3.1.1 Biodegradability (lability) and stability of organic matter

How many labile components of organic matter are lost during anaerobic digestion in a biogas plant can be demonstrated by determination of the degree of organic matter lability. For this purpose a number of methods can be used that are mostly based on resistance to oxidation or on resistance to hydrolysis. Oxidation methods are based on oxidation with chemical oxidants, e. g. with a solution of K2Cr2O7 in sulphuric acid at various concentrations — 6 M + 9 M + 12 M (Walkley 1947, Chan et al. 2001) or with a neutral solution of KMnO4 at various concentrations (Blair et al. 1995, Tirol-Padre, Ladha 2004). The degree of organic matter lability is evaluated from the amount of oxidizable carbon in per cent of its total amount in particular variously aggressive oxidation environments or the reaction kinetics of the observed oxidation reaction is examined while its characteristic is the rate constant of the oxidation process.

In 2003 was proposed and tested the method to evaluate the kinetics of mineralisation of the degradable part of soil organic matter by the vacuum measurement of biochemical oxygen demand (BOD) of soil suspensions using an Oxi Top Control system of the WTW Merck Company, designed for the hydrochemical analysis of organically contaminated waters (Kolar et al. 2003). BOD on the particular days of incubation is obtained by these measurements whereas total limit BODt can be determined from these data, and it is possible to calculate the rate constant K of biochemical oxidation of soil organic substances per 24 hours as the rate of stability of these substances. A dilution method is the conventional technique of measuring BOD and also rate constants. It was applied to determine the stability of soil organic substances but it was a time — and labour-consuming procedure. The Oxi Top Control method was used with vacuum measurement in vessels equipped with measuring heads with infrared interface indicator communicating with OC 100 or OC 110 controller while documentation is provided by the ACHAT OC programme communicating with the PC, and previously with the TD 100 thermal printer. Measuring heads will store in their memory up to 360 data sentences that can be represented graphically by the controller while it is also possible to measure through the glass or plastic door of the vessel thermostat directly on stirring platforms. The rate of biochemical oxidation of organic substances as the first-order reaction is proportionate to the residual concentration of yet unoxidised substances:

dy/dt = K (L — y) = KLz (1)

where:

L = total BOD y = BOD at time t Lz = residual BOD k, K = rate constants

By integrating from 0 to t of the above relation the following equation is obtained:

Lz = L. erK = L. 10-kt In general it applies for BOD at time t:

y = L (1 — 10-kt) (3)

where:

y = BOD at time t L = BODtotal

k = rate constant [24 hrs-1]

Used procedure is identical with the method of measurement recommended by the manufacturer in accordance with the Proposal for German Uniform Procedures DEV 46th Bulletin 2000 — H 55, also published in the instructions for BOD (on CD-ROM) of WTW Merck Company.

The decomposition of organic matter is the first-order reaction. In these reactions the reaction rate at any instant is proportionate to the concentration of a reactant (see the basic equation dy/dt). Constant k is the specific reaction rate or rate constant and indicates the instantaneous reaction rate at the unit concentration of a reactant. The actual reaction rate is continually variable and equals the product of the rate constant and the instantaneous concentration. The relation of the reaction product expressed by BOD at time t (y) to t is the same as the relation of the reactant (L — y) at time t and therefore the equations

(L — y) = L. e-kt (4)

and

y = L (1 — e-kt)

are analogical.

If in the graph the residual concentration of carbon is plotted on the y-axis in a logarithmic scale log (L — y) and the time in days from the beginning of experiment is plotted on the x — axis, we will obtain a straight line, the slope of which corresponds to the value — k/2.303.

The quantity of the labile fraction of organic matter can also be assessed by determination of soluble carbon compounds in hot water (Korschens et al 1990, Schulz 1990) and their quality by determination of the rate constant of their biochemical oxidation (Kolar et al. 2003, 2005a, b).

Hydrolytic methods are based on resistance of the organic matter different aggressive ways of hydrolysis that is realised at different temperature, time of action and concentration of hydrolytic agent, which is usually sulphuric acid. Among many variants of these methods the hydrolytic method according to Rovira et Vallejo (2000, 2002, 2007) in Shirato et Yokozawa (2006) modification was found to be the best. This method yields three fractions: labile LP1, semi-labile LP2 and stable LP3. The per cent ratio of these three fractions, the sum of which is total carbon of the sample Ctot, provides a very reliable picture of the degree of organic matter lability.

