Process Biochemistry 39 (2004) 971–975

Development of a solid phase kinetic assay for determination ofenzyme activities during composting

Carlos Peláeza,b, Alex Mejıab, Antoni Planasa,∗a Laboratory of Biochemistry, Institut Qu´ımic de Sarrià, Universitat Ramon Llull, Via Augusta 390, 08017 Barcelona, Spain

b Grupo GIEM, Instituto de Qu´ımica, Universidad de Antioquia, Medell´ın, Colombia

Received 24 February 2003; received in revised form 12 April 2003; accepted 8 June 2003

Abstract

Quantitative determination of enzyme activities can be used to evaluate the dynamics of the composting process. An enzyme activity assayin solid phase was developed in which the enzyme reaction is performed by mixing the compost sample and the corresponding substrate witha water content equivalent to the water retention capacity (WRC) of the compost sample. As opposed to common activity assays that extractand quantify soluble enzymes, this solid phase method evaluates total enzyme activities under conditions closer to those found in the actualcomposting process. Preliminary results on enzyme activities of a compost of poultry manure and sawdust include amylase, invertase, cellulase,phosphatase, protease, and dehydrogenase activities. With the exception of proteases, all increased from day 9 to 43 of the composting process.© 2003 Elsevier Ltd. All rights reserved.

Keywords:Manure composting; Enzyme activities; Solid phase method

1. Introduction

A biosphere is a closed macrosystem in which mat-ter recycling is essential for biomass generation[1]. Thisthermodynamic requirement driving biological processesallows the classification of biosystems in three main groupsaccording to their role in biogeochemical cycles: producers,consumers and decomposers[2]. Decomposition is consid-ered a general process applicable to any biological systemthat refers to the degradative breakdown of organic matter,whereas mineralization is a more specific term that refersto processes that release carbon as CO2 and nutrients in in-organic form (i.e. phosphate and ammonium). Most of themineralization reactions are performed by fungi and bacteriafrom the soil through the action of extracellular enzymes[3].

The composting reaction is a biochemical decomposi-tion of solid organic matter of the starting material undercontrolled conditions by the action of the same type ofmicroorganisms that are responsible for mineralizationprocesses in soil[4]. Macromolecules synthesized by anybiological system have a dual function when they are used

∗ Corresponding author. Tel.:+34-932-67-2000;fax: +34-932-05-6266.

E-mail address:aplan@iqs.edu (A. Planas).

by decomposer organisms: source of organic matter andsource of energy. Compounds such as lignin and celluloseare oxidized by microorganisms to produce metabolic en-ergy, as they are also carbon source for the biosynthesis oftheir own biomolecules.

From the point of view of trophic chains, compostingof solid organic waste involves three levels of consumerorganisms: first level (which are the true decomposers) iscomposed by microfauna, mainly actinomycetes, bacte-ria and fungi, and by macrofauna which includes beetlemites, earthworms, diptera in different stages, the mostactive being larvae, nematodes and snails. All these or-ganisms degrade organic matter of the starting material.Protozoa and arthropods form the second level which arefed with first level organisms, while the third level is com-posed of higher arthropods such as ants and beetles, whichare the predators of the second level organisms. In thisecological network, biotransformations of organic matterduring composting are mainly due to enzyme activitiesof first level microorganisms leading to mineralizationprocesses.

Most of the kinetic parameters commonly used to char-acterize the maturity of composts are physico-chemicalproperties such as C/N ratio, ashes, and cationic exchangecapacity[4,5]. Only recently, however, some attention has

0032-9592/$ – see front matter © 2003 Elsevier Ltd. All rights reserved.doi:10.1016/S0032-9592(03)00208-5

972 C. Pelaez et al. / Process Biochemistry 39 (2004) 971–975

been paid to enzyme activity measurements as indices ofthe course of the actual composting[5].

Enzymes in composts can be classified as intracellularenzymes, found inside living cells or in soil/compost dueto cell lysis, and extracellular enzymes purposely releasedfrom viable cells to catalyze the degradation of extracellu-lar polymeric substances. For instance, N biotransformationsare performed by extracellular hydrolases that depolymerizeproteins, aminopolysaccharides, and nucleic acids, as wellas hydrolyze urea[6]. Likewise, water insoluble organic par-ticles are degraded by the action of secreted enzymes tosmall water-soluble fragments that are then transported in-side the cells where they are used by microorganisms as asource of energy and/or biosynthetic precursors. These en-zyme activities vary in time as a consequence of a complexsequence of microorganism, where populations of bacteria,actinomycetes and fungi change in time depending on thespecific conditions during composting evolution[7,8].

