ENZYMATIC HYDROLYSIS OF ORGANIC WASTE MATERIALS IN A

SOLID – LIQUID SYSTEM

S.N. Jordana, and Mullenb, G. J.

a
National Centre for Freshwater Studies, Dundalk Institute of Technology, Co. Louth,

Ireland.
b
Department of Life Sciences, Schrödinger Building, University of Limerick, Co.

Limerick, Ireland.


Corresponding author. Tel: +353 429 381 872
Email address: siobhan.jordan@dkit.ie

1
 Abstract

In the current climate of increasing emphasis on environmental protection and

efficient waste management, regional management bodies and environmental

agencies are striving to achieve an economical and environmentally acceptable system

for the recycling of biodegradable organic wastes. Composting would appear to be a

cost effective solution to this problem but in its entirety, composting is an inherently

lengthy and variable process and is restrictive in terms of the demand on resources

and space in composting plants. The aim of this study was to compare a biological

composting process of solid residues with an enzymatic hydrolysis process of

residues. The length of time required to naturally compost three organic materials,

spent mushroom substrate (SMS), farmyard manure (FYM) and dairy wastewater

sludge (DWS) under optimal conditions was 42 days, 98 days and 84 days

respectively. In an attempt to accelerate this process, commercial enzymes were

added to the waste products in a heterogeneous solid - liquid system. The enzymes

utilised included a range of proteases, cellulases, ligninases, lipases and pectinases

, which are responsible for the hydrolysis of protein, cellulose, lignin, lipids and

carbohydrates respectively. Preliminary results indicate that all the organic materials

were stabilised within 9 hours and that the enzymes used would, therefore, improve

the efficiency of a waste management plant, if such a system were employed. Spent

mushroom compost has a mean N/P/K ratio of 20:10:10 recorded for composted

SMS, while a similar ratio of 20:10:20 was obtained for hydrolysed SMS. In contrast

composted farmyard manure has a N/P/K ratio of 30:0:30 and a ratio of 10:1:10 for

hydrolysed FYM. Finally, composted DWS has a N/P/K ratio of 20:1:30 while DWS

hydrolysate has a N/P/K ratio of 40:1:20, with the decrease in nitrogen in the

composted DWS attributed to the addition of wood chippings and sawdust as a

2
bulking agent. While all three materials have a considerable supply of plant nutrients,

the variability in nutrients could be overlooked when employed as a soil amendment.

Keywords: Enzymatic hydrolysis, composting, organic waste.

Introduction

Waste management has become increasingly important in recent years as European

law (99/31/EC) has stipulated that the quantity of organic waste discarded in landfills

must not exceed 35% by weight with reference to 1995 levels, not later than 2016

(EC, 1999). Consequently, a greater emphasis has been placed on recycling and the

reutilization of organic waste materials. Composting is considered to be a cost-

effective solution to the problem of effective waste disposal and this is witnessed in

the fact that thirteen compost production units are currently operating in Ireland.

The objectives of this study were to investigate the addition of commercial

extracellular enzymes to waste materials in an attempt to accelerate the degradation of

the associated organic matter and to compare and contrast naturally composted and

enzyme-hydrolysed compost with respect to selected physical and chemical

parameters. The organic waste materials; spent mushroom substrate (SMS), farmyard

manure (FYM) and dairy wastewater sludge (DWS) were utilised in this study, all of

which are produced in large quantities in Ireland annually.

Materials and Methods

Organic Waste Materials

SMS was obtained from a mushroom production farm in Co. Cavan, Ireland, that had

produced 3 flushes of mushrooms and was no longer capable of sustaining economic

3
levels of mushroom production. The SMS was composed of wheaten straw, horse

manure, poultry litter and gypsum. FYM was collected from a dairy farm in Co.

Limerick, Ireland, with hay being used as the only constituent of bedding. Finally,

DW sludge was obtained from a dairy industrial plant in Michelstown, Co. Cork,

Ireland. The dairy waste sludge was composed mainly of cheese whey, together with

washings from all the yards and instruments in the plant, which were treated using a

biological membrane reactor resulting in the degradation of COD, NH 4N and other

organic compounds (Anon. 2006). The resultant sludge was separated from the treated

effluent using an ultrafiltration membrane and the remaining water was drained off

into the local water system in conjunction with the requirements stipulated by the

EPA (Anon. 2006).

