Biol Fertil Soils (2014) 50:785794

DOI 10.1007/s00374-014-0903-1

Soil peroxidase regulates organic matter decomposition through improving

the accessibility of reducing sugars and amino acids
Lei Tian & Wei Shi
Abstract Peroxidase activity can be an important soil property for predicting soil
organic matter dynamics, but peroxidase control on organic matter degradation
has not yet been fully understood. We hypothesized that peroxidase could
enhance soil C and N mineralization via improving the bioavailability of reducing
sugars and amino acids. First, an arable soil (~13 g dry weight equivalent) was
amended with horseradish peroxidase at 0, 0.1, and 0.2 activity units g1 soil,
and then examined for peroxidase and hydrolase activities, waterextractable
phenolic content, and soil C and N mineralization over a 21-day incubation. Soil C
mineralization was enhanced by peroxidase addition, but it was not correlated
with the concentration of water extractable phenolics or hydrolase activity.
Exoglucanase and -glucosidase activities were significantly lower in soil
amended with peroxidase at 0.2 units than at 0.1 units. Second, peroxidase,
cellulase, protease or their combinations were added into sterile soils to examine
enzyme-catalyzed productions of soluble reducing sugars and amino acids.
Reducing sugar concentration was increased after peroxidase addition and this
effect was more pronounced when both peroxidase and hydrolases were added.
Thusproduced reducing sugars and amino acids were significantly correlated with
soil C and N mineralization (Pearsons correlation coefficient, r=0.65 and r=0.63,
P<0.01), respectively. Our results indicated that the effects of peroxidase on
organic matter degradation were not through the production of soluble phenolics
or the enhancement of hydrolase activity. Instead, by oxidizing phenolics soil
peroxidase liberated carbohydrates and proteins that were otherwise
encapsulated in soil humus and thus inaccessible to soil hydrolases and
microbes.
Keywords Peroxidase .Phenolics .Reducing sugars .Amino acids . Soil enzyme
activity . Soil C and N mineralization.
Introduction Peroxidases are ubiquitous and abundant oxidative enzymes in all
forms of life (Zmock and Obinger 2010). Fungi, particularly white- and brown-
rot fungi, are best known for producing extracellular peroxidases. Bacteria
belonging to actinomycetes and - and -proteobacteria are also recognized as
the producers of extracellular peroxidases (Ramachandra et al. 1988; Mercer et
al. 1996; Bugg et al. 2011). Despite differences in source and activity,
extracellular peroxidases, e.g., lignin- and manganese-peroxidases have all been
considered to play important roles in the condensation and polymerization of
phenolic and aromatic compounds during soil humus formation (Wagner and Wolf
1998). Peroxidases are also recognized for their roles in the oxidation and/or
degradation of aromatic substances, such as lignin and humus, and hence may
considerably affect soil organic matter dynamics. A meta-analysis has shown
that soil peroxidase in combination with soil pH, mean annual temperature, and
precipitation accounted for 32 % of the variation in soil organic matter content
across various ecosystems (Sinsabaugh 2010). While substantial data
demonstrate that oxidative enzymes can be a proximate control of soil organic
matter dynamics (Sinsabaugh et al. 2008), underlying ecological pathways
appear to be manifold.
Indirect regulation of hydrolytic enzyme activity represents one way that
oxidative enzymes control soil organic matter dynamics. Polyphenolic
compounds have been widely acknowledged as enzyme and microbial inhibitors
(Freeman et al. 1990; Wetzel 1992; Appel 1993; Fierer et al. 2001). Even phenolic
compounds of low complexity, such as monomers, dimers and trimmers, can be
toxic to soil microbes in some habitats (Fierer et al. 2001). Because oxidative
enzymes are responsible for phenolic degradation, they can help alleviate
inhibitory effects of phenolic compounds on hydrolytic enzyme activities. As a
result, an increase in oxidative enzyme activity may accelerate the
decomposition of soil organic matter. This appeared to be a prevailing
mechanism of organic matter reduction in wetlands following draining events
(Freeman et al. 2001). When O2 limitation is relieved, soil oxidative enzyme
activity increased, leading to rapid degradation of phenolic compounds. This
triggered the activity of soil hydrolytic enzymes and so the decomposition of soil
organic matter (Freeman et al. 2001).
