Botany, biomedical sciences, biology 01 B) Dr. Alvyra Slepetiene (Lithuanian Institute of Agriculture, biomedical sciences, agronomy-06 B) Defence of doctoral dissertation will take place during the public meeting of the Council of Agronomy Science on the 10 th of February, 2006 at 11 a.m. in room No. 322, the central building of the Lithuanian University of Agriculture. Address: Lithuanian University of Agriculture Student ų g. 11, LT- 53361 Akademija, Kauno raj., Lithuania Phone: ( 8-37) 752254, Fax. ( 8-37) 397500 The summary of the doctoral dissertation was distributed on the 10 th of January, 2006. The doctoral dissertation is available in libraries of Lithuanian University of Agriculture and the Lithuanian Institute of Agriculture.
LIETUVOS EM Ė S Ū KIO UNIVERSITETAS LIETUVOS EMDIRBYST Ė S INSTITUTAS Loreta Piaulokait ė Motuzien ė
AZOT Ą TRANSFORMUOJAN Č I Ų MIKROORGANIZM Ų PAPLITIMAS IR DINAMIKA Į VAIRAUS pH IR SKIRTINGAI TR Ę IAMAME BALKVAEMYJE
Daktaro disertacijos santrauka Biomedicinos mokslai, agronomija (06 B)
Disertacija rengta 2001 2005 metais Lietuvos emdirbyst ė s instituto V ė ai č i ų filiale. Darb ą r ė m ė Lietuvos valstybinis mokslo ir studij ų fondas. Mokslinis vadovas: Prof. habil. dr. Edmundas Lapinskas ( Lietuvos emdirbyst ė s institutas, biomedicinos mokslai, agronomija 06 B) Konsultantas: Doc. dr. Dalia Ambrazaitien ė ( Klaip ė dos universitetas, Lietuvos emdirbyst ė s institutas, biomedicinos mokslai, biologija 01B) Disertacija ginama Lietuvos em ė s ū kio universiteto Agronomijos mokslo krypties taryboje: Pirmininkas Prof. habil. dr. Pavelas Duchovskis (Lietuvos sodininkyst ė s ir darininkyst ė s institutas, biomedicinos mokslai, agronomija 06 B) Nariai: Prof. habil. dr. Algirdas Juozas Motuzas (Lietuvos em ė s ū kio universitetas, biomedicinos mokslai, agronomija 06 B) Prof. habil. dr. Algirdas Sliesaravi č us (Lietuvos em ė s ū kio universitetas, biomedicinos mokslai, agronomija 06 B) Habil. dr. Liudmila Tripolskaja (Lietuvos emdirbyst ė s institutas, biomedicinos mokslai, agronomija 06 B) Dr. K ę stutis Armolaitis (Lietuvos mik ų institutas, biomedicinos mokslai, ekologija ir aplinkotyra 03 B) Oponentai: Prof. habil. dr. Albinas Lugauskas, (Botanikos institutas, biomedicinos mokslai, biologija 01 B) Dr. Alvyra lepetien ė (Lietuvos emdirbyst ė s institutas, biomedicinos mokslai, agronomija 06 B) Disertacija bus ginama vieame Agronomijos mokslo krypties tarybos pos ė dyje, kuris vyks 2006m. vasario 10d. 11 val. Lietuvos em ė s ū kio universiteto Centrini ų r ū m ų 322 auditorijoje. Adresas: Student ų g. 11, LT- 53361 Akademija, Kauno raj., Lietuva Tel. ( 8-37) 752254, faks. ( 8-37) 397500 Disertacijos santrauka isiuntin ė ta 2006 m. sausio 10 d. Disertacij ą galima peri ū r ė ti Lietuvos em ė s ū kio universiteto ir Lietuvos emdirbyst ė s instituto bibliotekose
INTRODUCTION Soil degradation and variety are defined by specific climatic conditions, different soil formation time, relief, different soil texture and anthropogenic activities. Characteristic organism communities, which determine soil properties, are found in different soil groups depending on mentioned conditions /Bumblauskis et. al , 1998; Eitminavi č i ū t ė et. al , 1999; Krivoluckij, 1994/. Possibility of pollution increases when agriculture intensifies. Then, the significance of microbiological researches becomes distinct. Executing soil biological researches, it is necessary to understand the principles of microorganism functioning in natural ecosystems, to determine changes that occure and provide suitable regulation means. Though, the activity of microorganisms is described in general principles, but facing with specificity is inevitably, when researching organisms in concrete ecosystems. It is necessary to take into account general regularities such as succession, homeostasis and others and specific regularities, which influence microbial coenosis or their components (nitrogen fixation, rapid propagation, appropriation of ecological niche and others). It is important to exclude specific complex formation and organism functioning regularities, such as critical number of microorganisms and duplication of processes /Zvyagintsev, 1987/. Soil is