Effect of Slurry on Microorganisms and Chemical Properties of Soil

ZbI. l\likrobiol. 138 (19 83) , 501-509 [D ep artmen t of Soil Microbiology, Inst itute of Soil Scienc e a nd Agr icultural Che m ist ry, A cademy of Ag riculture, Poznan, Poland]

Effect of Slurry on Microorganisms and Chemical Properties of Soil H.

K ASZ UBIAK,

G. D URSKA, \V.

KACZMAREK ,

and G.

FILODA

With 3 Figures

Summary Field ex p erim en t s were con d uc te d in the soil of a flooded m ea do w to com p a re the effect of slurry with that of NPK fertilizers o n microbial number, bioma ss, a nd a ctiv it y as well as on t h e so il che m ical properties. In contr a ry with NPK, heavy dres sin g with slurry limited the bacterial b ioma ss, influencing aerobes m ai nl y, a n d increased both the a ctivi ty of urease and the soil p H . I n t he slu rry -t rea t ed soil sm a ller a m ou n t s of NO a were foun d in t he NPK-treated one. No co n sider a b le differ ences were observed b etween both soils in t he n u m ber of proteolytic micr oorgan ism s, the p o t ential activity of nitrifi cation, t h e con te nt o f NH 4 , N0 2 , total N, an d organic C.

Zusammenfassung Au f eine m iiberflute ten W iesenb oden a n der W arte wurden F eldv ersu che m it Giille - und Min eral d iingerv arianten d urchgefiihrt, urn ihren Ein fl u l3 a u f l\Iik roben za h l, -bi oma sse und -a k t ivitat so wie a u f v ers ch ie dene Boden eigen schaft en zu untersu ch en. Irn Gegen sa t z zur N P K -Diingu ng b eg ren ze n hohe Giillega b en d ie bakterielle Bioma sse, wob ei Aero be m ehr b eeinflul3 t w orden a ls An earo be , erh oh en die U reaseaktiv it iit und den p H -' Vert. 1m mi t Gii lle b ehand el t en Bod en wu r de wenige r Nitra t ge fu n de n als im mi t NPK ged u ngten Boden . Di e U n t ersch ie de in der Zahl d er p r oteoly ti sch en Mikroorganismen, in d er p otenti ellen Ak t iv itat d er Ni t r atbildun g und in d en Geh alt en a n NH4 - , N0 2 +, Gesa rnt-N und org anisch em K ohl enst off waren zwisch en den D iing u n gsv a ri anten se h r ge ri ng.

Slurry is sprea d on l and not only as a valuable source of nutrient for plant s, but also to di sp ose it. Owin g t o this fa ct slurry is often ap plied at very high d oses. The question arises how soil microorganism s respond t o that h ea vy dressing. Many expe rimen ts h a-ve been performed by us to solve thi s problem . Som e of them are described in this pap er presenting the results of three-year field experiments, conducted to compare the effect of slurry application with the effect of NPK fertilizers on the microorgani sm count s, their biomass and activit y , and on the nitrogen and carbon content and the ac t ivit y .

Materials and Methods Soil and enrich ment S t u d ies h ave b een ca r r ied out in 1977 -1979 in the soil of a fl ooded m eadow, situ ated on the b ank s of W a r t a riv er in Mied zy c hod a n d b el ong ing t o t h e Experiment al Station in Gorzyri. T h e soi l was sa n d y lo am, p H 5.8, co n tain ing a b ou t 2.5 % of organic carb on . The investigations were carried o u t in t h e experim enta l fa r m of t h e D ep artment of Nonr u m inating Ani mals, Academy of Agricu lture, Poznan . The m e adow wa s divid ed into plot s o f 0.4 h a a reas. The part icular pl ots receiv ed various do ses of slu rry, su p p lie d wit h N- an d P-sal t s. Co ntrol pl ots were dressed w ith NPK fertilizers (Table I).

502

H. KASZUBIAK et al.

Table 1. Enrichment of experimental objects with slurry and NPK Objects

Enrichment

kg Njha N

I

None (control soil)

2 3

Mineral salts

4 5 6

Slurry nitrogen and potassium salts

+

P 20S 0

0

250 375

125 188

+ 60 2) + 90 + 120

190 1 ) 285 380

95 143 190

o 230 345

+ 30 + 45 + 30

230 345 460

1) from slurry, 2) from mineral salts.

Dense cattle slurry, Norway salpetre (34 % wjw), superphosphate, and potassium salts (60 % wjw) were applied. The p. and K-salts were applied only once before the first cutting. The doses of slurry and salpetre were divided into three portions, applicated before each cutting.