Using chemically recalcitrant substances as the source of microbial C and
energy may represent another way by which oxidative enzymes control soil
organic matter dynamics. By oxidizing substances of high redox potential, such
as lignin, oxidative enzymes can produce phenolic compounds of low molecular
weight. These phenolic compounds may serve as growth substrates for soil
microbes as they are further degraded via consecutive steps of a ring cleavage
and subsequent conversion into aliphatic compounds for microbial metabolism
(Souto et al. 2000; Fenner et al. 2005). However, this mechanism may be less
pronounced in agroecosystems than forests where lignin-rich plant materials are
constantly added to the ground. Even when oxidative enzyme activities are
positively related to mass loss rates of lignin-rich plant litter in forests
(Sinsabaugh et al. 1992; Carreiro et al. 2000; SaiyaCork et al. 2002; DeForest et
al. 2004; Waldrop and Firestone 2004; Sinsabaugh et al. 2005), this mechanism
may not be the dominant one because microbes do not gain as much energy as
when degrading cellulose. It is more likely that by removing lignin, oxidative
enzymes help liberate cellulose in the lignocellulose complex and therefore
increase the mass loss rates of plant litter.
Oxidative enzymes may also regulate soil humus degradation by
improving microbial and enzyme accessibilities to carbohydrates and proteins,
which are otherwise protected by phenolic moiety-containing substances. This
concept was implicitly stated (Nadeau et al. 2007; Rezov et al. 2007), yet it
has not been explicitly tested. In this study, we examined if an oxidative enzyme
could enhance soil C and N mineralization by improving the bioavailability of
reducing sugars and amino acids. By soil amendments of oxidative and
hydrolytic enzymes, we evaluated the possible pathways of an oxidative enzyme
mediating organic matter degradation in an agricultural soil.
Agricultural land is intensively managed and often cropped with annual
herbaceous plants rather than perennial woody plants. As a result, it may not be
a favorable niche for phenol oxidase-producing microbes, such as white-rot fungi.
Perhaps, soil phenol oxidase in agricultural land is not as important to organic
matter degradation or soil C sequestration as in unmanaged ecosystems (e.g.,
forests). Compared to phenol oxidase, however, peroxidase can be produced by
a broad array of microbial genera and species (Bergbauer and Newell 1992;
Howard et al. 2003; Snchez 2009) and thus likely serves as the primary
oxidative enzyme for the degradation of soil humus. Our previous work in
agroecosystems also demonstrated that peroxidase rather than phenol oxidase
was correlated significantly with soil C and N mineralization (Tian et al. 2010).
Therefore, horseradish peroxidase was used in this study to examine how an
oxidative enzyme regulates soil organic matter degradation.
Horseradish peroxidase can oxidize aromatic compounds, although its
activity is likely lower than that of lignin peroxidase (Kurek and Monties 1994).
This enzyme has been found to degrade soil humic extract as wood- and soil-
associated fungi and bacteria did, indicating a significant involvement in soil
organic matter decomposition (Gramss et al. 1998). As the preferred electron
acceptor for peroxidase-mediated oxidations, H2O2 may be sufficient in soil
given that it can be generated by the activities of various enzymes, such as aryl
alcohol oxidase, glyoxal oxidase, galactose oxidase, and glucose oxidase
(Sinsabaugh 2010). As documented by Wang et al. (1990), soil H2O2 level was
indeed adequate for peroxidase to function because the addition of lignin
peroxidase with or without H2O2 into a sterile silt loam soil led to similar CO2
evolution. Therefore, no additional H2O2 was used in the following peroxidase
addition experiments.
Materials and methods
Soil sampling
Soil was sampled by coring techniques from an organic farming system of the
Center for Environmental Farming Systems (CEFS), located in Goldsboro, North
Carolina (35.23 N, 77.59 W). CEFS was established in 1999 and included five
farming systems: organic farming system, conventional farming system,
integrated crop-livestock system, plantation forestry system, and successional
system from abandoned agricultural field. A completely randomized block design
was used to arrange the five systems into each of three blocks, leading to total
15 plots. For organic farming, conventional farming and cropping phase of
integrated croplivestock system, crops were rotated annually, including corn
(Zea mays L.), peanut (Arachis hypogaea), cotton (Gossypium hirsutum),
soybean (Glycine max), wheat (Triticum aestivum), sweet potato (Ipomoea
batatas), and sorghum (Sorghum bicolor). Turkey litters were applied annually to
the organic farming system, and the amounts depended on rotated crops.