multifunctional open system, where close physical, chemical and biological processes occure. Negative phenomenons: soil acidification, the degradation of humus and increased contamination are observed because of technological progress, increased energetical potential and intensification of anthropogenic activities in agricultural sector /Andrejuk, 1992; Minejev et. al , 1990; Gunapala et. al , 1998/. Scientific researches proved, that quantitative and qualitative composition of microorganisms changed, when soil acidified. Azotobacter and cellulose decomposing bacteria (under aerobic conditions) were not discovered, and nitrifying and ammonifying bacteria spreaded not numerously in acid soils /Arlauskien ė , 1997; Bagdonien ė et. al , 1999; Lapinskas, 1998; Matarueva, 1998/. Researches, carried out in Lithuania and foreign countries, showed, that relatively small mineral fertiliser rates had positive influence on plant productivity, soil agrochemical properties and microbiological characteristics /Arlauskien ė , 1999; Deravin, 1991; Mahmod et. al , 1992; Plesevi č ien ė , 1995; ilnikov et. al , 1997/. Mineral fertilisers in limed soils not inhibit, but stimulate the spreading of ammonifying and nitrifying microorganisms, increases the activity of ferments /Minejev et. al , 1990/. Meanwhile, high mineral fertiliser rates have negative effect on soil agrochemical and biological properties, its ecological state, increases soil, water and atmospheric pollution. Some scientists propose, that liming increases anthropogenic load of the soil, not disturbing ecological equilibrium /Zvyagintsev, 1987/. However, microorganisms of different physiological groups react differently to mineral and organic fertilisers, pesticides /Arlauskien ė , 1999; Bagdonien ė et. al , 1999; Chappel et. al , 2000; Deravin, 1991; Kritaponyt ė , 1996; Mahmod et. al , 1992; Minejev et. al , 1998; Plesevi č ien ė et. al , 1994/. The number of microorganisms (ammonifying, mineral nitrogen assimilating, spore forming and micromycetes) in main physiological soil groups in Lithuania was determined. Some scientists indicate, that the number of microorganisms
did not decreased, when high fertiliser rates were used /Svirskien ė et. al , 2003/. High mineral fertiliser rates also did not have significant influence on the spreading of microorganisms in limed Albeluvisol . Though, certain changes were noticed in different years /Arlauskien ė , 1998/. The principles of microorganism complex formation and functioning not only explain the functioning into soil ecosystem, but also allow to forecast the development and vulnerability of microorganisms, and to solve biodiagnostic and bioindicational problems. Many issues still remain not sufficiently explained, when researching the changes and successions of microorganism coenosis. Contradictory treated researches are known. All the more, soil agrochemical properties were researched separately from its microbiological characteristics till now. Hypothesis. Different soil pH and fertilisation levels were anthropogenicaly formed in acid Dystric Albeluvisol in V ė ai č iai Branch of Lithuanian Institute of Agriculture. It is expected original, stable microorganism coenosis with settled principles of functioning were formed during 30 years. It particularly would affect the complex of microorganisms, participating in nitrogen compounds transformation processes. Therefore, it is expected the research would determine the influence of anthropogenic factors on the activity of microorganisms, witch participate in nitrogen compounds transformation processes. Research aim to determine the spreading, dynamics and activity of microorganisms, witch participate in soil nitrogen transformation processes, in different by pH and fertilisation level Dystric Albeluvisol ( Abd ). The following tasks were set: • To determine the spreading of ammonifying and mineral nitrogen assimilating microorganisms, spore forming bacteria and micromycetes. • To establish the dynamics of microorganisms, witch transform nitrogen compounds, in different by pH and fertilisation level Dystric Albeluvisol . • To investigate the influence of soil pH and mineral fertilisers combination