Of course, there are a lot of methods based on the study of organic matter biodegradability in anaerobic conditions. First of all, it is the international standard ISO CD 11734: Water quality — evaluation of the "ultimate" anaerobic biodegradability of organic compounds in digested sludge — Method by measurement of the biogas production, and particularly a very important paper using the Oxi Top Control measuring system manufactured by the German company MERCK for this purpose (Sussmuth et al. 1999).

Tests of methanogenic activity (Straka et al. 2003) and tests examining the activity of a microbial system (Zabranska et al. 1985a, b, 1987) are methods that can describe the degree of organic matter lability in its ultimate effect. Our long-time work experiences in the
evaluation of a huge amount of various analyses for the study of organic matter lability have brought about this substantial knowledge:

1. The study of the ratio of organic matter labile fractions, i. e. of their quantity, is always incomplete. A more authentic picture of the situation can be obtained only if information on the quality of this labile fraction is added to quantitative data. Such a qualitative characteristic is acquired in the easiest way by the study of reaction kinetics of the oxidation process of this fraction. The process of biochemical oxidation and the calculation of its rate constant KBio are always more accurate that the calculation of its rate constant of oxidation by chemical oxidants Kchem (Kolar et al. 2009a).

2. It applies to current substrates for biogas production in biogas plants that with some scarce exceptions the degree of organic matter lability is very similar in both aerobic and anaerobic conditions. In other words: organic matter is or is not easily degradable regardless of the conditions concerned (Kolar et al. 2006).

3. A comparison of various methods for determination of organic matter lability and its degradability in the anaerobic environment of biogas plant digesters and also for determination of digestate degradability after its application to the soil showed that hydrolytic methods are the best techniques. They are relatively expeditious, cheap, sample homogenisation and weighing are easy, and the results correlate very closely with methods determining the biodegradability of organic matter directly. E. g. with the exception of difficult weighing of a very small sample and mainly its homogenisation the Oxi Top Control Merck system is absolutely perfect and highly productive — it allows to measure in a comfortable way simultaneously up to 360 experimental treatments and to assess the results continually using the measuring heads of bottles with infrared transmitters, receiving controller and special ACHAT OC programme for processing on the PC including the graph construction. But its price is high, in the CR about 4 million Kc for the complex equipment. Hydrolytic methods require only a small amount of these costs and are quite satisfactory for practical operations (Kolar et al. 2008). However, for scientific purposes we should prefer the methods that determine anaerobic degradability of organic matter, designated by DC.

The substrate production of methane VCH4S [the volume of produced methane (VCH4c) after the subtraction of endogenous production of methane (VCH4e) by the inocula] was determined by an Oxi Top Control Merck measuring system.

The calculation is based on this equation of state:

n = p x V/RT (6)

where:

n = number of gas moles V = volume [ml]

P = pressure [hPa]

T = temperature [°K]

R = gas constant 8.134 J/mol °K

and the number of CO2 and CH4 moles in the gaseous phase of fermentation vessels is calculated:

nco2 g CH4 = (Ap x Vg/RT) x 10-4	(7)
0 1 и	(8)

where: p0 = initial pressure

Fermentation at 35° C and continuous agitation of vessels in a thermostat lasts for 60 days, the pressure range of measuring heads is 500 — 1 350 kPa and the time interval of measuring pressure changes is 4.5 min. Anaerobic fermentation is terminated by the injection of 1 ml of 19% HCl with a syringe through the rubber closure of the vessel to the substrate. As a result of acidification CO2 is displaced from the liquid phase of the fermentation vessel. The process is terminated after 4 hours. The number of CO2 moles is calculated from the liquid phase:

nCO2 l = {[p2 (Vg — VHCl) — pi x Vg]/RT} x 10-4 (9)