Besides gypsum for providing calcium, no other chemicals were applied to the
organic farming system. Soil was classified as Wickham sandy loam (fine-loamy,
mixed, semiactive, thermic Typic Hapludult) in one plot and Tarboro loamy sand
(mixed, thermic Typic Udipsamment) in the other two plots. On average, soil in
the organic farming system had 10.5 mg C g1 , 0.9 mg N g1 , 301 g
microbial biomass C g1 , 34.3 g microbial biomass N g1 , and pH 6.9.
Detailed information on the organic farming system has been reported previously
(Tian et al. 2010).
Twenty soil cores (2.5 cm [diam]10 cm [depth]) were collected randomly
in fall 2009 from each field plot of the organic farming system after corn was
harvested and then pooled, leading to a composite soil sample. Soil samples
were sieved (<2 mm), adjusted for soil moisture content to 45 % of water
holding capacity, and then stored at 4C prior to a series of laboratory
experiments.
Experimental designs
The regulatory role of peroxidase in soil C and N mineralization was
examined via soil addition of horseradish peroxidase, a plant peroxidase whose
oxidation pathways are similar to those of fungal lignin peroxidase (Kurek and
Monties 1994; Wong 2009). The first experiment was set up to examine the
impacts of peroxidase addition on soil C and N mineralization, water extractable
phenolic compounds, and soil hydrolytic enzyme activities. A stock solution of
horseradish peroxidase (Sigma P8250) at 3 unit ml1 was made by dissolving
the lyophilized powder in sterile distilled water. Here, a unit of horseradish
peroxidase is defined as the amount of enzyme for catalyzing the production of 1
mg of purpurgallin from pyrogallol in 20 s at 20 C (Sigma-Aldrich). The stock
solution was then added into soil samples (~13 g dry weight equivalent) to
produce three treatments: 0 unit g1 soil as the control, 0.1 units g1 soil as the
low peroxidase activity (i.e., Pox_L), and 0.2 units g1 soil as the high peroxidase
activity (i.e., Pox_H). Given that enzymes added to soil can be rapidly degraded
since they are energy and nutrient sources for soil microbial communities, the
role of control would be critical for the measurements on soil C and N
mineralization. The amounts of protein C and protein N added to the control
should be the same as those added in the other treatments. Thus, to make the
inputs of enzyme-associated C and other elements the same for the three
treatments, inactive horseradish peroxidase, which was made by boiling for up to
1 h until no activity was detected (see Determination of soil enzyme activities),
was also proportionally added to the soil, i.e., additions of inactive peroxidase
volumetrically equivalent to 0.2, 0.1, and 0 units g1 soil into the control, Pox_L,
and Pox_H, respectively. Soil samples were analyzed for soil enzyme activities
and water extractable phenolic compounds at 6 h, 3 days, 10 days, and 21 days
after the peroxidase addition. Soil C mineralization was measured 3, 10, and 21
days, and soil N mineralization was determined only at the end of the incubation.
A subset of soil treatments (i.e., control, Pox_L, Pox_H) was also used to examine
soil C mineralization during 36 and 69 days following another two consecutive
additions of horseradish peroxidase on day 3 and day 6, respectively. Again, the
amounts of protein C and protein N added to the control were the same as those
in the other treatments.
The second experiment was set up to examine the impacts of peroxidase
on water extractable reducing sugars and amino acids following the addition of
horseradish peroxidase, hydrolytic enzymes, or their combinations into sterile
soils. Sterile soils were used to minimize microbial consumption of reducing
sugars and amino acids. Thus, changes in those compounds could be attributed
to the activity and function of added enzymes. Soil samples were sterilized via
three consecutive autoclaving at 121C for 30 min to minimize indigenous soil
microbial and enzyme activities. Then, sterile soil samples were amended with
horseradish peroxidase at 0.1 units g1 soil, cellulase (Sigma C9422) at 1 unit
g1 soil, protease (Sigma P5147) at 0.1 units g1 soil, or combination of
horseradish peroxidase with cellulase or protease for total six treatments,
including (1) no enzyme addition (Control), (2) peroxidase addition (Pox), (3)
cellulase addition (Cel), (4) protease addition (Prt), (5) addition of both cellulase
and peroxidase, and (6) addition of both protease and peroxidase. Inactive
enzymes, which were made by boiling for up to 1 h until no detectable activity,
were also proportionally added into sterile soils, making the total amount of
inactive and active enzymes the same for all the treatments. For example, the
control treatment contained inactive peroxidase, cellulase, and protease
volumetrically equivalent to the active dose of these enzymes in the treatments
Cel, Pox, and Prt. This experiment was set up as a completely randomized design
with soil samples from three field plots as the three replicates. Soil samples, each
containing ~13 g dry weight equivalent of sterile soil and corresponding soil
enzymes were incubated for 5 days and then analyzed for the concentrations of
water extractable reducing sugars, amino acids, and phenolic compounds. After
5 days, 1 g of fresh soil was added into each enzyme-treated sterile soil sample,
incubated for an additional 7 days and then measured for soil C and N
mineralization. This sizable inoculation was used to promote rapid mineralization
of reducing sugars and amino acids.
