Org. Agr. DOI 10.1007/s13165-015-0135-1

Changes in P fractions after long-term application of biogas slurry to soils under organic farming Carmen Haase & Stefanie Wentzel & Reiner Schmidt & Rainer Georg Joergensen

Received: 26 August 2015 / Accepted: 7 October 2015 # Springer Science+Business Media Dordrecht 2015

Abstract In the present field study, the effects of applying biogas slurry for 25 years on soil microbiological and soil chemical properties were investigated in comparison with the application of raw cattle slurry. An onfarm research approach was used with pairwise comparison of neighboring fields, taking samples at a high spatial resolution from two biodynamic farms down to 10 cm. Texture, soil organic C (SOC), total N and P, microbial biomass C, N, and P as well as P fractions were determined by different extraction procedures. Application of biogas slurry had no significant effects on SOC, total N, or microbial biomass C, N, and P. Clay and pH effects outweighed site-specific differences for most properties. Biogas slurry increased the fractions of plant-available inorganic phosphate (+66 % more Olson-Pi) and of Ca-associated phosphates (+40 % 1 M HCl-Pi) at the expense of hardly soluble Fe and Al phosphates (-25 % 6 M HCl-Pi). This is in line with the observation that biogas slurry contains significantly more H2O-Pi and 1 M HCl-Pi than raw slurry. The longterm application of biogas slurry had no negative impacts on any of the soil properties analyzed.

C. Haase : S. Wentzel : R. G. Joergensen (*) Soil Biology and Plant Nutrition, University of Kassel, Nordbahnhofstr.1a, 37213 Witzenhausen, Germany e-mail: [email protected] R. Schmidt Extension Service Schwäbisch Hall, Eckartshäuserstr. 41, 74532 Ilshofen, Germany

Keywords Microbial biomass . Biogas slurry . Cattle slurry . Hedley P fractions . On-farm research

Introduction Biogas production is becoming increasingly important in Germany, due to strong government funding (Bachmann et al. 2011). Electric power produced by biogas plants increased from 0.19 MW in 2003 to 3.8 MW in 2014 (FNR 2014). Cattle slurries and maize silage are the most commonly used substrates (Möller and Müller 2012). Consequently, biogas slurries are increasingly used as a fertilizer for crop production, replacing to a large extent mineral fertilizer in conventional farming systems (Sieling et al. 2013) or nondigested raw animal slurries or farmyard manure in organic farming systems (Stinner et al. 2008; Terhoeven-Urselmans et al. 2009). While various studies have investigated the N fertilizing effects of biogas residues (Terhoeven-Urselmans et al. 2009; Fouda et al. 2013; Sieling et al. 2013), less is known about their effects on the soil P cycle (Bachmann et al. 2011; Möller and Müller 2012), although organic fertilizers are the most important P source in all farming systems with animal husbandry (Bach and Frede 1998; Oehl et al. 2004). The digestion of raw slurry in the biogas fermenter reduces the concentration of organic matter (Frear et al. 2011) and increases the pH (Sánchez et al. 2000), caused by higher NH4+ concentrations (Möller et al. 2008). Investigations show that changes in P fractions in

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animal manure caused by fermentation vary according to animal species and diet as well as storage and fermentation conditions (Loria and Sawyer 2005; Möller and Stinner 2010; Möller and Müller 2012). Another problem is that fermentation has contradictory effects on P availability. On the one hand, the release of inorganic phosphates (Pi) and Ca during fermentation reduces Pi availability by the formation of Ca-associated P i (Güngör and Karthikeyan 2005, 2008; Güngör et al. 2007). On the other hand, volatile fatty acids, such as acetate, are able to form Ca complexes with Pi in animal manures, further increasing Pi solubility (Sánchez et al. 2000; Güngör and Karthikeyan 2005). Short-term application of biogas slurry revealed positive effects on soil microorganisms and N supply to crops in comparison with a non-fertilized treatment (Terhoeven-Urselmans et al. 2009). This might be different in the long term, especially comparing biogas with raw slurry, caused by the differences in organic matter quality described above. The P fertilizer efficiency not only depends on the solubility of a specific fraction within these slurries but also on soil properties, especially pH and clay content (Hinsinger 2001; Negassa and Leinweber 2009; Bachmann et al. 2011), as well as on the activity and biomass of soil microorganisms (Oehl et al. 2004; Malik et al. 2013). In incubation experiments, soil microbial activity was lower after application of biogas residues in comparison with raw cattle slurry, due to the lower C input (Ernst et al. 2008; Chen et al. 2012). This is of particular concern in organic farming systems where soil fertility is of special importance (Scheller 2006, 2013). Several biodynamic farmers in the area of Hohenlohe, Baden-Württemberg, are pioneers in the production of biogas in Germany. This provides the opportunity to study the long-term effects of biogas slurry application by using an onfarm research approach, with pairwise comparison of neighboring fields, taking samples at a high spatial resolution (Wentzel et al. 2015). It has already been shown at the experimental sites that clay has strong effects on microbial activity, biomass, and residues (Wentzel et al. 2015). Soil pH has certainly a strong effect on the P availability to plants, but contradictory results exist on P fractions. Soil pH effects have been measured in some cases (Bar-Yosef 1991; Alt et al. 2011), but not in all (Pätzold et al. 2013), presumably as other factors, such as soil organic matter and clay, may be important too. Consequently, the objective of the present on-farm field study was to