The injection of 1 ml of 30% KOH into the rubber container in the second tube of the fermentation vessel follows. The sorption of CO2 from the gaseous phase of the vessel is terminated after 24 hours and the total number of CO2 moles in gaseous and liquid phases is calculated from a drop in the pressure in the vessel:

ncO2 l, CO2 g = {[рз (Vg — VhCI — Vkoh) — P2 (Vg — VHCl)J’RT} x 10-4 (10)

where:

Ap = difference in pressures [hPa]

Vg = the volume of the gas space of the fermentation vessel [ml] p1 = gas pressure before HCl application [hPa] p2 = gas pressure before KOH application [hPa] p3 = gas pressure after KOH application [hPa]

R = gas constant = 8.134 J/mol °K T = absolute temperature = 273.15 + X °C VHCl = the volume of added HCl [ml]

VKOH = the volume of added KOH [ml]

Based on the results, it is easy to calculate the number of CO2 moles in the gaseous phase and by the subtraction from nCO2 g CH4 the number of moles of produced methane:

nCH4 = (nCO2 g CH4 + nCO2 l) — nCO2 l CO2 g (11)

The total number of moles of the gases of transported carbon:

nCO2 g CH4 + nCO2 l = ntotal (12)

Baumann’s solution A + B in deionised water of pH = 7.0 is used as a liquid medium (Sussmuth et al. 1999).

The standard addition of the inoculum corresponds roughly to an amount of 0.3% by volume (aqueous sludge from the anaerobic tank of the digester). Instead of Baumann’s solution it is possible to use a ready-made nutrient salt of the MERCK Company for this system.

The operation of the Oxi Top Control measuring system was described in detail by Sussmuth et al. (1999).

Methane yield was calculated from the substrate production of methane VcH4S by division by the initial quantity of the added substrate:

where:

Verne = methane yield of C-source

Verne = methane yield of the added inoculum

S = substrate quantity at the beginning [g]

Lord’s test and other methods suitable for few-element sets and based on the R range of parallel determinations were used for the mathematical and statistical evaluation of analytical results including the computation of the interval of reliability.

Anaerobic degradability is given by the equation:

e

Dc =-£- .100
e

where:

es = total C content in the sample

eg = C content in methane released during the measurement of anaerobic degradability The value of Cg is computed from the substrate production of methane VeH4S:

e = ^2 p vch4S
g RT

(because 1 mol CH4 contains 12 g C) where:

K = temperature (°K)

R = gas constant

P = pressure

VeH4S = the volume of produced methane after the subtraction of endogenous production by the inoculum from total production

This method, which determines organic matter lability in anaerobic conditions, is so exact that it allows to investigate e. g. the digestive tract of ruminants as an enzymatic bioreactor and to acquire information on its activity, on feed utilisation or digestibility and on the influence of various external factors on the digestion of these animals (Kolar et al 2010a) or to determine the share of particular animal species in the production of greenhouse gasses (Kolar et al 2009b).

At the end of this subchapter dealing with the degree of organic matter lability and its changes after fermentation in a biogas plant these experimental data are presented:

A mixture of pig slurry and primary (raw) sludge from the sedimentation stage of a municipal waste water treatment plant at a 1 : 1 volume ratio was treated in an experimental unit of anaerobic digestion operating as a simple periodically filled BATCH-system with mechanical agitation, heating tubes with circulating heated medium at a mesophilic temperature (40°C) and low organic load of the digester (2.2 kg org. dry matter/m3) and 28- day fermentation.

Acid hydrolysis of sludge, slurry and their mixture was done before and after anaerobic fermentation. The hydrolysis of samples was performed with the dry matter of examined sludge and its mixture with pig slurry including the liquid fraction after screening the material through a 250-pm mesh sieve. The method of hydrolysis according to Rovira and Vallejo (2000, 2002) as modified by Shirato and Yokozawa (2006): 300 mg of homogenised sample is hydrolysed with 20 ml of 2.5 M H2SO4 for 30 min at 105°C in a pyrex tube. The
hydrolysate is centrifuged and decanted, the residues are washed with 25 ml water and the wash water is added to the hydrolysate. This hydrolysate is used to determine Labile Pool I

(LP I).