A third experiment was set up to test the impacts of phenolic compounds
of different molecular complexities on soil enzyme activities. A monophenol
group was represented by a mixture of equal moles of ferulic, p-coumaric,
phydroxybenzoic, vanillic and syringic acids; a diphenol group was composed of
equal moles of catechol, resorcinol and hydroquinone; and a polyphenol group
was represented by tannic acid. A stock solution at 24 mol ml1 was made with
50 mM acetate buffer, pH 5.0 for each phenolic group. Effects of phenolic
compounds on soil enzyme activities were tested in soil extracts, which were
made by shaking soil (~10 g dry weight equivalent) with 50 mM acetate buffer,
pH 5.0 in a weight/volume ratio of 1:2.5 for 1 h and then passing through a No.
42 Whatman filter. Here, soil extracts were used to minimize soil sorption of
added phenolic compounds so that their effects on enzyme activities could be
easily detected. It is possible that degrees of reactions in soil and soil extract
differed considerably. However, qualitative relations between phenolic
compounds and enzyme activities might be independent of test media and thus
phenolic impacts on soil enzyme activity could be inferred from soil extracts. This
experiment contained eight treatments, including (1) no addition of any phenolic
compound (Control), (2) addition of the monophenolic group (M), (3) addition of
the diphenolic group (D), (4) addition of the polyphenolic group (P), (5) addition
of equal moles of the monophenolic and diphenolic groups (M+ D), (6) addition
of equal moles of the monophenolic and polyphenolic groups (M+P), (7) addition
of equal moles of the diphenolic and polyphenolic groups (D+P), and (8) addition
of equal moles of monophenolic, diphenolic, and polyphenolic groups (M+D+P).
For each treatment besides the control, total concentration of added phenolic
compounds was 1.2 mol g1 soil. After amendments with different phenolic
compounds, soil extracts were incubated for 18 h in dark at 25C and then
analyzed for enzyme activities.
Measurements of water-extractable organic compounds and soil C and N
mineralization
Soil organic matter was water extracted based upon Zsolnays method (2003),
and then the extracts were analyzed for the concentrations of phenolic
compounds, amino acids, and reducing sugars (Hofman and Duek 2003;
DeForest et al. 2005).
 A base trap method was used to measure soil C mineralization for various
treatments (see the Experimental designs section). Briefly, a soil sample (~13
g dry weight equivalent) contained in a specimen container and 5 ml 0.5 M NaOH
in a scintillation vial were placed into a 1-l Mason jar for incubation. After the
incubation, the remaining NaOH was titrated with 0.2 M HCl to determine the
amount of CO2 evolution, i.e., C mineralization. Soil inorganic N (i.e., NH4 + - and
NO3 - N) was measured colorimetrically using a Lachat flowinjection analyzer
(QuikChem 8000; Lachat Instruments, Mequon, MI, USA) after soil samples were
extracted using 1 M KCl in a weight/volume ratio 1:5 and then soil slurries were
filtered through Waterman No. 42 filter papers. The difference between inorganic
N after and before the incubation was calculated as N mineralization.