Composting

The composting of each waste was carried out in individual small-scale (5 gallon)

drums. To provide adequate aeration, holes (0.5cm 2) were drilled in each drum and

the drums were tumbled every 3 days. A moisture content of 50 % was maintained for

SMS and FYM throughout the composting process. Due to the initial high moisture

content of the DWS sludge, a mixture of wood chippings and sawdust were

subsequently added in a ratio of 4:1, which successfully reduced the moisture content

to the recommended level of 50 % (Lohr et al. 1984).

Waste material pH (2:1 ratio, Jenway 3010 pH meter), moisture content, CO 2, NH3

(Solvita® maturity index test) and temperature (Tiny Tag® data logger) were

measured throughout the composting process. The process was deemed complete

when the CO2 and NH3 concentrations reduced to maturity index 7 or 8 (Solvita®),

4
which represented a matured compost. pH stabilisation and temperature decrease were

also noted during the maturation stage, indicating that all readily degradable organic

matter was decomposed (Bardos and Lopez-Real, 1988). The initial waste materials

and subsequent composts were analysed to evaluate their fertiliser merit.

Enzymatic hydrolysis

In order to hydrolyse the three organic materials, initial analyses were carried out to

determine the quantities of the various constituents present in each. Accordingly, the

moisture content and the carbohydrate, mineral, protein, fat and lignin concentrations

were determined. The mineral content was determined by the quantity of residue left

after ashing 2g of each waste overnight at 500 oC, in a Gallenkamp muffle furnace.

The amount of protein present was determined using the Kjeldahl method for plant

material (Rowell, 1994), whereas the concentration of fat was established using the

soxhlet method, which is a semi-continuous solvent extraction (Min and Steenson,

1998). The lignolytic material was calculated using a special plant analysis procedure,

which involved refluxing and ashing of the composts, as outlined by Byrne (1979).

Finally, the amount of carbohydrate present was determined by subtracting the

combined weights of crude protein, total fat and minerals from the weight of the

waste sample, the results of which are outlined in table 1.

Enzymes

From these results, the amount of each enzyme responsible for the hydrolysis was

then calculated. Each enzyme was added in excess of 2% volume, to ensure that if any

variability occurred, the particular enzyme would not be limiting in the reaction. Five

commercially prepared enzymes were used in total, all of which were supplied by

5
 Novo Industries, Bagsvaerd, Denmark. The amount of each enzyme used during the

hydrolysis process for each material is given in table 2.

Alcalase 2.4 L FG is a proteolytic food grade enzyme that is generated by the

submerged fermentation of a strain of Bacillus licheniformis. Pectinex Ultra SP-L is a

pectolytic enzyme produced from a strain of Aspergillus aculeatus and although it is

successful in the breakdown of hemicellulolytic materials, Celluclast 1.5 L was also

used in this study, to aid in the degradation of cellulosic substances. Celluclast® is

Feasability of Study
generated from the submerged fermentation of a selected strain of the fungus

Trichoderma reesei. NS 44064 was also utilised to aid the breakdown of lignolytic

Initial estimation costs of

materials. NS 44064 is a laccase (phenol oxidase) and is produced by the submerged

treating one tonne of SMS in this

fermentation of Aspergillus oryzae, which has been genetically modified. Finally,

Lipozyme® TL 100L is a lipolytic food grade enzyme prepared from Thermomyces

liquid – solid system is €803.90,
lanuginosus by the submerged fermentation of the genetically modified Aspergillus

which excludes any mechanical

oryzae (Anon. 2003).

or operational expenses.
Analytical Procedure

Hydrolysis experiments were carried out in 500ml reaction vessels (fig. 1). 50g of

each dried material (2mm) was hydrated with approximately 150ml of distilled water

and was allowed to equilibrate at 50°C for 1 h. The mixture was agitated throughout

this time and during the extent of the hydrolysis, with a mechanical over-head stirrer

(Heidolph Instruments, Schwabach, Germany). The pH was measured and adjusted to

between 9 and 10 using NaOH, before the addition of the enzyme lipozyme®. This

pH and temperature provide optimum conditions for lipozyme® to function

efficiently (Anon. 2003).