Sampling Soil samples were taken during the vegetative season 8-10 times on every 21st day, starting in the middle of April till the middle of October. Representative samples for each treatment consisted of 30-40 subsamples from 30 cm soil top layer taken along the diagonal lines of the plots.

Determinations In the representative soil samples the total bacterial biomass, the counts of microorganisms, growing in synthetic nutrient media under aerobic and anaerobic conditions, the counts of proteolytic bacteria, the activity of nitrifiers and of urease, the content of NH4 +, N0 2 - , N0 3 - , and total N, as well as the total organic C were determined as described below. 1. Total bacterial biomass Calcutations were made on the basis of the cell numbers. The fluorescent microscope and acridine orange as fluorochrome were used for the cell enumeration according to STRUGGER (1948). It was assumed that the average volume of the microbial cells was 0.22 ftm 3 (KASZUBIAK et al, 1977), their specific weight -1.1 (ZVYAGINTSEV and ROGACHEVSKIY 1973), and the water content in fresh weight - 70 % 2. Counts of microorganisms, growing in synthetic nutrient media under a e r obic and anaerobic conditions The estimations were made only in the third year of soil enrichment. The most probable number was calculated on the basis of microbial growth in liquid soil extract medium with 0.1 % glucose and yeast extract. Ten-fold dilutions of soil were made and 5 tubes for each dilution were set. Both aerobes and anaerobes were grown for 7 days at 28°C. Anaerobes were incubated in vacuum. 3. Counts of proteolytic bacteria Dilution plate method, using gelatine agar, was used for enumeration. The protein hydrolyse zones, formed around the bacterial colonies, were marked with Frazier's reagent after 2 days of incubation at 20 °C. The determinations were carried out during the first two experimental years. 4. Nitrifiers' activity Potential activity for nitrification was estimated. For that purpose 50 ml portions of inorganic medium, prepared according to WINOGRADSKI, were inoculated with 0,5 g of soil samples. After 14 days of incubation at 30°C the N0 3-N formed from NH4-N was measured colorimetrically.

Effect of Slurry

503

5. Urease activity The method by LLOYD and SCHAEFFE (1973) was employed, but the temperature of soil incubation with urea added was not 37° but 28°C. The enzymatic activity was expressed in fig NH 4·N producedjg dwt of soilj24 h of incubation. 6. Nitrogen content The NH4+, N0 2 - and N0 3 - nitrogen was measured colorimetrically according to the methods described by NOWOSIELSKI (1968). KJELDAHL'S method was applied for the total N determinations. 7. Organic carbon content TIURIN'S method, modified by KONONOVA (1968), was used. 8. Soil activity The measurements were done using pH-meter. 9. Additional note The microbiological analyses were made in five replicates, the chemical ones in triplicate. Interpretation of the data obtained was based mainly on the mean values for the vegetation seasons. The results were evaluated statistically. Calculations of the significance limits at the level of P = 0.05 were made. Moreover, variation coefficients "V" for the effects of slurry and NPK and for the changes, depending on the time of analyses, were established.

Results The parameters tested showed high fluctuation during the vegetation season (Tab. 2). The response of the microorganisms to the slurry or NPK soil treatment was observed. 1. Microbial counts, biomass, and activity.

In Table 3 the average numbers and the total biomass of bacteria, as well as the activities of nitrifiers and urease are given. Table 2. Variations of the parameters tested, depending on the soil enrichment and time of analyses Parameters

+NPK

+ Slurry

Variation coefficients VI) for

Bacterial biomass Aerobes Anaerobes Proteolytic bacteria Nitrifiers' activity Urease activity NH4+·N N0 3-·N Total mineral N Total N Total C pH

Enrichment

Time

18

12 178

30 16 6 9 91 37 1 2 1

112 94 17 56 39 37 5 7 4

-** -**

1) Averages for three vegetation seasons.