Determination of soil enzyme activities
The activities of soil enzymes, including peroxidase (EC 1.11.1.7), exoglucanase
(EC 3.2.1.4), -glucosidase (EC 3.2.1.21), -xylosidase (EC 3.2.1.37), -
glucosaminidase (EC 3.21.30), and protease (EC 3.4.24.31) were determined at
different incubation times by the rates of product appearance. All enzyme assays
were determined in quadruplicates against two types of controls (i.e., substrate
alone and soil slurry alone). Soil enzyme activity is expressed as mol of
products h1 g1 Soil samples (~10 g dry weight equivalent) were mixed with 50
mM acetate buffer (pH 5.0) in a weight/volume ratio 1:2.5 and then shaken at
200 rev min1 for 1 h. For the measurements of exoglucanase, -glucosidase, -
xylosidase, and -glucosaminidase activities, soil slurry samples (0.8 ml) were
pipetted into 2-ml Eppendorf tubes, which contained 0.2 ml of corresponding
substrate solutions (i.e., 2 mM p-nitrophenyl--D-cellobioside, 10 mM
pnitrophenyl--D-glucopyranoside, 2 mM p-nitrophenyl--xylopyranoside, and 2
mM p-nitrophenyl N-acetyl--Dglucosaminide, respectively, made with 50 mM
sodium acetate buffer at pH 5.0). After 1 h of incubation at 37C, except 2 h for
exoglucanase activity measurement, 0.2 ml of 0.5 M CaCl2 and 0.8 ml of 0.1 M
Tris buffer (pH 12.0) were added to stop the reaction and allow color
development. After centrifugation at ~11,700g for 4 min, optical density of the
suspension was measured at 410 nm to detect the released pnitrophenol (Tian et
al. 2010).
Peroxidase activity was measured with 3,3,5,5- tetramethylbenzidine (TMB) as
the substrate according to the method of Johnsen and Jacobsen (2008). Soil
slurry samples (0.2 ml) were pipetted into Eppendorf tubes that contained 0.4 ml
of TMB Easy solution (Fisher Scientific Inc.) and then incubated for 10 min at
room temperature. The enzymatic reaction was terminated by adding 0.8 ml of
0.3 M sulfuric acid. After centrifugation at ~11,700g for 4 min, optical density
of the suspension was measured at 450 nm and then soil peroxidase activity was
calculated based upon an extinction coefficient 59,000 M 1 cm1 .
Protease activity was assayed using the method described by Ladd and
Butler (1972). Briefly, 1 g of soil sample was weighed into a glass vial and then
2.5 ml of 0.2 M Tris buffer (pH 8.0) and 2.5 ml of a 2 % Na-caseinate solution
were added. The capped vials were incubated in a water bath at 50 C for 2 h.
Then, the remaining casein was precipitated with 5 ml of 10 % trichloroacetic
acid. In a 2-ml Eppendorf tube, 0.5 ml of the solution was mixed with 1 ml of 1.4
M Na2CO3 and 0.2 ml of 3-fold diluted FolinCiocalteu reagent. After 5 min of
incubation, the tube was centrifuged at ~16,400g for 1 min and the tyrosine
concentration was measured colorimetrically at 680 nm.
Data analysis
Analysis of variance (ANOVA) of a randomized block design was performed to
examine whether or not treatment effects were significant, with mean values
being compared via WallerDuncan k ratio at P<0.05 (the PROC GLM model, SAS
9.2; SAS Institute Inc., Cary, NC, USA). A composite soil sample was taken from
each of three field plots and was considered as the block and laboratory
treatments were randomized within individual soil samples. For any experiment
with multiple-time points, time was also considered as a factor and its interaction
with treatments was then examined. A linear regression was used to correlate
soil C mineralization with peroxidase activity. Pearsons correlation coefficient
was also calculated for elucidating the relationships of soil C and Nmineralization
with water extractable reducing sugars and amino acids, respectively.
Results
Soil C and N mineralization as affected by the addition of horseradish peroxidase
Peroxidase activity in soil was significantly enhanced by the addition of
horseradish peroxidase (P<0.05) and positively related to the activity units of
added horseradish peroxidase (Fig. 1). However, this enhancement lasted only
for ~3 days. Added horseradish peroxidase lost more than 60 % of its activity on
day 3 and almost all the activity on day 10. Thus, peroxidase activity in soil
measured 10 days later was similar regardless of different treatments.
Parallel with the change in soil peroxidase activity, soil C mineralization
differed only for the first 3 days after the addition of horseradish peroxidase
(P<0.05) (Table 1). Thereafter, no treatment effects were found on soil C
mineralization measured during 310 and 1021 days (data not shown). Soil N
mineralization measured during 021 days was also similar among the
treatments (data not shown). However, when horseradish peroxidase was
repeatedly added on day 3 and day 6, soil C mineralization measured during 36
and 69 days tended to be enhanced (Table 1). Regardless of different
treatments, soil C mineralization rates during 69 days were considerably lower
compared to the rates during 03 and 3 6 days. This is perhaps because the
readily degradable C compounds decreased over time. A regression analysis
showed that rates of soil C mineralization during 03, 36, and 69 days were
significantly correlated with added dose values of horseradish peroxidase
(Y=9.1+16.0X, R2 =0.20, P<0.05). Compared to the control, repeated addition of
horseradish peroxidase at 0.1 and 0.2 activity units increased soil C
mineralization by 17.6 % and 35.2 % during the 9-day incubation, respectively
(Table 1).