6
Lipozyme®, the enzyme responsible for the hydrolysis of fat, was added and the pH

was continuously monitored at 2.5-second intervals, using a Pasco pH data logger,

until the pH remained constant for at least one hour. The pH was adjusted to 6 using

HCl, which is optimal for the remaining enzymes (pectinex®, celluclast®, alcalase®

and laccase®) to function. The reaction was allowed to proceed until the pH remained

unchanged for one hour, indicating that most of the components had been hydrolysed.

Hydrolysate samples were then heated at 90°C, for 10 min to denature the enzyme

activities. Samples were then cooled and stored at 4°C until required for analysis.

Control runs were also carried out where no enzymes were added. Lignin, cellulose,

hemicellulose and organic matter content were determined on these control samples to

establish whether chemical breakdown may have transpired at high pH.

Analyses

All three organic materials (OM) were analysed to establish variations, if any,

between fresh, composted and hydrolysed composts and also to evaluate their stability

and the suitability of each for possible usage in horticulture or agriculture. Twenty-

four parameters, both physical and chemical, were measured in total.

Physical Analyses

The organic matter content was determined following overnight ashing at 500 oC, in a

Gallenkamp muffle furnace, while acid detergent lignin (ADL), acid detergent fibre

(ADF) and hemicellulose constituents were determined using an adapted procedure

outlined by Goering and Van Soest (1970). Particle size distribution (PSD) for each

compost was analysed on samples <0.53µm using laser diffraction on the Malvern

7
Mastersizer 2000® (Houghton et al. 2002). The value reported for this parameter, is

the d(4,3) value, which is the volume mean diameter of particles, which takes into

account the number and volume of particles in a particular medium. Finally, the

percentage germination index values were established using cress seeds (Hogg et al.

2002) and is an eminent test in determining the phytotoxicity of compost.

Chemical Analyses

Total nitrogen was determined using the Kjeldahl method (Rowell, 1994), total

phosphorus and potassium were also analysed as per Rowell (1994), with the

phosphorus filtrate analysed colorimetrically by UV spectrophotometry at 712nm

(UV Spect 2000) and potassium levels determined by atomic absorption

spectrophotometry (AAS) (Varian Spectra AA 1989) using an air-acetylene flame.

The remaining elements copper, zinc, iron, manganese, cadmium, chromium, nickel

and lead, along with the cations calcium, sodium and magnesium, were also

determined on the atomic absorption spectrophotometer, using a procedure developed

by Soon and Abboud (1993). The appropriate standard curves were achieved, using

commercially available AA standards. The standards were prepared within the range

for optimum working conditions, as outlined in the operator’s manual (Varian Spectra

AA 1989).

The electrical conductivity of the materials was determined using a procedure

developed by Bower and Wilcox (1965) using a Jenway 4200 portable conductivity

meter, while pH values were obtained using a Jenway 3010 pH meter, according to

Hendershot et al. (1993).

8
The C/N ratio, which is an important parameter in determining the stability of

compost, was calculated from the total nitrogen and organic matter values on the

assumption that organic matter contains 58% carbon (Haug, 1993). Finally, the degree

of hydrolysis (DH) values were quantified using the trinitrobenzenesulfonic (TNBS)

method (Adler-Nissen, 1979), which measures the degree to which the protein has

been hydrolysed by the enzyme alcalase®.

Statistical Analysis

The data obtained were analysed by independent-sample T-tests using the ANOVA

programme of SPSS, version 11.0 (SPSS, 2002).

Results & Discussion

Composting

The duration of the composting process for each of the three organic materials is

outlined in fig. 2. SMS aged in just over 6 weeks, at which stage a consistent pH of

6.6 was recorded for two weeks. This was in agreement with Lohr et al. (1984),

employing a similar composting method. The pH increased significantly from day 7

to day 30 (P<0.05) as is evident in fig. 2, which also coincided with an increase in

temperature to 39oC within the first 10 days (fig. 3). This was in accordance with the

findings of Lohr et al. (1984) and Maher et al. (1993). The volume of compost in the

composting drum deceased greatly due to the breakdown of organic matter by

microorganisms and the consequent loss in structure (Maher et al. 1993). The

resulting compost was dark brown / black in colour, was peat-like in texture and

odourless. This relatively short composting process was anticipated, as SMS is

9
already partly decomposed at the end of the mushroom cropping cycle as shown in

fig. 2 by the absence of stable pH values.