Studies addressing analysis of biochemical processes incomposting commonly use activity assay methods developedfor soil analysis, often restricted to the soluble enzyme frac-tion after extraction[9–12]. Commercial kits are availablefor evaluation of hydrolyzing enzyme activities, dehydroge-nases, ATP contents, ammonification and nitrification po-tential [6,13]. Other studies analyze purified enzymes fromcompost with the purpose of using them for the degradationof other substrates[14–16]. All these methods are based onend point assays often with low reproducibility.

We report here an enzyme activity assay for the determi-nation of extracellular enzymes (both soluble and anchoredto the matrix) in solid phase using a kinetic protocol (initialvelocities) instead of an end point method. Different enzymeactivities are measured by adding a specific substrate to acompost sample with a water content equivalent to its wa-ter retention capacity (WRC), thus performing the enzymereaction under conditions highly similar to those occurringduring the normal composting process, without previous ex-traction of soluble enzymes. At different time intervals, thesoluble enzyme product released from the specific substrateis extracted and determined spectrophotometrically. Enzymeactivity is expressed as initial velocity of product formation.

2. Material and methods

2.1. Reagents

Glycosidase activities were quantified by measuring re-ducing ends by the 3,5-dinitrosalycilic acid reagent (DNS)prepared by mixing solution A (1.682 g of DNS (Sigma),59 ml of 2M NaOH and 111 ml of water) and solutionB (50 g sodium potasium tartrate tetrahydrate in 125 mlof water) [17]. Ninhydrin reagent (Sigma) at 1% was usedfor determination of protease activity with ovoalbumin assubstrate[18]. The substrate for phosphatase activity mea-surements[19] was p-nitrophenylphosphate (Sigma), and

2,3,5-triphenyltetrazolium chloride (Sigma) was used for thedehydrogenases assay[20].

2.2. Composts

Poultry manure and sawdust were mixed at a C/N ratio of20, in an open static reactor (20 l) with mechanical mixingonce a week, and maintaining moisture content at approxi-mately 50%.

2.3. Water retention capacity (WRC)

A sample of compost is placed on a wet filter paper andweighed (Wi). Water is added and allowed to filter, then thewet sample is weighed again (Wf ). WRC is calculated as:WRC = (Wf −Wi)/Wi.

2.4. Sample preparation

Compost samples of approximately 10 g were taken fromthe middle of the bioreactor after mixing. Samples werefrozen at−20◦C and lyophilized. The solid was then ho-mogenized by grinding, and sieved through a 360�m mesh.

2.5. Enzyme activity assays

A solid phase assay follows two different general proto-cols depending on substrate–sample preparation. In methoda) the specific enzyme substrate was added as a solid tothe lyophilized compost sample. After homogenization bymixing, water was added to saturation, corresponding to theWRC previously determined. In method b) a solution of theenzymes substrate in water was added to the lyophilizedcompost sample to reach the same water content. Aliquotsof the resulting substrate–compost mixture were incubatedat 25◦C with gentle shaking (200 rpm in an orbital incuba-tor). Reactions were stopped at 15-min intervals for a totalof 2 h following specific protocols for each enzyme deter-mination. Analyses were done in triplicate.

2.5.1. Amylase, cellulase and invertase assays200± 2 mg (for amylase and invertase assays) or 50± 1

mg (for cellulase assay) aliquots of a 1:1 (w/w) mixture ofsubstrate (starch, saccharose or carboxymethyl cellulose, re-spectively) and lyophilized compost were weighed in Ep-pendorf tubes. 1 ml of deionized water was added to eachtube, homogenized by vortexing and incubated at 25◦C. Atdifferent time intervals, 500�l of water was added to the cor-responding tube, vigorously mixed by vortexing, centrifugedat 13 000 rpm for 1 min at room temperature. 250�l (amy-lase assay) or 500�l (invertase and cellulase assays) of thesupernatant were transferred to another tube containing thesame volume of the DNS reagent. The mixture was heatedat 100◦C in a water bath for 10 min, water were added (2ml for the amylase assay or 1 ml for the others), and theabsorbance at 540 nm was recorded (1 cm path length cu-

C. Pelaez et al. / Process Biochemistry 39 (2004) 971–975 973

bette). Initial velocities were calculated as the slope of thelinear plot Abs versus time, and enzyme activity in the sam-ple is expressed as�mol of glucose equivalents producedper min and per g of liophylized compost (dry matter).

2.5.2. Phosphatases assays700 �l of p-nitrophenyl phosphate (10 mM) were added

to a set of Eppendorf tubes containing 100± 2 mg oflyophilized compost each. After vortexing, the mixtureswere incubated at 25◦C. Reactions were quenched at differ-ent time intervals by adding 250�l of 1 M CaCl2 and 500�lof 2 M NaOH. 100�l of the supernatant were transferred toa cubette containing 2 ml of water, andp-nitrophenolate wasquantified by absorbance reading at 405 nm (ε = 1.9×104

M−1 cm−1). Phosphatase activity was expressed as�mol ofreleasedp-nitrophenol per min and g of lyophilized compost.