Horseradish peroxidase addition also affected soil hydrolytic enzyme activities
during the first 3 days and then these effects were diminished (Fig. 2). The -
glucosidase activity was greater (P<0.05) in the Pox_L than in the Pox_H or the
control. Exoglucanase activity was also higher (P<0.05) in the
Pox_L than in the Pox_H. Protease activity was similar between the Pox_L and Pox_H, but it
was lower (P<0.05) in the Pox_H than the control.
Similarly, the effect of horseradish peroxidase addition on the concentration of water
extractable soil phenolic compounds lasted only for 3 days (data not shown). On average,
phenolic concentration in the Pox_H was ~25 % greater (P<0.05) than the value, i.e., ~2.2
gCg1 soil in the Pox_L or the control.
Associations of soil C and N mineralization with reducing sugars and amino acids

Compared to the control (i.e., soil alone), protease or cellulose addition increased the
amount of water extractable reducing sugars by ~11 % and ~110 %, respectively (P<0.05)
(Fig. 3a, X-axis). Water extractable reducing sugars were ~25 % greater (P<0.05) in soils
amended with horseradish peroxidase than
 those without the addition of horseradish peroxidase (Fig. 3a, points above the 1:1
ratio). As expected, protease addition significantly increased the amount of water extractable
amino acids (P<0.05), but cellulose addition did not as compared to the control (Fig. 3b,
Xaxis). There was no statistical difference in the amount of water extractable amino acids
between soil alone and soil with the addition of horseradish peroxidase (Fig. 3b, the point
near the 1:1 ratio). When added to soils treated with cellulase or protease, however,
horseradish peroxidase increased the amount of water extractable amino acids by ~47 % and
~32 %, respectively (P<0.05) (Fig. 3b, points above the 1:1 ratio).
Soil C and N mineralization were determined via a 7-day incubation after enzyme-
treated sterile soils were inoculated. Compared to the control (i.e., soil alone), C
mineralization was increased by ~20 % by the addition of cellulase (P<0.05), but not by the
addition of protease (Fig. 4a, X-axis). While peroxidase addition did not improve C
mineralization in the control soil (Fig. 4a, the point near the 1:1 ratio), peroxidase addition
increased C mineralization in soil with cellulase and soil with protease by ~20 % (P<0.05)
(Fig. 4a, points above the 1:1 ratio). Nitrogen mineralization in the control, soil with cellulase
and soil with protease was significantly increased by the addition of peroxidase (P<0.05)
(Fig. 4b, points above the 1:1 ratio). Soil C and N mineralization were significantly correlated
with the concentrations of water extractable reducing sugars (Pearsons correlation coefficient
r=0.65, P<0.01) and amino acids (Pearsons correlation coefficient r=0.63, P<0.01) produced
after 5 days enzyme treatments in the sterile soil, respectively. No correlation was found
between extractable phenolic concentration and C or N mineralization (data not shown).
Fig. 3 Pairwise comparisons on the concentrations of water extractable reducing
sugars (a) and amino acids (b) in sterile soils amended with and without
horseradish peroxidase. Filled circle, filled upright triangle, and filled inverted
triangle represent comparisons for soil alone, soil with the addition of cellulase
and soil with the addition of protease, respectively. Soil alone: soil with the
addition of inactive cellulase, protease and horseradish peroxidase at 1, 0.1, and
0.1 units g1 soil, respectively; soil with cellulase: active cellulase addition at 1
unit g1 soil; soil with protease: active protease addition at 0.1 units g1 soil;
peroxidase treatment: active horseradish peroxidase addition at 0.1 units g1
soil.
Soil enzyme activities as affected by different types of phenolic compounds
Generally, hydrolytic enzyme activities were suppressed by the addition of
diphenols and polyphenols, but not by the addition of monophenols (Table 2). -
Glucosidase activity was decreased by ~45 % and ~93 % by the addition of
diphenols and polyphenols, respectively. Also, the activities of exoglucanase, -
xylosidase, and -glucosaminidase activity were decreased greatly by diphenols
and even completely inhibited by polyphenols.