on non-symbiotic nitrogen fixation. • To evaluate the succession of microorganisms, transforming nitrogen compounds. To research the direction of mineralization processes in different by pH and • fertilisation level Dystric Albeluvisol. To determine the connection between environmental conditions and soil • microbiological processes. Originality of the research. It is the first research in Western Lithuania when the dynamics of microorganisms, witch transform nitrogen compounds, is established in different by pH and mineral fertilisation Dystric Albeluvisol ( Abd ). The influence of different anthropogenic load (liming and mineral fertilising) on soil potential nitrogen
fixation possibilities, soil organic matter synthesis and destruction processes were investigated. Practical importance of the research. Taking into account the run of the microbiological processes, the research data allow farmers to select sustainable and environmental friendly farm management ways, what enable to grow ecologically clean production. Work approval. Major results were presented and considered at the conferences of PhD students Youth Seeks for a Progress 2001 and Youth Seeks for a Progress 2003 (LUA, Kaunas, 2001 and 2003), international scientific conferences Achievements and Tasks of Soil Science and Plant Nutrition in the Course of Integration into the EU (LUA, Kaunas, 2003), Mycology and Algology 2004 (Moscow, 2004) and Optimizing Agricultural output production: theory and praxis (Jelgava, 2005). Research data generalized in 1 scientific report. Published works. Main research data are presented in 4 publications, approved by the Lithuanian Scientific Publications List of the Department of Sciences and Higher Education and 5 conference proceedings. Volume of the work. The research was carried out according to the confirmed methodics and LIA work plans 2001-2004. Field trials and laboratorial methods were used. The dissertation is written in Lithuanian language. The doctoral dissertation consists of 70 pages, 21 tables and 30 drawings. It is constituted of introduction, 3 chapters, conclusions, list of the literature and list of the papers on dissertation theme. 151 literature sources are used in the dissertation. 2. MATERIALS AND METHODS Research place and scheme. The soil samples for the microbiological analyses of nitrogen compounds transformation were taken from stationary field trial named as Optimal reaction of Dystric Albeluvisols under different fertilisation, witch was arranged in 1976 in Vezaiciai Branch of Lithuanian Institute of Agriculture. Five-field rotation was used for the trial: 1) spring rape (2001), 2) permanent grasses with cover crop barley (2002), 3) 1 st year use permanent grasses (2003), 4) winter wheat (2004) and 5) oats. Different soil pH and mineral fertilisation levels were formed in the trial. Such trial scheme was used: Factor A (pH KCl ) pH < 4.7 (non-limed); pH 4.7-5.2; pH 5.2-5.7; pH > 6.7.
Factor B (fertilising) non-fertilised; N 45 P 39 K 57 ; N 135 P 117 K 171 .
The rates of mineral fertilisers (NPK) were calculated by the average fertilising rates during crop rotation. Average annual rate was N 45 P 39 K 57 and correspondingly 3 rates - N 135 P 117 K 171 . pH levels were maintained liming repeatedly each five years. Trial soil is Dystric Albeluvisol (Abd) /FAO-Unesco, 1997; Buivydait ė et. al, 2001/. Soil texture is light loam on medium loam. Depth of carbonate effervescence is 1.5-2.0 meters. The arable soil layer before trial arrangement was very acidic (pH KCl 4,1-4,4), hydrolytic soil acidity - 47-59 m-ekv.kg -1 , total absorbed bases - 22-29 m-ekv.kg -1 , bases saturation degree - 27,8-36,8 %, the amount of mobile aluminium - 50-77 mg kg -1 . The soil was with low amounts of mobile P 2 O 5 (53-112 mg kg -1 ), high amounts of mobile K 2 O (238-290 mg kg -1 ) and medium amounts of humus (~ 2%) / Č iuberkien ė , 1997; Č iuberkien ė et. al , 2003; Mavila, 1998/. The time of research execution. The spreading and activity of physiological groups of microorganisms, participating in soil nitrogen transformation, was determined during 2001-2004 years. The researches were done before plant vegetation resumed and after