2.5.3. Proteases assayTo a series of Eppendorf tubes containing 50± 1 mg of

lyophilized compost, 1 ml of ovoalbumin (1% in water) wasadded, and the mixtures incubated at 25◦C. At different timeintervals, 500�l of water were added to each tube, mixedby vortexing and centrifuged. 250�l of supernatant weretransferred to a tube containing 500�l of ninhydrin reagent,and the mixture was heated at 100◦C for 10 min. After 1:10dilution in water, absorbance at 570 nm was measured. Ac-tivity was expressed as�mol of amino groups produced permin and g of lyophilized compost using glycine as standard.

2.5.4. Dehydrogenases assay500 �l of 2,3,5-triphenyltetrazolium chloride (TPTZCl,

1% in water) were added to a series of Eppendorf tubescontaining 50± 1 mg of lyophilized compost. At differenttime intervals reactions were quenched by adding 1 ml ofmethanol. After mixing and centrifugation, 500�l of super-natant were diluted with 500�l of water, and absorbancewas measured at 485 nm (ε = 6997 M−1 cm−1). Dehydro-genase activity was expressed as�mol of formazan formedper min and g of lyophilized compost.

All data were analyzed usingstatgraphics Plus 5.1 (Sta-tistical Graphics Corp.) software. Linear regressions Absversus time were accepted forr≥0.95 andP-values for theslopes≤0.005.

3. Results and discussion

3.1. Samples preparation

The first issue to be addressed for developing an enzymeactivity assay in compost is the sample preparation protocol.The product to be quantified is formed from the added sub-strate to the compost sample, but at the same time it may bea substrate for the microorganisms present in the compostsample, thus being depleted during the time of measure-ment. Under normal conditions, microorganism populations

of 109–1010 cells per g of compost are present. For in-stance, when monitoring starch hydrolysis after adding thesubstrate (100 mg of starch) to an intact compost (100 mg),95–100% of the glucose equivalents produced during thefirst 6 h of incubation is consumed by endogenous microor-ganisms in the next 14-h incubation time. It is, therefore,essential to assure that living microorganisms that may bepresent in the assay do not interfere with products of theenzyme activity being monitored. In a first set of trials, dif-ferent concentrations of sodium azide as biocide (100–2000ppm) were added to the compost sample to decrease micro-bial activity. However, it was not effective since the numberof viable cells was not significantly reduced at these ratherlow azide concentrations. Higher concentrations could notbe used due to their inhibitory effect on enzymes to bemeasured, in particular glycosidases[21].

The sample preparation procedure that gave best resultswas freezing and lyophilization of the compost. Biologicalactivity is stopped and samples can be stored for a few daysbefore analysis. New biomass production when perform-ing the enzyme activity assays is drastically slowed down,avoiding interference due to microorganism growth duringthe short analysis time (<2 h).

3.2. Development of a solid phase activity assay

The aim of the solid phase method is to measure enzymeactivities under conditions close to water saturation with amoisture content similar to that in normal composting. TheWRC of the lyophilized compost sample is determined, andthe assay mixture of substrate and compost is adjusted tothis water content, resulting in a monophasic system. Theenzyme substrate is mixed as a solid with the lyophilizedcompost sample and water is added up to its WRC value(method a, cf.Section 2), or a substrate solution is added tothe compost to obtain the substrate–compost mixture withthe same water content (method b). At different incubationtime intervals, the reaction is quenched and the enzyme prod-uct released is extracted and quantified by the correspondingspectrophotometric method.

Poultry manure and sawdust at an initial C/N ratio of 20and moisture content of 50% was composted in a 20 l aer-obic reactor. Samples were taken at different days of com-posting to evaluate enzyme activities according to the solidphase assay reported here. The enzyme activities determinedwere: amylase, cellulase and invertase activities by measur-ing the increase of reducing power when using starch, car-boxymethyl cellulose and sucrose as substrates, respectively,by the DNS method; phosphatases by measuring the releaseof p-nitrophenol fromp-nitrophenylphosphate as substrate;proteases by measuring the generation of�-amino groupsfrom hydrolysis of ovoalbumin as substrate by the ninhydrinreagent; and dehydrogenases by monitoring the reduction ofa triphenyltetrazolium salt as substrate to the correspondingformazan product.Table 1shows the results of absorbancereadings for each assay (kinetic assay measuring increase in

974 C. Pelaez et al. / Process Biochemistry 39 (2004) 971–975

Table 1Absorbance readings in the kinetic activity assays for two samples at days 9 and 43 of composting