Interestingly, suppressive effects of di- and poly-phenolic compounds on
hydrolytic enzyme activities were alleviated to some degree by a combined
addition with monophenolic compounds. Exoglucanase activity was even
increased by

Fig. 4 Pairwise comparisons on C mineralization (a) and N mineralization

(b) in soils amended with and without horseradish peroxidase. Filled circle, filled
upright triangle, and filled inverted triangle represent comparisons for soil alone,
soil with the addition of cellulase and soil with the addition of protease,
respectively. Soil alone: soil with the addition of inactive cellulase, protease and
horseradish peroxidase at 1, 0.1, and 0.1 units g1 soil, respectively; soil with
cellulase: active cellulase addition at 1 unit g1 soil; soil with protease: active
protease addition at 0.1 units g1 soil; peroxidase treatment: active horseradish
peroxidase addition at 0.1 units g1 soil.
the addition of M+P and D+P as compared to the control treatment.
Discussion
Peroxidase mediates the degradation of soil organic matter presumably
through its controls on the release of phenolic compounds. Simple phenolic
compounds, such as monophenols, can be utilized and mineralized by soil
microbes (Sparling et al. 1981; Blum and Shafer 1988; Blum 1998). We
postulated that peroxidase might also regulate the mineralization of soil organic
matter through its controls on the accessibility of other bioavailable compounds,
such as carbohydrates and proteins.
It is generally believed that soil organic matter is composed of high
molecular weight polymers, i.e., phenolic-based moieties associated covalently
with carbohydrates and proteins (Stevenson 1982). But increasingly, soil organic
matter has been recognized as a supramolecular structure, i.e., molecular
aggregates of various compounds via non-covalent bonds (Wershaw 1999;
Piccolo 2001). Phenolic compounds are deemed critical in the formation of
supramolecular substances due to their amphiphilic nature and therefore
interactions with carbohydrates and proteins (Haslam 1998). Nevertheless, both
polymer and supramolecular models support that soil organic matter is full of
interactions of phenolic moieties with carbohydrates and proteins. Thus, when
soil peroxidase oxidizes phenolic moieties (Mangler and Tate 1982; Dari et al.
1995; Gramss et al. 1999; Rezov et al. 2007), carbohydrates and proteins can
be released and become available for hydrolytic enzymes and soil microbes. This
pathway of peroxidase for organic matter degradation appears to be implied by a
study in desert soil where the combined addition of peroxidase and cellulase
increased the concentration of soil reducing sugars more than the addition of
cellulase alone (Nadeau et al. 2007).
As the substrates for microbial metabolisms, carbohydrates and proteins
can be present in soil in two states, i.e., freely accessible to soil hydrolytic
enzymes or encapsulated in phenolic complexes and soil structures (Knicker and
Hatcher 1997; Krull et al. 2001). If carbohydrates and proteins are in the free
state, they would be depolymerized by cellulase and protease to respective
oligomers, dimers and monomers. As a result, water extractable organic
compounds would be increased following the addition of hydrolytic enzymes into
soil. Indeed, we observed that the concentrations of water extractable reducing
sugars and amino acids increased significantly following the addition of cellulase
and protease, respectively. In contrast, encapsulation into phenolic complexes
prevents carbohydrates and proteins from being accessed by hydrolytic
enzymes. Hayes and Malcolm (2001) reported that easily -degradable
compounds and humic materials were tightly associated and could not be easily
separated out. These associations can protect easily degradable compounds
from biodegradation (Lichtfouse et al. 1998; Zang et al. 2000; Fan et al. 2004).
Only when oxidative enzymes such as peroxidase degraded humic substances,
the associations between easily degradable compounds and humic substances
can be disrupted, making otherwise protected compounds available for
hydrolytic enzymes and soil microbes. We did find that the concentrations of soil
reducing sugars and amino acids were increased by the addition of horseradish
peroxidase in sterile soils, in particular by the addition of horseradish peroxidase
in combination with cellulase or protease. Although protease could degrade other
enzymes (Renella et al. 2002), protease did not reduce the activity and function
of added horseradish peroxidase in this study. These reducing sugars and amino
acids could be rapidly mineralized as shown by significant
 correlations with soil C and N mineralization, which were measured
via a 7-day incubation after enzyme-treated sterile soils were inoculated
with soil microbes. Our results indicated that peroxidase regulated organic
matter degradation through controls on releasing carbohydrates and/or
proteins that were otherwise bound to humic substances.