cropping in 2001 2002, and during all vegetation period in 2003 2004 (Table 1). Table 1. The dates of soil sampling for microbiological analyses. V ė ai č iai, 2001-2004 Research time 2001 2002 2003 2004 Before plant 28-May 23-May 19-May 06-May vegetation resumed After 5 weeks 23-June 10-June After 10 weeks 28-July 15-July After 15 weeks 10-September 29-August 01-September 19-August After 20 weeks 06-October 23-September After 25 weeks 10-November 04-November Climatic conditions. G. Selianinov hydrothermal coefficient (HTC) was calculated to characterize meteorological conditions /Bukantis, 1994/. Despite the precipitation deficit in May of 2001 (precipitation didnt fall for three weeks), plants developed normally, because soil moisture was sufficient. Trial was carried out in drained soil. Therefore the abundant amount (157 mm) of precipitation in June (HTC 4.5) did not have negative influence. The condition of excess moisture was determined in September (HTC 5.45). Total amount of precipitation in 2001 year was 1007 mm. The 2002 spring was early. April was warm (1.5 °C warmer than long-term annual temperature). April-May months were warmer and drier than usual therefore slight draught (hydrothermal coefficient HTC = 0.46) in May occurred. Plant growing conditions in June-July months were normal. Very strong draught (HTC = 0.01) in August occurred. October temperature was lower than long-term annual temperature. 2003 year was favourable for grown plants and microorganisms by the amounts of warmth and moisture. May was wet if comparing to 2001-2002. July was dry (HTC 0.80), the amount of precipitation was lower by 37.4 mm than long-term average amount. The
amounts of precipitation in August and October were higher by 27.7 mm and 49.2 mm respectively than long-term average amount. 2004 May was dry (HTC 0.57). The long-term average amount of precipitation was lower in July and August, but exceeded it in September and October in 2004. The temperature was by 2 ° C higher than long-term annual temperature in August. Both higher and lower precipitation amounts could form unfavourable conditions for the development of soil microorganisms.
HTC 6 5 4 3 2 1 0 May June July August September 2001 0,82 4,50 1,61 1,48 5,45 2002 0,20 1,34 2,29 0,01 1,51 2003 1,20 1,66 0,80 2,26 1,72 2004 0,57 1,71 1,07 1,16 4,66 Figure 1. Hydrothermal coefficients of vegetation periods in 2001 2004 years Methods of laboratory analysis. Soil samples for determining microbiological parameters were taken from the arable soil layer (from 0-20 cm depth) in 3 replicates. The spreading of physiological groups of microorganisms was determined in soil samples with natural moisture, calculating the number of colonies forming units (c. f. u.) in one gram of oven-dry soil. The spreading of ammonifying microorganisms was determined by dilution method in albumen (using peptone) agar medium (X 3 ) /Rachno, 1964/ and spreading of mineral nitrogen assimilating microorganisms in ammonia salts medium, witch have starch ammonia agar (KAA) /Metody po č venoj mikrobiologiji, 1991/. The number of spore forming bacteria were determined on the mixture of food and neutral mash agar (S), constituted at equal volume parts /Miustin et. al , 1954 /, of micromycetes on acid mash agar medium (AMAr) /Aristovskaja et. al , 1962/. The coefficient of mineralization - immobilisation M/H = KAA/X3 was calculated in order to evaluate the structural-functional ratio of bacterium in the soil. M/H is the ratio between the number of amonificating (M) and mineral nitrogen assimilating (H) microorganisms. This ratio defines the direction of mineralization-humification processes in the soil. In general, the direction of these processes may be expressed as follows: (mineralization) M/H<1 ← M/H = 1 → M/H>1 (humification) /Andrejuk et. al, 1992/. The processes of mineralization and humification are in balance M/H=1 in