investigate the effects of applying biogas slurry for 25 years on soil chemical properties and on soil microbial biomass indices, with a special focus on soil microbial biomass P and soil P fractions (Hedley et al. 1982; Khan and Joergensen 2012). The underlying hypotheses were (1) that long-term application of biogas slurry increases the contribution of Ca-associated phosphates to total P in soil in comparison with raw cattle slurry and (2) that clay content and especially soil pH override organic fertilizer effects on the distribution of P fractions.

Materials and methods Site and soils The investigation was performed on two biodynamic farms at Kirchberg, located in the northeast of BadenWürttemberg (Germany), with a mean annual temperature of 8.7 °C and a mean annual precipitation of 772 mm. One farmer had a biogas plant and the other used raw cattle slurry. Both farms have practiced organic farming for at least 40 years and the farm with the biogas plant has used the biogas slurry as a fertilizer for 15 years. Both farms had two neighboring fields at two sites, enabling the pairwise comparison (Wentzel et al. 2015). The soils were classified as Clayic Cambisol at site I (with biogas slurry: 49°13′0.62″ N, 9°57′31.83″, 388 m asl; with raw slurry: 49°12′53.36″ N, 9°57′36.15″, 380 m asl) and as Haplic Luvisol at site II (with biogas slurry: 49°13′3.25″ N, 9°58′35.33″, 409 m asl; with raw slurry: 49°13′44.32″ N, 9°59′ 16.89″, 427 m asl) according to the WRB classification system (FAO 2014). In November 2010, volumetric soil samples were taken 4 weeks after fertilization from nine independent points per field at 0–10 cm depth, using steel cores. All samples were analyzed separately, after sieving (<2 mm) and homogenization. They were stored in polyethylene bags at 4 °C until the biological analyses were started. Sub-samples of sieved soils were dried and finely ground for chemical analyses. The two fields with biogas slurry application were fertilized on average with 30 m3 ha−1 a−1 and additionally received 6 t fresh weight composted farmyard manure ha−1 a−1. The two fields with raw slurry application were fertilized on average with 30 m3 ha−1 a−1 and additionally received 13 t fresh weight farmyard manure ha−1 a−1. The crop rotation consisted of red clover

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(Trifolium pratense)/grass, potatoes (Solanum tuberosum), winter wheat (Triticum aestivum), winter barley (Hordeum vulgare), alfalfa (Medicago sativa), and winter wheat. Winter wheat was sometimes replaced by dinkel (Triticum spelta), winter barley by winter rye (Secale cereale), and potatoes by carrots (Daucus carota subsp. sativus). More information on the crop rotation can be obtained by Wentzel et al. (2015). Legumes as main crops did not receive any organic fertilizer. The biogas slurry consisted of 98 % raw cattle slurry and of 2 % whole crop silage (rye, barley, rape). All fields were moldboard plowed at 0– 20 cm depth.