Peroxidase might also stimulate soil C mineralization via depolymerizing
substances of high redox potentials (e.g., lignin). However, this is unlikely to
occur in our study because horseradish peroxidase is not as efficient at oxidizing
lignin as lignin peroxidase (Wong 2009). Furthermore, if this depolymerization
represents the major pathway of soil C mineralization following the addition of
horseradish peroxidase, we would expect the increase in soluble phenolic
concentration and its positive correlation with peroxidase activity. However, we
found that soluble phenolic concentrations were similar between the control and
Pox_L, while soil C mineralization rates between the two treatments were
significantly different. Although soluble phenolic concentration was greater in
Pox_H than in Pox_L, this difference was unlikely to be the cause for the different
rates of soil C mineralization between Pox_H and Pox_L. As shown by the soil
extract experiment, monophenols had little impacts on hydrolase activities,
whereas diphenols and polyphenols could significantly reduce hydrolyase
activities. Hence, lower activities of - glucosidase and exoglucanase in Pox_H
than in Pox_L suggest that Pox_H treatment produced more soluble diphenols
and polyphenols than Pox_L did. Compared to monophenols, complex phenolic
compounds were less likely to be assimilated and then metabolized by soil
microbes in a short term. Thus, our results disputed the pathway that the
peroxidase mediated soil C mineralization through depolymerization of lignin and
thereby release of simple phenolic compounds.
It is also possible that horseradish peroxidase addition frees soil organic
matter-bound hydrolytic enzymes and therefore stimulates soil C mineralization.
Given the protein nature, soil enzymes may interact with phenolic moieties of
soil organic matter, making them more thermally stable and resistant to
proteolysis in soil (Burns 1982; Gianfreda and Bollag 1996; Allison 2006;
Nannipieri et al. 2012). But this interaction can reduce soil enzyme activity
and/or affinity of soil enzymes for specific substrates (Ruggeiro and Radogna
1988; Gianfreda and Bollag 1996; Allison 2006; Ceccanti et al. 2008; Nannipieri
et al. 2012). By oxidizing phenolic compounds and thus freeing organic matter-
bound enzymes, horseradish peroxidase addition might stimulate the activities of
hydrolytic enzymes involved in organic matter degradation. However, our data
did not support this pathway because the addition of horseradish peroxidase at
the high dose had no stimulatory effects on the activities of glucosidase and
exoglucanase and even reduced soil protease activity.
It is well known that complex phenolic compounds, such as polyphenols,
can be detrimental to microbial growth due possibly to their roles of inhibiting
enzyme activity and sequestering organic substrates and trace elements needed
for microbial metabolisms (Fierer et al. 2001; Joanisse et al. 2007; Rosas et al.
2008). We examined the inhibitory effects of phenolic compounds of different
complexities on soil enzyme activities and found that even diphenols could
significantly inhibit soil enzyme activities. When soil was amended with
horseradish peroxidase, a mixture of mono-, di-, and polyphenolic compounds
could be produced. However, the composition of these phenolic compounds
might vary with the dose of horseradish peroxidase, causing different impacts on
soil hydrolytic enzyme activity between Pox_L and Pox_H. Nonetheless, inhibitory
effects of phenolic compounds on soil enzyme activities further disputed the
argument that peroxidase stimulated soil C mineralization by releasing soil
organic matter-bound enzymes and therefore increased soil enzyme activities.
Through well-controlled enzyme-addition laboratory incubation
experiments, this study improves our understanding on peroxidase-mediated soil
organic matter degradation. When peroxidase oxidizes phenolic moieties of
humus, simple phenolic compounds can be produced for microbial assimilation.
Simultaneously, peroxidase oxidation can improve the accessibility and thus
bioavailability of compounds, such as sugars and proteins that are otherwise
bound to soil humus. Apparently, it is difficult to maintain soil enzyme activities
as demonstrated in this study and others (Allison 2006; Sinsabaugh et al. 2008),
because a variety of soil processes and conditions, such as binding onto colloidal
surfaces, lack of proper coenzymes, and presence of metal inhibitors may cause
enzyme activities reduced (Nannipieri et al. 2012). Thus, information on ecology
of soil peroxidase activity is needed for better understanding of spatial and
temporal variations in soil organic matter in agricultural soil.
Acknowledgements We thank Ms. Emily Dell, Ms. Melissa Bell and Dr.
Jianjun Dai for helping in soil collection and preparation, and the NCSU
Environmental and Agricultural Testing Service laboratory for total soil C and N
analyses. This study was financially supported by the USDA-SSARE grant and the
North Carolina Agricultural Research Services.