Farmyard manure was deemed to be successfully composted after 14 weeks, as the

compost had no odour, was dark in colour, and the pH had stabilised at 7.62 (fig. 2).

The temperature had also dropped below ambient levels. As is evident in fig. 2, the

pH increased from 8.18 to 8.65 with the initial days of composting, whereas it

decreased from 8.54 to 8.34 in the study undertaken by Wang et al. (2004). The

duration of the composting process may have been attributable to the initial higher

organic matter content of the FYM and the particle size of the hay present, resulting in

a subsequent reduction in surface area for microbial attack (Crawford, 1983).

Following the addition of the bulking agent, the length of time required to stabilise

DW sludge was 12 weeks (fig. 2). Das et al. (2003) suggested that the addition of

bulking agents such as bark / wood chippings would help establish optimal moisture

levels, maintain air-filled porosity, and generally improve the overall structure of the

compost. This resulted in the amplification of microbial activity within the compost

and the concomitant reduction in odour concentration (Das et al. 2003), all of which,

should facilitate the rapid composting of the organic material which was noted in this

study (fig. 3).

Enzymatic Hydrolysis

10
Interpretation of fig. 4 shows clearly that the time taken for the pH to stabilise

following hydrolysis was about eight hours. Following the initial addition of

lipozyme®, the pH increased swiftly within the first two hours, and then levelled off,

illustrating that the hydrolysis of lipids within the samples was complete within four

hours, which coincidentally was within the recommended reaction time specified on

the lipozyme® product sheet (Anon. 2003). On addition of the remaining enzymes,

the pH remained around 6 for the first 30min and then dropped significantly for all

three materials. The pH subsequently remained constant with the notable exception of

SMS, where the pH increased slightly before stabilising.

The SMS hydrolysate was dark brown in colour, and was quite viscous, with minute

solid particles remaining. The final pH of the SMS was pH 6.1, but when the compost

suspension was brought back to room temperature the pH recorded was pH 6.48,

which is only slightly lower than the findings of Lohr et al. (1984), who recorded a

pH of approximately 6.7 for SMS aerobically composted over 60 days.

The FYM hydrolysate was somewhat similar to that of SMS, as it was quite viscous,

with some solids remaining, and was slightly darker in colour. However the pH of the

hydrolysed FYM was 4.61, which is extremely low for mature compost (Crawford,

1983). The DWS hydrolysate was also darker in colour, however a slight odour

persevered indicating that perhaps the DWS was not fully hydrolysed. The addition of

sawdust prior to hydrolysis may prove successful in this case, as it may reduce the

odour significantly in advance and/or the inclusion of enzymes more specific to the

various components present in DWS.

Physical and Chemical Analysis

11
The variation of properties for all fresh organic materials, and the changes they

undergo following hydrolysis and natural composting, are outlined in tables 3, 4 and

5. Evidently, there is great variation between all three materials, particularly among

the cations and the fertiliser elements N, P and K, while all heavy metals are

consistently low, and well below the recommended standards (Hogg et al. 2002).

The organic matter content of the three organic materials decreased hydrolysis and

composting. Changes in the organic matter content during composting is most likely

to be due to the microbial utilisation of readily decomposable substances within the

organic matter, such as sugars, starches, lipids, pectin, amino acids, and nucleic acids

(Bardos and Lopez-Real, 1988).

The range of pH values determined for fresh and composted FYM and hydrolysed

SMS, suggest that these materials would successfully raise the pH of soil, if employed

as a soil conditioner or amendment (Wang et al. 1984). By comparison, the pH of the

hydrolysed FYM and DWS are quite low; nevertheless, it is within the recommended

pH range (4 - 7) for compost (Verdonck, 1988).