Microbial soil properties Microbial biomass C and microbial biomass N were estimated by the fumigation-extraction method (Brookes et al. 1985; Vance et al. 1987), using the preextraction procedure of Mueller et al. (1992), as modified by Mayer et al. (2003). For pre-extraction, 6 g moist soil was extracted for 30 min by oscillating shaking at 200 rpm with 20 ml 0.05 M K2SO4 and filtered (hw3; Sartorius Stedim Biotech, Göttingen). Then, fumigated and non-fumigated portions of 2 g soil were extracted for 30 min again with 8 ml 0.5 M K2SO4 and filtered. Organic C and total N in these 0.5 M K2SO4 extracts were measured using a multi N/C 2100S automatic analyzer (Analytik Jena, Germany). Microbial biomass C was calculated as EC/kEC, where EC =(organic C extracted from fumigated soils)−(organic C extracted from non-fumigated soils) and kEC =0.45 (Wu et al. 1990). Microbial biomass N was calculated as EN/kEN, where EN =(total N extracted from fumigated soils) −(total N extracted from non-fumigated soils) and kEN =0.54 (Brookes et al. 1985). Soil microbial biomass P was also measured by the fumigation-extraction method (Brookes et al. 1982) as described by Joergensen et al. (1995). Fumigated, nonfumigated, and non-fumigated and spiked portions of 3 g soil (on an oven-dry basis) were extracted with 0.5 M NaHCO3, pH 8.5 (see P fractionation). Microbial biomass P was calculated as EP/kEP/recovery, where EP =(PO43−-P extracted from fumigated soil)−(PO43−-P extracted from non-fumigated soil) and kEP = 0.40 (Brookes et al. 1982). Recovery of added P (25 μg g-1 soil) to account for P adsorption during extraction was calculated as follows: 1−((PO43−-P extracted from non-

fumigated and spiked soil)−(PO43-P extracted from nonfumigated soil))/25. P fractionation of soil and slurries Soil phosphorus was fractionated according to a modified Hedley method (Hedley et al. 1982, 1994; Tiessen and Moir 1993), integrated into the procedure for determining microbial biomass P (Brookes et al. 1982). The sequential fractionation procedure was started by extracting 3 g (on an oven-dry basis) chloroformfumigated field moist soil (see microbial biomass P) for 30 min oscillating shaking at 200 rpm with 60 ml 0.5 M NaHCO3, pH 8.5, for determining inorganic phosphate (Pi). Then, three 60 ml extraction steps with 16 h oscillating shaking at 170 rpm followed, using 0.1 M NaOH for determining Pi and Po again (Redel et al. 2007), 1 M HCl for determining Pi, and finally 6 M HCl for determining Pi and Po (Hedley et al. 1994; Liu et al. 2008). After each extraction step, the suspension was centrifuged at 3000×g for 10 min before roughly 50 % of the supernatant was filtered (hw3). Pi of all extracts was measured colorimetrically in the filtered extracts at 882 in a multi-plate reader (FLUOstar Omega; BMG Labtech, Offenburg, Germany), using ammonium heptamolybdate and ascorbic acid (Murphy and Riley 1962) as described by Joergensen et al. (1995). Total P in the extractable fractions was determined after adjusting the pH in a 20-ml aliquot of the centrifuged but unfiltered extract to 1.0, using 4.5 M H2SO4 for the 0.5 M NaHCO3 soil extracts, 4 M NaOH for the 1 M HCl extracts, and 16 M NaOH for the 6 M HCl extracts. Then, 10 ml of the pH-adjusted extract were transferred into 25-ml glass tubes, mixed with 1 ml 5 % K2S2O8 solution, and heated for 35 min at 120 °C. The Po was estimated as the difference between total P minus Pi. Concentrations of total P in soil but also in slurries were determined by HNO3/pressure digestion as described by Chander et al. (2008), followed by ICP-AES analysis (Spectro Analytical Instruments, Kleve, Germany). The phosphorus from six biogas and six raw slurry samples obtained from the current two farms were analyzed according to a modified fractionation method as proposed by Jorgensen et al. (2010). Freeze-dried and pulverized slurry material of 350 mg was weighed into 70-ml centrifuge tubes and extracted with 35 ml H2O for 16 h at 175 rpm. Then, this suspension was centrifuged at 4000×g for 10 min before the supernatant was

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filtered. An aliquot of this extract was acidified with 4 M H2SO4 to pH 1, and filtered again before Pi and Po was measured as described above. The residue was extracted again with 35 ml 1 M HCl and centrifuged at 4000×g for 10 min before the supernatant was filtered.