71

**) Not calculated, the control soil was not analysed. 33

Zbl. Mikrobiol., Bd. 138

Enrichment

Time

22

20 139 186 84 55 32 49 47 46 9 9 3

-** -** 35 17 23 19 68 20 3 4 4

504

H. KASZUBIAKet al.

Table 3. Microbial biomass, number, and activity on the soils, treated with slurry and NPKI) Parameters tested

Year of enrichment

Control

+NPK

+ Slurry

kg Njha 0

250 5.3 4.0 3.8

4.5 3.9 3.5

250

375

500

Bacterial biomass (mg dwtjg dwt)

1 2 3

Aerobes (10 6 jg dwt)

3

22

44

35

17

Anaerobes (10 6 jg dwt)

3

8

3

8

11

Proteolytic bacteria

1 2

107 83

52 160

96 106

161 87

52 65

105 68

1 Nitrifiers' activity (,ugN03-·Njmlmedium) 2

118 116

151 115

173 169

94 136

151 136

143 135

1 Urease activity (,ug NH4 +-Njgdwtj24h) 2 3

50 41 61

51 55 57

58 53 56

59 68 64

76 76 116

65 99 76

(10 3 jg dwt)

4.9 4.9 3.9

375

4.4 3.5 3.2

3.3 2.2 2.6

3.2 2.4 2.3

1) Averages for the vegetation season.

-

No determination.

1.1. Total bacterial biomass

In the soil under study the microorganisms, visible under the microscope, were very abundant - up to 91,5 X 109 cellsjg dwt. Their biomass showed a relatively small fluctuation under the influence of climatic conditions. Slurry application decreased the bacterial biomass distinctly, and that unfavourable effect occurred as early as in the first year of dressing. It became more intense in the second year and was a little smaller in the third one after the spring flood of the meadow. Smaller values for the bacterial biomass in the slurry-treated soil were statistically significant at slurry doses from 375 to 500 kg Njha. Immediately after that dressing many dead cells were found in the soil. The differences noted between the NPK-treated and the control soil were statistically insignificant at all fertilization levels. 2.1. Microorganisms growing in the laboratory media under aerobic and anaerobic conditions Contrary to the bacterial biomass the number of microorganisms, able to grow in the laboratory media, changed considerably in the vegetation season. Nevertheless, a visible effect of slurry on the microorganisms, growing in the presence of oxygen, was revealed in nearly all samples (Fig. 1). At the dose of 250 kg Njha those microorganisms were better promoted by slurry than by NPK. However, when the dose of slurry was increased up to 375 kg Njha their number diminished, while the mineral salts gave a positive effect at the same fertilization level. The reaction of anaerobes to the soil enrichment was not so clear like that of .aerobes.

Effect df Slurry

505

A. AEROBES

20 250kg N/ha

375kgN/ha

50

B. ANAEROBES

250kgN/ha

375kgN/ha

TIMES OF AN!'.LYSIS Fig. 1. N u mbers of a erobes a n d a naer obes in t he t h ird year of soil en ric hm en t w it h slu r r y (.) an d N P K ( 0) , u sed in do ses of 25 0 an d 375 k g N /h a .

1.3. Prot eolytic ba cter ia The number of those mi croorganism s depended on the time of a nalyses to a hi gher degree than on the soil enrichment . The changes of the population s were manifested both by increasing a nd decreasing of cell number (Fig. 2). No relation ship was found betw een t ype or dose of su bst a nces ad de d to the soil a nd the abunda nce of t he pr ot eolyti c ba ct eri a.

104. Nit rifiers' activity The effect of soil enrichment wa s very slight and th e change s of the ac t ivity resulted mainly from the sens it ivit y of nitrifiers to ' climatic cond itions. Always in th e middl e of Oct ober the pot ential a ct ivi ty of those mi croorganism s fell fr om several hundreds or t en s to a few fl g of NOs - -N , produced in 1 ml of their raw cultures derived from the st udied soil. 3S·

506

H. KASZUBIAK et al.

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~500

,\~

C5 8: 400

ao

, , Slurry , 'added

,,','l,'\

I

kJ300 ~

Slurry added

a~ [jj

ct ~ ~

, , I ,

I

I

100

l

~

f2200 ,:

•

,

••• '1,

-.

\ .•• ,.1.......

,

I \ , • , I t.\ ' i ~I If , ,.Ii' i ...,',~,

Slurry added

••

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~

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t: ' ----- --.,-, - --......:...\-\ - - - - - '. - -':---'---t \

: ..•..