In comparison, the EC values, which is a measure of the total dissolved salts in the

residue, increased slightly for the SMS and FYM composted materials and it is

hypothesised that this increase is due to the release of salts during the decomposition

of organic matter (Lohr et al. 1984) and is also attributed to the mass of the compost

as it becomes denser during composting. In relation to the EC values measured for the

hydrolysed composts, there was a vast increase in the magnitude of EC, due primarily

to the addition of sodium hydroxide, required for pH change. This increase resulted in

12
imbalances amongst nutrients, so as a preventative measure, further research is

required into enzymes that work at a more neutral pH and will thus eliminate the use

of any chemicals for pH adjustment. Elevated EC levels will almost certainly have a

negative effect on the growth of salt sensitive plants, and perhaps, on the growth of

young seedling. In a standard commercial growing medium, the EC levels are usually

between 1.2 - 1.5 mS cm-1 (Maher et al. 2000), however almost all the organic

materials analysed in this study were above this threshold value, with the exception of

DWS compost, while EC values differed significantly (P<0.01) for all composts.

Each compost has more than adequate amounts of the fertiliser elements nitrogen,

potassium and phosphorus present and with the exception of nitrogen, their values

varied significantly during the composting and hydrolysing processes (tables 3, 4 and

5). A decrease was noted in phosphorus levels for all composted and hydrolysed

materials, which is of particular interest given recent emphasis on nutrient

management in agriculture (Kurz, 2005). Of all the macronutrients and heavy metals

analysed, little variation was noted between composts, with sodium being the solitary

exception. Levels of sodium were amplified for all hydrolysed samples, owing to the

NaOH additions during hydrolysis. These amplified values may be a factor in the poor

germination index values obtained overall for hydrolysed compost. A germination

index (GI) of 90% is recommended for mature compost (Hogg et al. 2002), but this

only occurred in two of the samples, namely fresh FYM and composted DWS.

However, as hydrolysed samples are in solution, they will not be solely used as a

growing medium, but merely as a soil conditioner.

13
Lignocellulose with is composed of lignin, cellulose and hemicellulose, forms the

foremost part of biomass in nature, and subsequently its degradation is fundamental

within the carbon cycle (Tuomela et al. 2000). Levels of lignocellulitic material were

relatively high in each compost analysed, with straw, hay, wood chippings and

sawdust being primary providers of such components. Lignin, cellulose and

hemicellulose values appeared to increase during both the composting process and

hydrolysis. This is probably due to the fact that the more readily degradable carbon

sources have been depleted, and most of the residual compost is lignocellulitic in

nature. Lignocellulitic components are not easily degradable and can only be naturally

hydrolysed through the secretion of extracellular enzymes that are produced by only a

small number of compost microorganisms. Overall, lignin appears to be the most

resistant component of microbial attack (Crawford, 1983). There were significant

differences evident in these components between most of the compost samples, in

particular between all composted and hydrolysed materials (P<0.001), with the

exception of lignin values for SMS. Overall, the lignolytic components of the

hydrolysates are considerably lower in amount than those of their corresponding

composted samples, which demonstrates the success of the laccase® and pectinex®

over naturally produced enzymes excreted by compost microbes. The possibility that

the high pH of the hydrolysates may chemically breakdown the cellulitic material was

not applicable, as the cellulitic values of the control samples were significantly higher

than the hydrolysates (table 6).

Particle size distribution

Particle size distribution is an essential feature of the composting process, as

microbial activity is greatly improved when particle sizes are minute, resulting from

14
an increased surface area. However, if the particle size is too minute, void spaces are

then reduced, resulting in a reduced rate of gaseous diffusion (Crawford, 1983). The

particle size distributions carried out in this study were carried out on samples

>0.53µm, as particles greater than this size may abrade the cell lens. The d(4,3) values

for fresh, composted and hydrolysed SMS are outlined in fig. 5, and significant

variations (P<0.05) in particle size was observed for all treated substrates. An increase

in particle size occurred for composted and hydrolysed SMS, which was probably due

to the release of humic acids during hydrolysis and the composting process (Grabbe,

1988), resulting in the particles binding together, to form a more stable complex.

Similar to the particle size distributions in SMS, the composted FYM showed a

greater increase in particle volume and size, when compared to both fresh and

hydrolysed materials (fig. 6). There was a significant difference (P<0.01) noted

between all treated substrates and again these could be due to the differences in

biodegradability of each material (Houghton et al. 2002).

Finally, for the DW sludge, the fresh DWS proved to have the largest volume of

particles present (fig. 7), and differed significantly (P<0.01) from composted and

hydrolysed DWS. This indicates that the breakdown in organic matter during

hydrolysis and composting influenced the particle size of the materials, but not the

percent volume <0.53µm.