Table 1 P fractions in raw and biogas slurries, obtained from the two farmers

General soil properties

Raw

Slurry

H2O-Pi H2O-Po (μg g−1 dry weight)

1 M HCl-Pi

Total P

Biogas

1110 a

220 a

3270 a

7500

850 b

0b

3170 b

7680

CV (±%)

All analyses were carried out in triplicate. Soil pH was measured in water (ratio 1 to 2.5). Total C and total N were determined after combustion by gas chromatography using a Vario EL (Elementar, Hanau, Germany) analyzer. Soil organic C (SOC) was measured as total C minus carbonate C, which was measured gas-volumetrically after the addition of HCl (Blume et al. 2011). Statistics The results presented in the tables are arithmetic means and expressed on an oven-dry basis (about 24 h at 105 °C). After testing the normal distribution of the data using the Kolmogorov-Smirnov test, the significance of site and treatment effects was tested by a two-way ANCOVA (analysis of covariance), using slurry and site as factors as well as clay content and soil pH as covariates to analyze their effects on the dependent variables. The relationships between the different soil properties were analyzed by principal component analysis (PCA) using the orthotran/varimax rotation to achieve either small or large component loading and an eigenvalue of 0.1 as the lower limit. All statistical analyses were performed using SPSS (version 19).

Results Total P did not differ between the slurry types (Table 1). In the biogas slurry, total P consisted of 44 % 1 M HClPi, 15 % H2O-Pi, and 3 % H2O-Po, whereas 1.0 M HClPi, H2O-Pi, and H2O-Po contributed 41 %, only 9 %, and even nothing, respectively, to total P in the raw slurries. The soil pH varied around 6.7 at both sites and negatively affected the total P content (Table 2). Site I had higher contents of sand, clay, SOC, total N, and total P than site II. These higher contents of SOC and total N were mainly caused by the higher clay content (36 vs. 23 %), as revealed by the analysis of covariance. Biogas slurry application resulted on average in 12 % higher

3.0

33

1.8

2.0

CV mean coefficient of variation between replicate samples (n=6). Different letters indicate a significant difference within a column (t test, P<0.05)

total P contents compared with raw slurry, exclusively due to site I. The microbial biomass C content ranged around 620 μg g−1 soil and was on average highest at site I, whereas microbial biomass N and P were highest at site II (Table 3), leading to roughly 20 % higher microbial biomass C/N and C/P ratios at site I. All microbial indices were significantly affected by the clay content, whereas soil pH only affected microbial biomass N and the microbial biomass C/N and C/P ratios. Biogas slurry application did not affect any microbial property except the microbial biomass C/P ratio, which was significantly increased, mainly due to the clayey site I. The P fractions were dominated by 1 M HCl-Pi, 0.1 M NaOH-Pi, and residual P, exceeding on average 20 % total P, respectively (Table 4), whereas 6 M HCl-Pi and -Po varied between 5.8 and 14.9 % total P. Microbial biomass P, Olson-Pi, and Olson-Po ranged from 1.6 to 5.6 % total P. The clayey site I exhibited higher percentages of the inorganic fractions (Olson-Pi, 0.1 M NaOHPi, and 1 M HCl-Pi), but lower percentages of microbial biomass P and most organic fractions (0.1 M NaOH-Po and 6 M HCl-Po) as well as 6 M HCl-Pi and residual P than the loamy site II. The main differences in P fractions were again caused by the clayey site I with biogas application. Clay had especially strong positive effects on the contribution of Olson-Pi and 0.1 M NaOH-Pi to total P as well as negative effects on microbial biomass P and residual P. A lower soil pH significantly increased the percentages of Olson-Po, microbial biomass P, 0.1 M NaOH-Po, and residual P. Biogas slurry application resulted in significantly 66 % more Olson-Pi and 40 % more 0.1 M HCl-Pi as well as 25 % less 6 M HCl-Pi in comparison with raw slurry application, clearly demonstrated by the ratio of biogas to raw slurry effects in the respective soil P fractions.