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TIMES OF ANALYSIS Fig. 2. Number of proteolytic bacteria in the second year of soil enrichment with slurry, and NPK used in doses of 350 kg NJha. • - - . Slurry • . . . . • NPK

Slurry added Slur ry added

I

Slurry added

TIMES OF ANALYSIS Fig. 3. Urease activity in the second year of soil enrichment with slurry and NPK, used in doses of 375 kg NJha. • - - - . Control . - - . Slurry • . . . . • NPK

Effect of Slurry

507

1.5. Urease ac t ivit y The differ en ces between the urea se ac tiv ity in the NPK·treated and control soils were statist ically in significant, bu t in the ca se of slurry treatment a high er lev el of t he enzy me was found . In t he first and the third year of dressing the significant in crem ents wer e noted at do ses of 375 a nd 500 kg N fha, whil e in the second one also at a dose of 250 kg N fha . Howev er, the change s of the urease act ivity, occurring in the vege· t ation seas on, wa s not closely r elated to the times of slurry applica t ion (Fig. 3). 2. Cha nge s of ch emi c al pr op erti e s of th e s o il 2.1. Min eral nitrogen The NH 4 +·N conte nt changed with time. In the slurry.treated soil the mean va lues for the vegetation seaso n wer e usuall y not higher than in the cont rol (Tab. 4). The NH 4 +·N was nitrified without any di sturbances. If N0 2 appea red it wa s always found in t ra ces only. The differen ces noted between the total miner al N contents inthe soil of particular objects resulted m ainly from various amounts of N 0 3 - -N. In the first year of enr ichment the total mineral N content was sm all er in the slurry than in the NPK·treated soil. In the seco nd year the reserve was true , but in the third year NPK fertilizers yielded a new more inorganic N than the slurry . T a bl e 4. Am mon iu m an d n itra te n it rogen in t h e soil, treated with slu rry an d NPK N itrog en fo rm (m g /100 g d wt )

N H4

N0 3

T o t al m inera l

Year of enrichment

Con t rol kg

+ NPK

+ Slurry

~/h a

0

250

375

25 0

375

500

l.l

1.0 2. 1 2.1

1.0 2.8 1.8

1.1 2.4 2.5

1 2 3

1.1 2.0

1.1 2.0

:l.5

3.1

2.4 3.0

2 3

0.4 0.3 1.4

1.9 1.2 4.5

2.5 2.6 7. 6

0.9 2.9 3.5

1.2 0.8 3.8

1. 5 1.6 3. 8

1 2 3

1.5 2.3 4.9

3.0 3.2 7.6

3.6 3.0 10.2

1.9 5.0 5.6

2.2 3.6 5. 6

2.5 4. 0 6.3

2.2 . Total nitrogen and organic carbon In the soil of all experimental plots high fluctuation s of the total N and organic C conte nt were observed during the vegetation season. They ranged up to 20 %, but the a verage remained n early the same. The deviation was limited to some perc ents only . The res ults of Corg : N tota1 ratio ca lculat ions, sho wn in Ta ble 5, gave no clear evidence of its dep enden ce on the slurry or NPK soil enrichme nt . 2.3. Soil aci dity Slurry made the soil m ore a lkaline. That effect wa s noted as earl y a s in the first year of dressing (Tab. 6). In the next year s the alkalinati on did not increa se and wa s always limi t ed to several t ent h of p H unit. The pH increa sed parall elly with the slurry d ose, bu t only up t o 375 kg N /ha . At 500 kg N /h a lower p H va lues were noted .

508

H.

KA.SZUBIA.K

et al.

Table 5. Carbon nitrogen ration in the soil, treated with slurry and NPK Year of enrichment

1 2 3

Control

+ NPK

+ Slurry

kgN/ha 0

250

375

250

375

500

10.4 9.5 8.8

10.3 9.6 10.0

9.4 9.4 8.0

10.1 9.4 8.0

9.9 9.9 9.0

9.9 9.9 9.0

Table 6. pH of the soils, treated with slurry and NPK Year of enrichment

1 2 3

Control

+NPK

+ Slurry

kgN/ha 0

150

375

250

375

500

5.5 5.9 6.2

5.6 5.9 6.3

5.7 5.9 6.3

5.9 6.2 6.4

6.2 6.5 6.8

6.0 6.2 6.5

Diseussion The data obtained in the first two experimental years indicated that the main trend of changes, occurring in the soil after slurry treatment, was observed as early as in the first year of the dressing, then the changes became more intense. However, the results of the third experimental year showed that in the case of a flooded meadow the effect of slurry may be limited by the flood of land. One should consider in such case a pollution of the adjacent environment (for example-river) with slurry. Determining the microbial biomass, only the bacteria were taken into account. Blue and green algae were omitted because of their relatively small abundance in the cultivated soils of our climatic zone. Data on the fungal biomass were not obtained. Recently, however, we performed incubation experiments with the soil taken from the experimental meadow (KASZUBIAK and LIDKA, unpublished). We found the fungal biomass many times smaller than the bacterial one. It ranged from 0.02 to 0.1 mg dwtjg dwt. of soil, and those fluctuations depended mainly on the time of measurements, not on the slurry treatment. The facts mentioned above led us to the conclusion that the diminishing of bacterial biomass, observed in the field experiments after heavy slurry dressing, limited the total microbial biomass. In our studies one could expect that, beside the diminishing of biomass of the bacterial community, the group of the microorganisms, readily growing in the laboratory media, would develop in the presence of slurry, as observed by CZEKALA and Dnnosz (1975). That bacterial group consisted mainly of strains with high reactivity fo freshly added organic matter (KACZMAREK et al. 1973). However, while we estimated the numbers of microorganisms, growing in the laboratory media, the sensitivity of their aerobic form on slurry in the heavy-dressed soil was observed. That phenomenon resulted probably from the sedimentation of the coarser particles of slurry on the soil surface layer (McALLISTER 1977).