Time (min) Phosphatase Amylase Invertase Proteasea Dehydrogenaseb

Day 9 Day 43 Day 9 Day 43 Day 9 Day 43 Day 9 Day 43

0 0.072± 0.007 0.12± 0.01 0.8± 0.06 0.74± 0.02 0.26± 0.02 0.2± 0.01 0.11± 0.01 0.11± 0.0115 0.10± 0.009 0.24± 0.02 0.84± 0.08 0.84± 0.08 0.285± 0.003 0.24± 0.02 0.14± 0.01 0.21± 0.00430 0.12± 0.01 0.35± 0.03 0.89± 0.05 1.02± 0.02 0.31± 0.02 0.31± 0.01 0.165± 0.005 0.30± 0.00145 0.16± 0.01 0.46± 0.03 0.93± 0.05 1.19± 0.09 0.330± 0.005 0.35± 0.02 0.19± 0.01 0.385± 0.00460 0.18± 0.01 0.6± 0.02 0.97± 0.04 1.31± 0.09 0.35± 0.02 0.40± 0.02 0.220± 0.003 0.491± 0.002

a Protease activity at day 43 was not detected.b Dehydrogenase activity at day 9 was not detected

absorbance for 1 h at 15 minintervals) for compost sam-ples at days 9 and 43 of the process. All assays were donein triplicate for statistical analysis.Fig. 1 plots the kineticresults showing that linear progress curves are obtained inall cases for 1 h assay under these experimental conditions.The corresponding enzyme activities of each sample are ob-tained from the slopes of Abs versus time linear plots andexpressed in enzyme units (U g−1 compost) inTable 2. Sinceenzyme activities are obtained as rates of product forma-

Fig. 1. Enzyme kinetic assays of a compost of poultry manure and sawdust at days 9 and 43 of the process. Initial velocities are calculated as the slopesof the linear reaction progress for 1 h.

tion, the progress curve Abs versus time has to be linear inthe time interval of measurements. Assay conditions mustbe modified by decreasing the compost concentration and/orreducing the time intervals to ensure linearity. As opposedto end point methods, this kinetic method gives more pre-cise and reproducible activity values since they are obtainedas initial velocities of product formation.

By comparing the results at days 9 and 43 of the com-posting process, phosphatase and amylase activities increase

C. Pelaez et al. / Process Biochemistry 39 (2004) 971–975 975

Table 2Enzyme activities at days 9 and 43 of composting

Enzyme Activity (�mol min−1 g−1)a

Day 9 Day 43

Phosphatase 0.034± 0.002 0.180± 0.005Amylase 1.25± 0.02 6.26± 0.32Cellulase n.d. n.d.Invertase 0.27± 0.01 0.94± 0.05Protease 1.55± 0.06 n.d.Dehydrogenase n.d. 0.053± 0.001

n.d., not detected.a Enzyme activities in�mol of product formed per min per g of

lyophilized compost sample.

5-fold, and invertase activity increases 3.4-fold, whereas pro-teases are detected at day 9, but activity decreases below thedetection limit at day 43. Dehydrogenases show the oppo-site behavior, not being detected at day 9 but showing highactivity at day 43. For this composting mixture, cellulaseactivity was not detected.

Phosphatase and dehydrogenase activities are commonlydetermined in soil analysis, with values of 10−3–10−2 U g−1

for alkaline phosphatase and values of 10−5–10−4 U g−1

for dehydrogenase[22,23]. Because of the higher microbialactivity during composting, phosphatase and dehydrogenaseactivities are one to three orders of magnitude higher thanin soil [24]. Tiquia et al.[13] reported that dehydrogenaseactivity of a compost of yard trimmings is maximal at thebeginning of the composting period and it decreased as com-posting proceeded. Since dehydrogenase activity measuresthe overall population of heterotrophic microorganisms, theirresults suggest that the starting material at the beginning ofcomposting was an aged material that already has high mi-crobial activity. The poultry manure used in this study wasa fresh material with low microbial content, so dehydroge-nase activity was observed to increase during compostingwhile the accumulated microbial population increased.

The enzyme assay methodology on solid phase describedhere provides assay conditions closer to the actual compost-ing process as compared with common extractive protocolsto assay enzymes in solution. Not only secreted enzymesand soluble enzymes released by cell lysis are measured, butalso those attached to the matrix that would not be solubi-lized using extractive methods. The protocol is also devel-oped as a kinetic method instead of an end-point method,giving more accurate and reproducible initial velocities toevaluate specific activities. Work is in progress to applythis methodology for the dynamic evolution of a number ofco-composting processes of cattle and agricultural waste.

Acknowledgements

C.P. acknowledges funding from the Universidad de An-tioquia, Colombia.

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