The washed residue is dried at 60°C and hydrolysed with 2 ml of 13 M H2SC>4 overnight at room temperature and continuous shaking. Such an amount of water is added that the concentration of the acid will be 1 M, and the sample is hydrolysed for 3 hours at 105°C at intermittent shaking. The hydrolysate is isolated by centrifugation and decantation, the residue is washed again with 25 ml of water and the wash water is added to the hydrolysate. This hydrolysate is used for the determination of Labile Pool II (LP II). The residue from this hydrolysis is dried at 60°C and Recalcitrant Pool (RP) is determined from this fraction.

Ctot is determined in all three fractions.

Degradability of organic matter of the test materials was studied by modified methods of Leblanc et al. (2006) used to examine the decomposition of green mulch from Inga samanensis and Inga edulis leaves. These authors conducted their study in outdoor conditions (average annual temperature 25.1°C) and we had to modify their method in the cold climate of this country. At first, the liquid phase of sludge, slurry and mixture was separated by centrifugation; the solid phase was washed with hot water several times and separated from the solid phase again. By this procedure we tried to separate the solid phase from the liquid one, which contains water-soluble organic substances and mineral nutrients. Solid phases of tested organic materials were mixed with sandy-loamy Cambisol at a 3:1 weight ratio to provide for inoculation with soil microorganisms and volume ventilation of samples with air. After wetting to 50% of water retention capacity the mixtures at an amount of 50 g were put onto flat PE dishes 25 x 25 cm in size. The material was spread across the surface of the dish. Cultivation was run in a wet thermostat at 25°C, and in the period of 2 — 20 weeks dishes were sampled in 14-day intervals as subsamples from each of the four experimental treatments. The agrochemical analysis of the used topsoil proved that the content of available nutrients P, K, Ca and Mg according to MEHLICH III is in the category "high" and pKKCl = 6.3. After drying at 60°C for 72 hours the content of lipids, crude protein, hemicelluloses, cellulose, lignin, total nitrogen and hot-water-insoluble dry matter was determined in the dish contents.

After twenty weeks of incubation organic substances were determined in the dish contents by fractionation into 4 degrees of lability according to Chan et al. (2001).

The content of hemicelluloses was calculated from a difference between the values of neutral detergent fibre (NDF) and acid detergent fibre (ADF), lignin was calculated from ADF by subtracting the result after lignin oxidation with KMnO4. Because ADF contains lignin, cellulose and mineral fraction, it was possible to determine the cellulose content by ashing the residue in a muffle furnace and by determination of mineral fraction. These methods were described by Van Soest (1963), modifications used by Columbian authors (Leblanc et al. 2006) were reported by Lopez et al. (1992).

Ion exchange capacity [mmol. chem. eq./kg] was determined in dry matter of the examined materials according to Gillman (1979), buffering capacity was determined in samples induced into the H+-cycle with HCl diluted with water at 1 : 1 and washed with water until the reaction to Cl — disappears. In the medium of 0.2 M KCl the samples were titrated to pH = 7 with 0.1 M NaCH and buffering capacity was calculated from its consumption.

Tab. 1 shows the analyses of a mixture of pig slurry and primary sludge used in the experiment. Cbviously, compared to the values reported in literature our experimental materials had a somewhat lower content of organic substances in dry matter, and perhaps this is the reason why anaerobic fermentation reduced the content of organic substances by 39% only although the usual reduction by 45 — 65% for primary sludge was expected as reported in literature (Pitter 1981) and by 40 — 50% for pig slurry (StehHk 1988). As a result of the organic dry matter reduction the content of nutrients in sludge after anaerobic fermentation is higher, nitrogen content is lower by about 20%. In this process organic nitrogen is converted to (NH^)2CO3, which partly decomposes into NH3 + H2O + CO2 and partly passes into the sludge liquor. Roschke (2003) reported that up to 70% of total nitrogen might pass to the ammonium form at 54% degradation of organic substances of dry matter. Even though concentrations of the other nutrients in dry matter of the aerobically stabilised sludge increased as a result of the organic dry matter reduction, their content in the sludge liquor also increased (Tab. 2).