Org. Agr. Table 2 Soil chemical and physical properties of the four different arable fields taken from two sites, probability values for first-order effects of the two-way ANCOVA, using site and slurry as factors and clay and soil pH as covariates Site

Slurry

Soil pH (H2O)

Sand (%)

Silt

Clay

SOC (mg g−1 soil)

Total N

Total P

I

Biogas

7.0

19

47

34

25

2.3

1.21

I

Raw

6.6

16

46

38

28

2.6

0.97

II

Biogas

6.3

5

71

24

22

1.6

0.69

II

Raw

6.7

5

74

21

17

2.1

0.72

P values Site

NS

<0.01

<0.01

<0.01

NS

NS

<0.01

Slurry

NS

NS

NS

NS

NS

NS

<0.01

Clay

NS

ND

ND

ND

<0.01

<0.01

NS

pH

ND

ND

ND

ND

NS

NS

<0.01

CV (±%)

4.8

19

13

19

13

14

8.1

CV mean coefficient of variation between the replicate samples within one field (n=9), ND not determined, NS not significant; interactions were all non-significant, when considered by the ANCOVA analysis

Principal component analysis revealed a close relationship between 0.1 M NaOH-Pi, Olson-Pi, 1 M HCl-Pi, and 0.5 M Olson-Po, forming factor 1 (Table 5). Residual P and microbial biomass P were closely related to SOC, microbial biomass C but also clay, forming factor 2, whereas 0.1 M NaOH-Po could not be definitely assigned to one

factor. The fractions 6 M HCl-Po and HCl-Pi were negatively related, forming their own factor 3.

Discussion Slurry composition

Table 3 Properties of the soil microbial biomass at the four different arable fields taken from two sites, probability values for first-order effects of the two-way ANCOVA, using site and slurry as factors and clay and soil pH as covariates Site

Slurry

Microbial biomass C N (μg g−1 soil)

P

C/N

C/P

I

Biogas

640

93

32

6.9

21.7

I

Raw

675

91

43

7.5

16.1

II

Biogas

630

103

39

6.1

16.2

II

Raw

530

89

35

6.0

15.5

P values Site Slurry

0.01 NS

Clay

<0.01

pH

NS

CV (±%)

18

<0.01 0.07 <0.01 0.02 19

<0.01

0.01

NS

0.01

0.08

0.01 0.02

<0.01

0.01

NS

0.01

25

6.0

0.04 20

CV mean coefficient of variation between the replicate samples within one field (n=9), NS not significant; interactions were all non-significant, when considered by the ANCOVA analysis

Biogas slurry contained significantly more H2O extractable Pi and 1 M HCl extractable Pi than raw slurry, which was reflected by an increased contribution of Olson-Pi and M HCl extractable Pi after the long-term application of biogas slurry to the arable sites. The fraction of POlson is certainly labile und usually referred to as plant available, whereas the 1 M HCl extractable Pi is assigned as Ca-associated phosphate (Hedley et al. 1982; Tiessen and Moir 1993) and considered as stable P fraction (Negassa and Leinweber 2009). It remains unknown whether all biogas slurries have similar effects on soil P fraction considering the high variability of in the bulk compositions of biogas slurries, caused by the differences in substrates (Möller and Müller 2012; Voelkner et al. 2015). It remains also unknown to what extent the quality of biogas slurries shows temporal variability. The total P concentrations of the current raw and biogas slurries are in the middle of the range from 1.5 to 16 mg P g−1 dry weight given in the literature (Field et al. 1984; Barnett 1994; Sharpley and Moyer 2000; Turner and Leytem 2004; Toor et al. 2005; Jorgensen

Org. Agr. Table 4 P fractions of soils in % total P at the four different arable fields taken from two sites, probability values for first-order effects of the two-way ANCOVA, using site and slurry as factors and clay and soil pH as covariates Site

Slurry

P-Olsona Pi

Po

Microbial

0.1 M NaOH

1 M HCl

6 M HCl

Biomass P

Pi

Pi

Pi

Po

Residual Po

P

(% total P) I

Biogas

4.8

1.8

2.6

27.7

5.7

34.8

7.5

5.8

15.7

I

Raw

2.7

1.6

4.4

20.6

7.5

27.7

II

Biogas

1.9

2.0

5.6

14.7

8.8

26.2

12.4

7.8

22.6

12.0

12.2

II

Raw

2.3

1.8

4.9

20.9

8.1

22.1

14.9

25.7

10.4

21.4

<0.01

NS

<0.01

<0.01

0.02

Slurry

0.01

NS

0.07

NS

0.07

Clay

0.01

NS

<0.01 0.02

P values Site

pH

NS

0.02

CV (±%)