Effect of Slurry

509

While the microbial counts and biomass declined, the activity of urease increased, although a high positive correlation between those parameters was found by NANNIPIERI et al. (1978). One could suppose that the large amounts of urease were introduced together with the slurry into the soil. If that was true, the fluctuation of urease activity, occurring in the vegetation season, would be closely related to the time of dressing. However, that dependence was not observed here. The increase of urease level in the slurry-treated soil was also noted by MIKLASZEWSKI (1978). Frequent occurrence of that phenomenon suggests to take precautions in the use of urea fertilizers after slurry application to avoid the accelleration of urea decomposition. A narrow C: N ratio of a slurry indicates danger to humus degradation in the heavy-dressed soils. That process is of frequent occurrence in the incubated soil samples, but in the field experiments the persistent increments of soil organic matter may occur, as reviewed by MACKOWIAK (1977). In our experiments the soil organic matter content did not increase. Moreover, a higher level of the mineral nitrogen was found in the slurry-amended than in the NPK-treated soil in the second year of enrichment. That evidenced a stimulatory effect of slurry on the breakdown of organic substances. Acknowledgement Thanks are due to L. BEDNAREK, B. PAETZ, and M. STOCKA for their technical assistance.

References CZEKALA, J., and Dll!BOSZ, K.: Dzialanie gnojowicy w glebie. 1. Przemiany wegla i azotu oraz dynamika drobnoustrojow. Roczniki Akademii Rolniczej w Poznaniu. 82 (1975), 33-41. KACZMAREK, W., KASZUBIAK, E., and GUZEK, E,: Comparison of changes in the number of microorganisms in the soil by the plate and microscopic procedures. Pol. J. Soil Sci. 6 (1973), 133-139. KASZUBIAK, E., KACZMAREK, W., and Pll!DZIWLK, Z.: Comparison of different methods for estimating the productivity of microorganisms in soil. Ekol. Pol. 25 (1977), 289-296. KOKONOVA, M. M.: Organicheskoe veshchestvo pochvy. Akademia Nauk SSSR, Moscow 1968. LLOYD, A. B., and SCHEAFFE, M. J.: Urease activity in soils. Plant and Soil 39 (1973),71-80. MACKOWIAK, OZ.: Zmiany w skladzie chemicznym gleb pod wplywem nawozenia gnojowica. Trans. Symp. Sklad chemiczny gleb i wed glebowych w warunkach wykorzystania gnojowicy do celow nawozowych. JUNG Baborowko (1977),7-40. McALLISTER, .T. S. V.: Spreading slurry on land. Soil Sci. 123 (1977),338-343. MIKLASZEWSKI, S.: Wplyw gnojowicy na akbywnose ureazy w glebie. Trans. Symp. Rola substancji organicznych w metabolizmie glebowym. Badgoszcz-Golub Dobrzyn (1978), 53-54. NANNIPIERI, P., JOHNSON, R. L., and PAUL, E. A.: Criteria for measurements of microbial growth and activity in soil Soil BioI. Biochem. 10 (1978), 223-229. NOWOSIELSKI, 0.: Metody oznaozania potrzeb nowozenia, ed. PWRL, Warszawa 1968. STRUGGER, S.: A fluorescent microscope examination of bacteria in soil. Can. J. Res., Sec. C 26 (1948),188-193. ZVYAGINTSEV, D. G., and ROGACHEVSKIY, L. M.: Plotnost' (udielniy vies) kletok mikroorganizmov Mikrobiologiya 42 (1973), 892-898. Eingegangen am 15. 6. 1982 Author's address: Dr. EENRYK KASZUBIAK, Prof., Dept. of Soil Microbiology, Academy of Agriculture, Wolynska 35, 60 - 637 Poznan, Poland