TNBS Analysis

The degree of hydrolysis (DH) for SMS was 86.3%, while, for FYM and DWS, the

DH values measured were 3.5% and 38.14% respectively. This indicates that the type

15
of substrate in SMS appeared to be more suited to the alcalase® enzyme while DWS

and FYM were not so suitable even under optimal conditions as a reduced number of

peptide bonds were cleaved during proteolysis.

Summary

The addition of commercial enzymes in a heterogeneous solid - liquid system proved

successful in aiding the breakdown of organic materials. However, some adjustments

may be necessary in order for this process to work more efficiently and cost

effectively. These include the possibility of acquiring enzymes that function at a more

neutral pH and yield a high degree of hydrolysis, which would eliminate the use of

any chemicals for pH adjustment. Furthermore, if this system was employed at pilot

scale, the recycling of the commercially available enzymes could prove a successful

cost cutting measure. As it stands, the hydrolysates obtained in this study could also

be used in the bioenergy industry, where fermentation of the hydrolysates could

successfully lead to the generation of ethanol and other useful by-products.

The possibility of nutrient removal, in a similar way to that employed in wastewater

treatment plants, is also an option as the organic waste hydrolysates are in solution. It

would also require a critical observation to the C/N rate and NH 4 concentrations.

Furthermore, the fact that the hydrolysed compost is in solution may also be

beneficial to farmers who wish to use this compost as a soil amendment or conditioner

since conventional injection or band spreaders are widely available on farms

nowadays.

16
The economic feasibility of employing the hydrolysis methodology on a pilot scale

has not yet been explored, yet it is anticipated that aerated system similar to that

functioning in water treatment facilities may be suitable. Moreover, stringent

temperature controls and monitoring will be required. Initial enzyme estimation costs

of treating one tonne of SMS in this liquid – solid system is €803.90, which excludes

any mechanical or operational expenses. This cost effectiveness of this system will

improve particularly when; the system works more efficiently and through the

recycling of these enzymes.

Conclusions

The length of time required to naturally compost three organic materials, spent

mushroom substrate (SMS), farmyard manure (FYM) and dairy wastewater sludge

(DWS) under optimal conditions was 42 days, 98 days and 84 days respectively.

DWS has great potential for the production of high-quality compost as it yielded a

germination index of 99.9 % following the addition of wood chippings.

Furthermore, enzymatic hydrolysis is an effective methodology for the treatment of

organic waste components, as stable hydrolysates were obtained within nine hours.

The hydrolysates obtained have high fertiliser merit, which were comparable to those

of their corresponding composts. Spent mushroom compost hydrolysate had a mean

N/P/K ratio of 20:10:20, farmyard manure hydrolysate had a N/P/K ratio 10:1:10,

while DWS hydrolysate had a ratio of 40:1:20.

17
The hydrolysis of organic manures in general has the potential to generate vast

quantities of reducing sugars, which could be further fermented into ethanol (Wen et

al. 2004) and this area of research is relatively unexplored.

Acknowledgements

The authors would like to acknowledge Dairygold, Michelstown, Cork, Monaghan

Mushrooms, Cavan and Mr. Patrick Dundon for the supply of the organic waste

materials and to Novo Industries, Bagsvaerd, Denmark, for the contribution of the

enzymes utilised in this study.

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(Bidlingmaier, W. and L’Hermite, P. Eds) CEC, Brussels, p 83-90.

Wang, P., Changa, C.M., Watson, M.E., Dick, W.A., Chen, Y. and Hoitink, H.A.J.

2004. Maturity indices for composted dairy and pig manures

Soil Biology and Biochemistry, 36 (5), p 767-776.

Wang, S.H.L., Lohr, V.I. and Coffey, D.L. 1984. Growth response of selected

vegetable crops to spent mushroom compost application in a controlled

environment. Plant and Soil, 82, p 31-40.

Wen, Z., Liao, W. and Chen, S. 2004. Hydrolysis of animal manure lignocellulosics

for reducing sugar production. Bioresource Technology, 91 (1), 31-39.

Tables

Table 1 Summary of percentage composition of the study materials

Moisture Carbohydrate Minerals Fat Protein Lignin
% % % % % %
SMS 50.6 65.6 20.3 3.37 8.20 2.49
FYM 86.7 74.3 9.52 3.98 6.84 5.33
DWS 92.6 17.6 27.5 20.0 34.7 0.25
SMS: Spent mushroom substrate; FYM: Farmyard manure; DWS: Dairy wastewater
sludge.