	Pig slurry	Primary sludge	Mixture of slurry and sludge before methanisation	Mixture of slurry and sludge after methanisation
Organic substances	65.1 + 2.6	62.7 + 2.4	64.1 + 2.4	36.9 + 1.5
Total nutrients	N	6.2 + 0.2	2.6 + 0.1	3.9 + 0.2	3.1 + 0.1
P	1.6 + 0.1	0.7 + 0.0	1.1 + 0.0	1.3 + 0.1
K	2.3 + 0.1	0.2 + 0.0	1.2 + 0.0	1.2 + 0.0
Ca	2.8 + 0.1	2.6 + 0.1	2.5 + 0.1	2.8 + 0.1

Table 1. The analysis of experimental pig slurry and primary sludge, mixture of pig slurry and primary sludge before methanisation in a digester and after methanisation in % of dry matter (pig slurry and primary sludge were mixed for anaerobic digestion at a 1:1 volume ratio). (Sample size n = 6, interval of reliability of the mean for a significance level a = 0.05)

	[%]	[%]	[mg/l]	[mg/l]
	A	B	Before fermentation	After fermentation
Total N	8.40	55.20	246.2 + 14.7	994.7 + 59.6
Ammonia N	52.60	90.80	153.7 + 8.4	907.2 + 48.2
Total P	12.20	25.30	134.5 + 8.7	176.3 + 11.6
Total K	19.90	28.10	172.9 + 10.4	184.1 + 11.0

Table 2. The analysis of the liquid fraction (sludge liquor) of a mixture of pig slurry and primary sludge from a waste water treatment plant (1 : 1) before fermentation and after fermentation in mg/l. The values A and B express % in the liquid phase of the total amount of sludge before and after fermentation (Sample size n = 5, interval of reliability of the mean for a significance level a = 0.05)

Taking into account that the amount of water-soluble nutrients in the sludge liquor and organic forms of N and P dispersed in the sludge liquor in the form of colloid sol (but it is a very low amount) is related not only to the composition of the substrate but also to technological conditions of anaerobic digestion, digester load and operating temperature, it is evident that the liquid fraction of anaerobically stabilised sludge contains a certain amount of mineral nutrients, approximately 1 kg N/m3, besides the others, although differences in the concentration of P and K in the liquid fraction before and after fermentation are generally negligible. It is a very low amount, and there arises a question whether the influence of the liquid fraction on vegetation is given by the effect of nutrients or water itself, particularly in drier conditions.

After anaerobic digestion the solid phase of sludge still contains a high amount of proteins and other sources of organic nitrogen that could be a potential pool of mineral nitrogen if the degradation of sludge after fermentation in soil is satisfactory.

Material	Proportion
LP I	LP II	RP
Primary sewage sludge	68 + 5	23 + 2	9 + 1
Pig slurry	59 + 5	15 + 2	26 + 2
Mixture of primary sludge and pig slurry at a 1:1 volume ratio	63 + 5	20 + 2	17 + 1
Mixture of primary sludge and pig slurry at a 1:1 volume ratio after methanisation	18 + 2	16 + 1	66 + 5

Table 3. Proportions of the three pools of carbon in experimental materials, as determined by the acid hydrolysis method of Rovira and Vallejo (2002), (Sample size n = 4, interval of reliability of the mean for a significance level a = 0.05), (Materials including the liquid fraction were used)

The results of hydrolysis in Tab. 3 prove that pig slurry has 59% of its total carbon in LP I, which indicates great lability, corresponding to the hydrolysability of cereals and grasses according to Shirato and Yokozawa (2006). Primary sewage sludge is still better from this aspect, having almost 70% C in LP I. The degree of lability of the sludge and slurry mixture is relatively high and corresponds to the component ratio. After methanisation carbon content in LP I of the sludge and slurry mixture decreases to less than a third of the original amount and carbon of non-hydrolysable matters increases even almost four times in the RP fraction. The sum of LP I and LP II, i. e. the labile, degradable fraction of carbon compounds of the sludge and pig slurry mixture, was reduced by anaerobic digestion from 83% to 34%, that means approximately by 50%. These are enormous differences and they prove that mainly very labile organic substances are heavily destroyed by the anaerobic process even though a reduction in the content of organic substances during anaerobic fermentation is lower (by 39% in our experiment).

