Warning: include_once(../article_type.php): failed to open stream: No such file or directory in /home/suxhorbncfos/public_html/ctbeb/CTBEB.MS.ID.556002.php on line 115
Warning: include_once(): Failed opening '../article_type.php' for inclusion (include_path='.:/opt/alt/php56/usr/share/pear:/opt/alt/php56/usr/share/php') in /home/suxhorbncfos/public_html/ctbeb/CTBEB.MS.ID.556002.php on line 115
Is Laccase Enzyme an Answer for Sustainable
Thatch Management in Turfgrass Systems: A Review
Sudeep Singh Sidhu1,2*
1Department of Crop and Soil Sciences, The University of Georgia, USA
2North Florida Research and Education Center, University of Florida, USA
Submission: March 01, 2019; Published: April 09, 2019
*Corresponding author: Sudeep Singh Sidhu, University of Florida, North Florida Research and Education Center, Quincy, FL 32351, USA
How to cite this article: Sudeep S S. Is Laccase Enzyme an Answer for Sustainable Thatch Management in Turfgrass Systems: A Review. Curr Trends
Biomedical Eng & Biosci. 2019; 19(1): 556002. DOI: 10.19080/CTBEB.2019.19.556002
Use of laccase enzyme as a potential technique to manage thatch layer in turfgrass systems has been investigated in recent years. Thatch layer is a layer of organic matter formed between the soil and the green turfgrass. Presence of lignin, a recalcitrant component of organic matter, acts as the rate limiting step in organic matter degradation. Detrimental effects of a thick layer of thatch has been well documented which merits its effective management. Several costly, labor intensive, and destructive cultural practices are in use by industry to effectively control thatch. The destructive nature of these cultural practices presented an opportunity to investigate non-destructive and ecofriendly methods to manage thatch. This article reviewed unique characteristics of laccase enzyme, its ability to effectively degrade lignin, and development of laccase as a potential technique to effectively manage thatch layer in turfgrass systems.
Lignin, a major component of plant cell wall, acts as a protective matrix and limits microbial degradation of cellulose and hemi-celluloses which are readily degraded by microbes . Heterogenous and complex structure of lignin macro-molecule which is due to random coupling of three monomers makes it resistant to microbial degradation and hence a rate limiting step in organic matter decomposition [2-5]. Formation of thatch layer in turfgrass systems is accelerated when organic matter accumulation rate exceeds its degradation rate. Excessive thatch layer accumulation leads to physical conditions in thatch which are detrimental to turfgrass . Cultural management practices such as core aeration, vertical mowing, and grooming are destructive in nature and adversely impact turf quality.
Several non-destructive studies in the past utilized glucose, cellulase, mixture of amino acids, and microbial inocula to enhance organic matter decomposition in thatch layer. These studies proved futile as they were targeting decomposition of cellulose and hemi-cellulosic components instead of lignin [1,7]. Certain white-rot fungi produce extra-cellular enzymes known as lignolytic enzymes which are capable of lignin degradation . Mechanism of most of the lignolytic enzymes such as lignin peroxidases (LiP; EC220.127.116.11), manganese peroxidases (MnP; EC 18.104.22.168), and versatile peroxidases (VP; EC 22.214.171.124) is hydrogen peroxide dependent . The requirement of using hydrogen peroxide, a strong oxidant, to carry out the reaction of these enzymes makes them unusable in turfgrass systems
due to phytotoxicity. Laccases (Lac; EC 126.96.36.199) have a unique mechanism and presented the opportunity to be utilized in turfgrass systems as a direct surface application . This article reviewed complex structure of lignin, structure and unique oxidation mechanism of laccase enzyme that makes it suitable to be used in turfgrass systems, and developmental progress of laccase as a technique to manage thatch layer.
Lignin is the second most abundant organic substance next to cellulose and the major contributor to lignocellulosic recalcitrance to microbial degradation. Lignin is a three-dimensional amorphous polymer which is composed of three lignin monomers: p-caumaroyl, coniferyl, and sinapyl alcohols. The corresponding lignin monomers are known as p-hydroxy phenyl, guaiacyl and syringyl units, respectively, and often abbreviated as H, G, and S lignin . The ratio of G:S:H is generally 70:25:5 in the lignocellulosic materials . The recalcitrant nature of lignin is attributed to its heterogeneous complex structure, which is derived from random oxidative coupling of lignin monomers and cross-linking of polymers. A lignin macromolecule contains monolignols randomly bonded by C-O-C and C-C linkages including β-O-4, β-5, β-β, 5-5, 4-O-5, and β-1 bonds [2,3].
Fungal laccases occur as monomeric or dimeric protein structures with four copper atoms per molecule. The monomeric protein structures have a molecular mass of 50 to 100 kDa .