Table 2 Quantity of each enzyme added (µl) to the study materials

Lipozyme® Pectinex® Celluclast® Alcalase® Laccase®
(µl) (µl) (µl) (µl) (µl)
SMS 84.1 1641 1641 205 62.1
FYM 99.5 1858 1858 171 133
DWS 500 439 439 867 6.13
SMS: Spent mushroom substrate; FYM: Farmyard manure; DWS: Dairy wastewater
sludge.

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Table 3 Physical and chemical properties of fresh, composted and hydrolysed SMS
on a dry matter basis
Fresh Fresh Composted Fresh/ Hydrolysed Composted/
Parameter
/Composted Hydrolysed Hydrolysed
OM (%) 68.3 *** 36.1 ** 65.5 ***
pH 6.42 *** 7.12 *** 6.48 *
EC (mS cm-1) 5.32 ** 7.05 *** 13.4 ***
Total N (g kg-1) 20.7 *** 23.2 *** 19.1 ***
Total P (g kg-1) 8.21 *** 5.13 *** 7.20 *
Total K (g kg-1) 20.1 *** 12.9 *** 21.6 ***
C/N ratio 19.2 *** 8.99 *** 20.8 ***
Total Ca (g kg-1) 27.4 *** 27.3 *** 30.5 ***
Total Mg (g kg-1) 7.29 *** 9.31 *** 6.77 ***
Total Na (g kg-1) 2.65 * 2.55 *** 120.4 ***
Total Cu (g kg-1) 0.022 * 0.041 *** 0.046 **
Total Zn (g kg-1) 0.020 *** 0.013 *** 0.015
Total Fe (g kg-1) 0.029 * 0.061 *** 0.077 **
Total Mn (g kg-1) 0.128 *** 0.074 *** 0.059 **
Total Cd (g kg-1) 0.058 NS 0.077 NS 0.076 NS
Total Cr (g kg-1) 0.013 NS 0.023 NS 0.030 NS
Total Ni (g kg-1) 0.018 NS 0.010 NS 0.018 NS
-1
Total Pb (g kg ) 0.024 NS 0.007 NS 0.000 NS
Lignin (%) 27.0 *** 21.8 *** 21.0 NS
Cellulose (%) 52.4 NS 52.4 *** 44.9 ***
Hemicellulose (%) 45.7 *** 39.4 *** 35.4 ***
d(4,3) 20.6 *** 22.0 *** 21.1 *
Germination Index (%) 18.0 *** 53.1 *** 0.00 ***
Composts significantly different at P<0.001 (***), P<0.01 (**) and P<0.05 (*) levels. NS: Not
significant.

Table 4 Physical and chemical properties of fresh, composted and hydrolysed FYM
on a dry matter basis
Fresh Fresh Composted Fresh/ Hydrolysed Composted/
Parameter /Composted Hydrolysed Hydrolysed
OM (%) 91.9 *** 75.9 *** 52.2 ***
pH 7.39 *** 7.95 *** 4.66 ***
EC (mS cm-1) 2.38 *** 4.84 *** 20.8 ***
Total N (g kg-1) 14.4 *** 25.3 *** 7.61 ***
Total P (g kg-1) 2.02 *** 0.028 *** 1.11 ***
Total K (g kg-1) 44.6 *** 27.3 *** 14.1 ***
C/N ratio 37.1 *** 17.4 *** 39.9 ***
Total Ca (g kg-1) 27.2 *** 27.9 *** 21.1 ***
Total Mg (g kg-1) 9.31 *** 9.77 *** 7.30 ***
Total Na (g kg-1) 5.27 *** 9.21 *** 346.4 ***
Total Cu (g kg-1) 0.321 * 0.223 *** 0.527 ***
Total Zn (g kg-1) 1.980 *** 1.073 *** 0.425 ***
Total Fe (g kg-1) 0.528 *** 0.375 *** 1.26 ***
Total Mn (g kg-1) 6.10 *** 1.35 *** 2.44 ***
Total Cd (g kg-1) 0.095 NS 0.077 NS 0.097 NS
Total Cr (g kg-1) 0.031 NS 0.028 NS 0.055 NS
Total Ni (g kg-1) 0.006 NS 0.023 NS 0.027 NS
Total Pb (g kg-1) 0.000 NS 0.000 NS 0.000 NS
Lignin (%) 5.42 *** 21.1 *** 2.17 ***
Cellulose (%) 62.1 *** 54.8 *** 18.0 ***
Hemicellulose (%) 51.1 *** 41.8 *** 12.5 ***
d(4,3) 24.5 *** 28.4 *** 56.5 ***
Germination Index (%) 91.8 *** 47.9 *** 8.25 ***