natural and conditionally undisturbed soil biotopes (e.g. in forests) /Bagdanavi č ien ė , 1994/. The fixation power of soil nitrogen was established in 2004, evaluating the activity of ferment nitrogenase (classification No. 188.8.131.52.) by gas chromatograph CHROM-5 using acetylene. The activity of nitrogenase is expressed in μ MN 2 1g of soil per 1 hour /Ambrazaitien ė , 1999/. Soil chemical analyses were done from the same soil samples as microbiological analyses, using the following methods: pH KCl by potentiometer (ISO 10390), total nitrogen - by Kjeldal (ISO 11261), nitrate and ammonium nitrogen by colorimetric (ISO 14256, ISO 13878) methods, organic carbon by dry burning with Hereaus apparatus. Laboratory analyses were done in Agrochemical Research Centre of LIA and in the Agrobiologic laboratory of V ė ai č iai Branch of LIA. Data was treated using Excel2003, Statgraphics and ANOVA for Excel vers.4,0. Two- and three-way analyses of variance were executed. Correlation - regression dependences were calculated using STATISTICA. Data statistically evaluated by Fishers (F) criterion, the coefficient of correlation (r), average error S x and standard deviation S. The significance level of equation was determined using F criterion. The significance of data is noted: significant at * - 95 % and at ** - 99 % level /Tarakanovas et. al , 2003 /. 3. RESULTS AND DISCUSSION The evaluation of the succession of microorganisms, transforming nitrogen compounds. The number of researched microorganisms depended not only on environmental conditions, but also on the organic matter transformation time. This state was made when the spreading dynamics of microorganisms, transforming nitrogen compounds, in different by pH and mineral fertilisation Albeluvisol was evaluated. The largest number of ammonifying bacteria was found after five weeks from the beginning of plant vegetation (in June). Later their activity became weaker. The largest number of mineral nitrogen assimilating microorganisms and the lowest number of micromycetes were found after 10 weeks from the beginning of plant vegetation. The micromycetes were most active after 20 weeks from the beginning of vegetation (in October). After 25 weeks from the beginning of plant vegetation, the most abundantly spreaded spore forming bacteria. The influence of soil pH and mineral fertilisation on microorganisms, transforming nitrogen compounds, is shown in Figure 2-4. It was determined, that the activity of ammonifying and mineral nitrogen assimilating microorganisms in non-fertilised Albeluvisol was more intensive when soil pH was close to the neutral. The micromycetes better spreaded in very acidic soil. The ammonifying microorganisms spreaded best in average and low acidity soil, fertilised with conditionally low NPK rate (N 45 P 39 K 57 ). The largest number of mineral nitrogen assimilating microorganisms was in close to neutral Albeluvisol . The activity of spore forming bacteria was the most intensive in low acidity and close to neutral soil.
The number of microorganisms pH<4.7 The number of microorganisms H 4.7-5.2 18000 18000 16000 16000 14000 14000 12000 12000 10000 10000 8000 8000 6000 6000 4000 4000 2000 2000 0 0 0 5 10 15 20 25 0 5 10 15 20 25 Weeks Weeks The number of microorganisms pH 5.2-5.7 The number of microorganisms pH> 6.7 18000 18000 16000 16000 14000 14000 12000 12000 10000 10000 8000 8000 6000 6000 4000 4000 2000 2000 0 0 0 5 10 15 20 25 0 5 10 15 20 25 Weeks Weeks ¡ Ammonifying bacteria(x1000) S Spore forming bacteria (x10) z Micromycetes (x10) Mineral nitrogen assimilating bacteria (x1000) Figure 2. The dynamics of microorganisms, participating in soil nitrogen transformation processes, in different by pH non-fertilised Albeluvisol in 2003 -2004 The number of microorganisms H<4.7 The number of microorganisms H 4.7-5.2 18000 18000 16000 16000 14000 14000 12000 12000 10000 10000 8000 8000 6000 6000 4000 4000 2000 2000 0 0 0 5 10 15 20 25 0 5 10 15 20 25 Weeks Weeks The number of microor anisms pH 5.2-5.7 The number of microor anisms H> 6.7 1800018000 1600016000 1400014000 12000 12000 10000 10000 8000 8000 6000 6000 4000 4000 2000 2000 0 0 0 5 10 15 20 25 0 5 10 15 20 25 Weeks Weeks ¡ Ammonifying bacteria(x1000) S Spore forming bacteria (x10) z Micromycetes (x10) Mineral nitrogen assimilating bacteria (x1000) Figure 3. The dynamics of microorganisms, participating in soil nitrogen transformation processes, in different by pH Albeluvisol fertilised with N 45 P 39 K 57 in 2003 -2004 15