The process of laccase catalysis occurs in presence of oxygen in
1) Reduction of type I Cu by substrates;
2) Electron transfer from type I Cu to type II and III Cu trinuclear
3) Reduction of oxygen to water at the trinuclear cluster .
Efficacy of laccase enzyme is dependent on its redox potential.
Laccase produced from fungal sources have higher redox
potential and is used in several biotechnological and environmental
studies [13,15,16]. Redox potential of laccase enzyme
from different fungi is reported from 450 mV to 800 mV . Low
redox potential of fungal laccase restricts its ability to oxidize
non-phenolic compounds . However, addition of low molecular
weight substances, known as mediators, increase the substrate
range of laccase enzyme to non-phenolic groups, benzyl
and alyl alcohols and ethers [18-20] which contribute the major
fraction of the lignin macromolecule [11,12]. As laccase enzyme
requires oxygen instead of a strong oxidant as part of its mechanism
to oxidize organic molecules, this property makes laccase
unique to be utilized in agricultural systems.
A novel non-destructive approach was developed at The
University of Georgia, Griffin Campus to utilize laccase enzyme
alone or along with mediators as a direct application on
several turfgrasses maintained as home lawns or recreational
turf [21,22]. For all the studies, laccase from fungus Trametes
versicolor was used due to its high redox potential (800 mV).
The results of these studies will be discussed in the following
A greenhouse study was conducted on ‘Crenshaw’ creeping
bentgrass (Agrostis stolonifera L.) pots. Treatment application
included direct spay of laccase enzyme with or without a mediator,
guaiacol. Treatments were applied once every two weeks
which included 40-mL solution of laccase at activity levels 0,
0.206, and 2.06 units cm-2 and 10-mL solution of guaiacol at 0
and 0.1 M concentration. Samples were analyzed after two and
nine months of treatment application to observe the impact of
laccase enzyme on thatch layer and turfgrass quality. No significant
differences were observed for any of the treatments after
two months of application. After nine months of application,
laccase treatment at lower activity level of 0.206 units cm-2 was
not effective. However, laccase treatment at activity level of 2.06
units-2 reduced thatch layer thickness by 45% when compared
to control pots. Treatment with 2.06 units cm-2 laccase with and
without guaiacol reduced organic matter content in the top 2.5
cm thatch layer by 25.9 and 30.3 mg·g-1, respectively compared
to control (Figure 1). Laccase treatment also reduced lignin content
by 19.0 mg·g-1 compared to control. No adverse effect was
observed on turfgrass quality for the duration of the experiment.
Application of mediator guaiacol had slight impact on overall efficacy
of laccase enzyme when samples were analyzed after nine
months. This study provided answers in terms of laccase activity
and application duration needed to impact turfgrass thatch layer
and if there were any adverse phytotoxicity effects of direct application
This study was conducted on dead creeping bentgrass pots to
observe the impacts of laccase application on thatch layer composition.
In this study, growth of creeping bentgrass was ceased
by herbicide Roundup Pro. A week later, grass was clipped down to thatch layer and covered by a black plastic sheet to avoid stimulation
of re-growth. As discussed in the previous study, laccase
and guaiacol were applied directly on thatch once every two
weeks. However, in this study laccase enzyme was applied at 0,
2.06, and 20.6 units cm-2 with the 10-mL solution of guaiacol at
0 and 0.1 M concentration. Samples were analyzed after two and
six months of treatment application. After six months, all treatments
with or without guaiacol significantly impacted thatch
layer characteristics. Organic matter content in that top 2.5 cm
reduced by 24.7% and thatch layer thickness reduced by 57.2%
compared to control with laccase treatment of 20.6 units cm-2
(Figure 2). A significant increase in saturated hydraulic conductivity
was observed with laccase treatments. A significant reduction
in monosaccharide components of structural cellulose and
hemicellulose carbohydrates were observed. This reduction in
the sugar content can be attributed to the opening of biomass
structure by breaking down of lignin macromolecule. However,
increase in both acid-soluble and -insoluble lignin components
were observed. This increase in lignin components in thatch can
be attributed to the over all reduction in structural carbohydrate
(sugar) content where lignin and structural carbohydrates are
the major components of plant cell wall. Lignin content is calculated
as a percent of total lignin and carbohydrates and is thereby
dependent on the carbohydrate content. This study supported
the original assumption of using lignin degrading enzyme.
In the previous greenhouse study , it was evident that
laccase, when applied at the activity level of 2.0 units cm-2 once
every two weeks, was effective in reducing organic matter buildup
and reducing thatch layer thickness. A two-year field study
was conducted on creeping bentgrass to evaluate the effectiveness
of laccase under field conditions and to optimize the rate
and frequency of application. Laccase application was also compared
with prevalent cultural practice used in industry, core-aeration
followed by sand topdressing. Laccase enzyme was applied
as a 410-mL solution over an area of 0.185 m2 for all the treatments.