Tab. 4 shows the analysis of raw materials (sludge and pig slurry) and their mixture before and after anaerobic fermentation while Tab. 5 shows the analysis of their liquid fraction. The same results (Tab. 4) are provided by the incubation of the solid phase of sludge, pig slurry and their mixture before and after anaerobic fermentation when incubated with soil at 25°C and by the contents of lipids, crude protein, hemicelluloses, cellulose, lignin, total nitrogen and hot-water-insoluble dry matter; the same explicit conclusion can be drawn from the results of the fractionation of organic matter lability of the experimental treatments after 20- week incubation with soil according to Chan et al. (2001) shown in Tab. 5. A comparison of the results in Tab. 3 and 5 indicates that as a result of the activity of microorganisms of the added soil in incubation hardly hydrolysable organic matter was also degraded — differences between the most stable fractions F 3 and F 4 in Tab. 5 are larger by about 73% after anaerobic fermentation while in the course of acid chemical hydrolysis the content of non-hydrolysable fraction was worsened by anaerobic fermentation because it increased by about 290%. But it is a matter of fact that the soil microorganisms are not able to stimulate the anaerobically fermented sludge to degradation as proved by more than % of total carbon in fraction 4.

	I Before incubation (25° C)	II After incubation (25°C, 20 weeks)
A	B	C	D	A	B	C	D
Lipids (petroleum ether extractable compounds) [%]	8.60 ± 0.69	14.27 ± 1.14	10.82 ± 0.86	2.01 ± 0.15	7.97 ± 0.65	13.50 ± 1.09	10.39 ± 0,85	2.08 ± 0,17
Proteins (Berstein) [%]	13.43 ± 1.30	17.95 ± 1.62	15.31 ± 1.60	8.50 ± 0.93	11.81 ± 1.20	16.10 ± 1.53	13.89 ± 1.42	8.50 ± 0.98
Hemicelluloses [%]	1.82 ± 0.19	5.03 ± 0.73	3.32 ± 0.61	0.70 ± 0.60	1.43 ± 0.11	4.23 ± 0.51	2.89 ± 0.30	0.69 ± 0.10
Cellulose [%]	7.45 ± 0.92	11.18 ± 1.33	9.61 ± 1.05	6.03 ± 0.95	5.42 ± 0.82	9.27 ± 0.98	7.96 ± 0.94	6.05 ± 0.83
Lignins [%]	4.84 ± 0.62	5.16 ± 0.84	4.99 ± 0.75	5.18 ± 0.92	4.83 ± 0.91	5.18 ± 1.07	4.98 ± 0.84	5.20 ± 0.91
Total N [%]	1.59 ± 0.06	2.70 ± 0.11	2.29 ± 0.10	1.07 ± 0.04	1.51 ± 0.06	2.50 ± 0.11	2.14 ± 0.09	1.08 ± 0.05
Hot-water insoluble dry matter [%]	98.25 ± 2.94	98.26 ± 2.95	98.25 ± 2.95	98.23 ± 2.92	89.05 ± 2.67	85.17 ± 2.60	87.26 ± 2.58	98.20 ± 2.93
Ion exchange capacity [mmol chem. eq./kg]	48 ± 3	55 ± 3	53 ± 3	145 ± 9	50 ± 3	61 ± 4	55 ± 4	168 ±10
Buffering capacity [mmol chem. eq./kg]	62 ± 4	69 ± 4	65 ± 4	157 ± 9	65 ± 4	72 ± 4	70 ± 4	179 ± 11

Table 4. The content of selected organic substances (%) and ion exchange and buffering capacity of the solid phase of primary sludge (A), pig slurry (B), sludge and pig slurry mixture at a 1:1 ratio before fermentation (C) and after fermentation (D) before and after 20 weeks of incubation with sandy-loamy Cambisol topsoil at a 3:1 ratio at 25°C in dry matter (Sample size n = 4 /hot-water-soluble dry matter n = 7/, interval of reliability of the mean for a significance level a = 0.05)