20

21

0.01

0.09

0.01

<0.01

<0.01

NS

NS

NS

NS

NS

NS

<0.01

NS

0.01

NS

NS

NS

15

14

17

19

21

0.01

23

0.01

0.04 13

CV mean coefficient of variation between the replicate samples within one field (n=9), NS not significant; interactions were all nonsignificant, when considered by the ANCOVA analysis a

0.5 M NaHCO3 extractable P from non-fumigated soils

et al. 2010). This huge range is caused by differences in livestock species, feeding management, co-substrates, and storage conditions (Sharpley and Moyer 2000). Also, unclear separation of terms “manure” and “slurry” may contribute to this range. Slurry contains a dry Table 5 Oblique solution primary pattern matrix of the principal component analysis for different soil chemical and biological properties (orthotran/varimax transformation; bold—definite assignation to a certain factor Variable

Factor 1

Factor 2

Factor 3

0.1 M NaOH-Pi

0.99

−0.10

−0.13

Olson-Pi

0.96

−0.06

0.18

1 M HCl-Pi

0.90

0.05

−0.09

Olson-Po

0.86

0.00

0.23

−0.15

0.95

0.01

Residual P

0.00

0.91

0.03

SOC

0.32

0.82

0.00 −0.16

Microbial biomass C

Clay Microbial biomass P 0.1 M NaOH-Po

0.31

0.80

−0.34

0.79

0.09

0.42

0.47

0.17

6 M HCl-Po

−0.05

0.00

0.81

6 M HCl-Pi

−0.06

−0.02

−0.82

Eigenvalue

5.2

2.9

1.4

Variance

0.43

0.24

0.12

matter content below 12 % according to van Kessel and Reeves (2000), whereas manures contain less water and additionally often some bedding material. Low Po concentrations were found in the H2O extracts, whereas no Po was found in the 1 M HCl extracts. Also, Turner and Leytem (2004) did not find any Po in the HCl fraction, i.e., this fraction apparently consists, not only in soils but also in slurries, almost exclusively of Caassociated phosphates.

Slurry effects on P fractions and other soil properties The long-term application of biogas slurry in a biodynamic farming system had no negative impacts on the soil biological and chemical properties analyzed in comparison with raw slurry, although a loss in vitality has been feared (Scheller 2006, 2013). This supports the view stated by Wentzel et al. (2015), based on a larger group of biodynamic farms in the same area. Biogas slurry effects might be masked by the relatively high SOC and microbial biomass contents of the two sites in comparison with other German sites (Anderson and Domsch 1989; Höper and Kleefisch 2001). Biodynamic organic farming has generally positive effects on soil fertility and soil quality indices, as stated in long-term field experiments (Raupp 2001; Mäder et al.

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2002) and shown by data obtained in soil monitoring programs (Höper and Kleefisch 2001). In contrast to the contents of soil organic matter and microbial biomass indices, the long-term application of biogas slurry affected the composition of P fractions at both sites, independently of the total P content. In agreement with our hypothesis stated in the “Introduction” section, long-term application of biogas slurry increased the fractions of plant-available phosphate (Olson-Pi) and of Ca-associated phosphates (1 M HCl-Pi). This increase occurred mainly at the expense of 6 M HCl-Pi. This fraction is especially high in Ferralsols (Redel et al. 2007; Khan and Joergensen 2012), due to its very stable associations with Fe and Al at mineral surfaces and P occluded in sesquioxides (Hedley et al. 1982; Blake et al. 2003). Hot acid soluble Pi constitutes a part of the residual P pool, which is not considered as plant available (Tiessen and Moir 1993; Crews and Brookes 2014). Dilute acids such as 1 M HCl or 0.5 M H2SO4 mainly extract Ca-associated Pi, some occluded Pi released on dissolution of sesquioxides, but little or no Po (Perrott et al. 1989; Cross and Schlesinger 1995; Blake et al. 2003). However, the potential consequences for the observation that biogas slurry applications has subtle changes in the contribution of extractable P percentage of total P for plant growth and soil fertility remain unclear. The close relationship between the fractions of OlsonPi and 1 M HCl-Pi (r=0.84, P<0.01) suggests that OlsonPi, often considered as plant-available phosphate, is regenerated from Ca-associated phosphates. This view is in line with the observation by Pätzold et al. (2013) that the fractions of Olson-Pi and 1 M HCl-Pi were strongly enriched after seven decades of P fertilizer application. In agreement with the current results, Pätzold et al. (2013) also observed a strong decrease in 6 M HCl-Pi. However, they also observed strong decreases in Olson-Po, 6 M HCl-Po, and residual P. This is likely caused by different P fertilization rates in different treatments, contrasting the current situation, where similar amounts of P were applied. An exception is probably the increase in total P content at the clayey site I with biogas slurry application. This indicates a higher P input by fertilization, which is probably not only caused by biogas slurry application but by different P application rates in the land-use history. Such differences might mask any difference in further fertilization effects. The pairwise comparison of sites is based on the assumption that the neighboring sites had identical soil chemical and soil microbiological properties at the time