Laccase was applied at five activity levels 0 (control), 0.5,
1.0, 2.0, and 4.0 units cm-2 at an application frequency of once
every two weeks. It was observed that all the levels of laccase
treatments were effective in in reducing thatch layer thickness
(Figure 3), organic matter content, and carbohydrate content in
comparison to control. Lignin content decrease for two lower
activity levels of laccase but increased for the higher two levels
when compared to control. This increase in the lignin content
in higher two levels could be attributed to a higher reduction in
carbohydrates in these treatments. The study suggested that laccase
application as low as 0.5 units cm-2 is as effective in managing
thatch when applied once every two weeks when compared
to 2.0 units cm-2 treatment suggested by the greenhouse studies.
The effective rate of laccase from the previous greenhouse
study, 2.0 units cm-2 was applied at four different application
frequencies. The application frequencies were once every 2, 4,
8, and 12 weeks. Laccase applied at all application frequencies
was effective in managing thatch layer but it was observed that
effectiveness of laccase was significantly higher when it was applied
once every 2 and 4 weeks as compared to once every 8 and
12 weeks (Figure 3). It was concluded that one application of
laccase at activity level of 2.0 units cm-2 once every 4 weeks may
be sufficient for efficient reduction in thatch layer.
Plots that received cultural management treatments were
core-aerated and sand top dressed twice yearly. Core-aeration
was accomplished with 1.27 cm tines spaced at 5.0 by 5.0 cm.
Immediately after core-aeration plots were top dressed with
1134 g of sand. Laccase applications associated with cultural
management treatments were applied at 2.0 units cm-2 applied
every 4 weeks. Reduction in thatch layer thickness similar in cultural
management and laccase treatments (Figure 3). However, a
further reduction in thatch layer was observed when laccase was
applied in combination with cultural management treatment
(Figure 3). Application of laccase in combination with core-aeration
and sand topdressing may lead to a reduction in number
of cultivations necessary to keep thatch layer at desired levels.
This study was not a factorial design and did not provide the
information for a single minimum effective laccase treatment
in terms of rate of laccase activity and frequency of application.
This was due to the great demand on the available resources.
Information gained from this study was later used to design a
much smaller factorial experiment to provide minimum effective
rate and frequency of laccase application. The results from this
factorial study are not published yet.
A two year study was conducted on ultra-dwarf bermudagrass
(Cynodon dactylon L., ‘TifEagle’) green, and zoysiagrass
(Zoysia japonica Stued., ‘Meyer’) maintained as a home lawn to
observe the influence of laccase enzyme applications on thatch
development. Laccase solution was applied bi-weekly at the activity
levels of 0 (control) and 2.0 units cm-2. Response to laccase
enzyme applications by both turfgrass species was recorded by
measuring physical and chemical properties of thatch layer after
six months of treatment applications within each year. A significant
18-22% and 21-30% reduction in thatch layer thickness
was observed for bermudagrass and zoysiagrass, respectively.
Organic matter content (0-2.5 cm) decreased by 23-24% (Figure
4), while saturated hydraulic conductivity increased by 19-30%
for bermudagrass in both years. Acid-soluble and-insoluble lignin
reduced in both the grass species after laccase treatments.
The results indicate that bi-weekly application of laccase on bermudagrass
and zoysiagrass has positive impact on thatch management.
A field experiment on creeping bentgrass was conducted to
evaluate the residual effects on thatch accumulation after ceasing
laccase applications. A significant reduction in thatch layer
thickness was observed at 6, 12, and 18 months after treatment
initiation when laccase was applied at different rates and frequencies
for six months. Residual effects of laccase application
were observed as a reduction in thatch layer thickness and no
additional accumulation of thatch after six months of treatment
cessation. At 18 months after treatment initiation, a significant increase in thatch layer was observed in plots where treatments
had ceased for 12 months. This study suggested that either 12
applications of laccase at activity level 0.5 units cm-2 applied
once every two weeks (six months) or six applications of laccase
at 2.0 units cm-2 applied once every 4 weeks (six months) will be
sufficient for managing thatch for a year.
There are several evidences from literature that support the
efficacy of laccase enzyme from Trametes versicolor L. to provide
sustainable thatch management in turfgrass systems. Laccase
when applied at certain rates of activity level and at certain frequency
of application for six months in a year is sufficient for
thatch control. Future research is needed to further minimize
the amount of laccase used, either in terms of laccase activity or
in terms of frequency of application to help reduce the cost and
number of application treatments per year for this non-destructive
thatch management approach.