	Unfermented primary sludge	Unfermented pig slurry	Mixture A	Mixture B	Soil only
Fraction 1 (12 N H2SO4)	59.84 + 7.18 (32.00)	55.38 + 6.52 (28.40)	54.09 + 6.50 (30.05)	2.65 + 0.30 (2.60)	1.30 + 0.17 (7.22)
Fraction 2 (18 N — 12 N H2SO4)	42.45 + 5.13 (22.70)	35.76 + 4.26 (18.34)	34.22 + 4.10 (19.01)	9.28 + 1.10 (9.07)	0.80 + 0.09 (4.44)
Fraction 3 (24 N — 18 N H2SO4)	27.34 + 3.28 (14.62)	20.18 + 2.53 (10.35)	20.30 + 2.42 (11.28)	11.13 + 1.33 (10.91)	3.70 + 0.44 (20.56)
Fraction 4 (TOC = 24 N H2SO4)	57.37 + 6.85 (30.68)	83.67 + 10.01 (42.91)	71.39 + 8.55 (39.66)	78.97 + 9.40 (77.42)	1.22 + 1.42 (67.78)

Table 5. The fractionation of organic carbon (g/kg) of primary sludge, pig slurry, and sludge and slurry mixture at a 1:1 ratio before fermentation (A) and after fermentation (B) in a mixture with sandy-loamy Cambisol (3 : 1) in dry matter after 20 weeks of incubation at 25°C by the modified Walkley-Black method according to Chan et al. (2001) with a change in H2SO4 concentration. (The values given in brackets are % of the C fraction in total dry matter carbon) (Sample size n = 5, interval of reliability of the mean for a significance level a = 0.05)

The table results document that 20-week incubation decreased more or less the per cent content of examined organic substances except lignin (total N 5 — 8%, cellulose 17 — 25%, hemicellulose 13 — 22%, proteins 9 — 12%, lipids 4 — 7%, and the content of hot-water — insoluble dry matter by 10 — 15%) factually in all experimental treatments except the treatment of the anaerobically fermented mixture of primary sludge and pig slurry where a reduction in these matters is low or nil. Hence, primary sludge, pig slurry and their mixture can be considered as organic fertilisers but only before anaerobic fermentation. We recorded a substantially lower degree of degradation of selected organic substances in sludge, pig slurry and their mixture during incubation with 25% of sandy-loamy soil (5 — 25%) than did Leblanc et al. (2006) with phytomass of Inga samanensis and Inga edulis leaves, who reported about 50% degradation of total mass, hemicelluloses and nitrogen in mass. We are convinced that it is caused by a very different content of hemicelluloses in our materials compared to the materials used by the above-mentioned authors. No easily degradable hemicelluloses are present in sewage sludge or in pig slurry any longer, and obviously, only more stable forms pass through the digestive tracts of animals and humans. It is also interesting that after anaerobic fermentation and after 20-week aerobic cultivation at 25°C only the compounds (lipids + proteins + hemicelluloses in mixture II D account roughly for 11%) that could be considered as labile remained in the mixture of slurry and sludge. These are apparently their more stable forms as confirmed by the results in Tab. 5 which illustrate that to approximately 11% of organic carbon compounds it is necessary to add the % proportions of the first and second fraction on the basis of oxidisability according to Chan et al. (2001). Literary sources report that the sum of lipids, proteins and hemicelluloses in the anaerobically stabilised sludge from municipal waste water treatment plants amounts to 13% — 39.6% of dry matter, so it is quite a general phenomenon.

The ion exchange capacity of sludge, pig slurry and their mixture before fermentation, before incubation and after incubation is very low and does not reach the values that are typical of sandy soil. It is increased by anaerobic fermentation along with incubation markedly but practically little significantly to the level typical of medium-textured soils. The same relations were observed for buffering capacity, which is not surprising. The results document that degradability of the organic part of anaerobically stabilised sludge worsened substantially and that it cannot be improved very markedly by the use of soil microorganisms and soil.

We have to draw a surprising conclusion that sludge as a waste from the processes of anaerobic digestion is a mineral rather than organic fertiliser and that from the aspect of its use as organic fertiliser it is a material of much lower quality than the original materials. We cannot speak about any improvement of the organic material by anaerobic digestion at all. Their liquid phase, rather than the solid one, can be considered as a fertiliser. If it is taken as a fertiliser in general terms, we do not protest because besides the slightly higher content of mineral nutrients available to plants (mostly nitrogen) it has the higher ion exchange capacity and higher buffering capacity than the material before anaerobic fermentation, but this increase is practically little significant.