of starting biogas slurry application. However, even long-term experiments often suffer from the problem that the situation at the experimental start was insufficiently documented (Joergensen et al. 2010; Heinze et al. 2010). In most cases, only a representative bulk sample was taken initially, where sampling of each single plot would have been required. One main advantage of on-farm research by comparing neighboring sites is the possibility to answer actual practical questions on long-term management effects on the basis of best practice by farmers (Probst et al. 2008; Bowles et al. 2014). Clay and pH effects on soil properties Site-specific differences in SOC and total N contents were outweighed by positive clay effects on these two properties and also all microbial biomass indices were significantly affected by clay. However, the relationships between clay and microbial indices were more complex, especially for microbial biomass N and P, probably caused by other site characteristics and differences in management. This suggests that microbial soil properties respond more sensitively to soil management than chemical soil properties (Powlson et al. 1987). Positive clay effects on microbial biomass C have been observed by Müller and Höper (2004) as well as Bach et al. (2010), whereas no clay effect could be detected in the meta-study of Kallenbach and Grandy (2011) on the microbial biomass C/N ratio. One reason for this absence of clay effects may be soil pH-related differences in nutrient, especially in P availability, as indicated by pH effects on microbial biomass N, C/N, and C/P. Especially the two microbial elemental ratios are known to be strongly affected by the P availability to soil microorganisms (Anderson and Domsch 1980; Salamanca et al. 2006). Clay had positive effects on the contribution of the more easily extractable inorganic P fractions Olson-Pi and 0.1 M NaOH-Pi to total P but negative effects on that of microbial biomass P and residual P. A higher contribution of 0.1 M NaOH-Pi but not of Olson-Pi to total P has also been observed by Alt et al. (2011) in clayey soils in comparison with silty soils. A decrease in soil pH in the range from 7.2 to 6.3 had solely negative effects on the organic P fractions Olson-Po and 0.1 M NaOH-Po as well as on microbial biomass P and residual P again. The contribution of easily extractable organic P, i.e., the fractions associated with soil organic surfaces and colloids such as humic and fulvic acids

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(Hedley et al. 1982; Blake et al. 2003), to total P decrease with decreasing soil pH. This contrasts the results for acid sandy soils (Slazak et al. 2010), but also that for nearly neutral silt and clay loams (Alt et al. 2011). One reason for the current results may be a lower formation rate of organic P components, but the exact reasons cannot be explained by the current data.

Conclusions In comparison with raw slurry, the long-term application of biogas slurry in biodynamic farming systems increased the contribution of the inorganic fractions of plant-available P and of Ca-associated P to total P at the expense of hardly extractable inorganic P, i.e., mainly Fe and Al phosphates. The presence of clay controls the contribution of extractable inorganic P fractions to total P. A decrease in soil pH increased the microbial biomass C/P ratio and decreased the contribution of the easily soluble organic P fractions to total P. The longterm application of biogas slurry had no negative impacts on any of the soil properties analyzed. Acknowledgments We would like to thank Karl Kuch and Gerhard Kuch for access to their sites and the information on their management systems. The technical assistance of Gabriele Dormann and Ann-Kathrin Becker is highly appreciated. This project was supported in part by a grant of the Research Training Group 1397 “Regulation of soil organic matter and nutrient turnover in organic agriculture” from the German Research Foundation (DFG).

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