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Composts significantly different at P<0.001 (***), P<0.01 (**) and P<0.05 (*) levels. NS: Not
significant.

Table 5 Physical and chemical properties of fresh, composted and hydrolysed DWS
on a dry matter basis
Fresh Fresh Composted Fresh/ Hydrolysed Composted/
Parameter /Composted Hydrolysed Hydrolysed
OM (%) 71.9 *** 92.5 *** 70.0 ***
pH 6.46 ** 6.62 *** 4.64 ***
EC (mS cm-1) 6.21 *** 1.75 *** 13.4 ***
Total N (g kg-1) 60.4 *** 20.2 *** 43.9 ***
Total P (g kg-1) 0.13 *** 1.33 *** 1.32
Total K (g kg-1) 8.91 *** 26.3 *** 17.2 ***
C/N ratio 6.91 *** 26.6 *** 9.26 ***
Total Ca (g kg-1) 22.6 *** 7.85 *** 39.7 ***
Total Mg (g kg-1) 7.78 *** 0.769 *** 9.11 ***
Total Na (g kg-1) 6.19 *** 2.48 *** 235.5 ***
Total Cu (g kg-1) 0.098 ** 0.028 *** 0.048 ***
Total Zn (g kg-1) 0.753 *** 0.525 ** 0.933 ***
Total Fe (g kg-1) 2.65 *** 2.76 *** 2.61 **
Total Mn (g kg-1) 0.679 *** 0.190 NS 0.706 ***
Total Cd (g kg-1) 0.059 NS 0.108 NS 0.127 NS
Total Cr (g kg-1) 0.019 NS 0.000 NS 0.052 NS
Total Ni (g kg-1) 0.008 NS 0.000 NS 0.028 NS
Total Pb (g kg-1) 0.000 NS 0.000 NS 0.000 NS
Lignin (%) 16.6 *** 10.40 *** 14.3 ***
Cellulose (%) 13.47 *** 65.5 *** 55.5 ***
Hemicellulose (%) 12.32 *** 26.5 *** 16.5 ***
d(4,3) 33.3 *** 31.1 *** 50.2 ***
Germination Index 0.00 *** 99.5 0.000 ***
(%)
Composts significantly different at P<0.001 (***), P<0.01 (**) and P<0.05 (*) levels. NS: Not
significant.

Table 6 Organic matter properties of control samples without enzyme addition

SMS FYM DWS
Organic matter (%) 67.7 84.0 67.4
Lignin (%) 21.5 5.73 38.3
Cellulose (%) 15.8 42.6 13.6
Hemicellulose (%) 11.3 44.4 34.4
SMS: Spent mushroom substrate; FYM: Farmyard manure; DWS: Dairy wastewater
sludge.

23
Figures

Fig. 1 Schematic diagram of reaction vessel for the hydrolysis of the organic wastes. A: Motor / speed
regulator, B: pH data logger, C: Mechanical stirrer, D: Reaction vessel, E: Thermometer, F: Water
bath.

Fig. 2 Changes in pH with time in aerobically composted SMS, FYM and DWS

Fig. 3 Changes in temperature and maturity index with time in aerobically composted SMS,
FYM and DWS. MI: Maturity index (Solvita®); Temp: Temperature (oC).

24
Fig. 4 pH changes during the hydrolysis of three organic waste materials

Particle size (µm)

Fig. 5 Particle size distribution (d(4,3)) for fresh, composted and hydrolysed SMS.

Particle size (µm)

Fig. 6 Particle size distribution (d(4,3)) for fresh, composted and hydrolysed FYM.

Particle size (µm)

Fig. 7 Particle size distribution (d(4,3)) for fresh, composted and hydrolysed DWS.

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