Journal of
Yeast and Fungal Research

  • Abbreviation: J. Yeast Fungal Res.
  • Language: English
  • ISSN: 2141-2413
  • DOI: 10.5897/JYFR
  • Start Year: 2010
  • Published Articles: 125

Full Length Research Paper

The potential use of Lentinus edodes to manage and control water hyacinth in Zimbabwe

Nompumelelo Sibanda
  • Nompumelelo Sibanda
  • Department of Crop Science and Post-harvest Technology, School of Agricultural Sciences and Technology, Chinhoyi University of Technology, Private Bag 7724, Chinhoyi, Zimbabwe.
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Oziniel Ruzvidzo
  • Oziniel Ruzvidzo
  • Department of Botany, School of Biological Sciences, North-West University, Private Bag X2046, Mmabatho, 2735, South Africa.
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Cuthbert J. Zvidzai
  • Cuthbert J. Zvidzai
  • Department of Food Science and Technology, School of Agricultural Sciences and Technology, Chinhoyi University of Technology, Private Bag 7724, Chinhoyi, Zimbabwe.
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Arnold B. Mashingaidze
  • Arnold B. Mashingaidze
  • Department of Crop Science and Post-harvest Technology, School of Agricultural Sciences and Technology, Chinhoyi University of Technology, Private Bag 7724, Chinhoyi, Zimbabwe.
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Tshegofatso B. Dikobe
  • Tshegofatso B. Dikobe
  • Department of Botany, School of Biological Sciences, North-West University, Private Bag X2046, Mmabatho, 2735, South Africa.
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Mutsa M. Takundwa
  • Mutsa M. Takundwa
  • Department of Botany, School of Biological Sciences, North-West University, Private Bag X2046, Mmabatho, 2735, South Africa.
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David T. Kawadza
  • David T. Kawadza
  • Department of Microbiology, School of Biological Sciences, North-West University, Private Bag X2046, Mmabatho, 2735, South Africa.
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Lebogang M. Katata-Seru
  • Lebogang M. Katata-Seru
  • Department of Chemistry, School of Physical and Chemical Sciences, North-West University, Private Bag X2046, Mmabatho, 2735, South Africa.
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Sibonani S. Mlambo
  • Sibonani S. Mlambo
  • Department of Biotechnology, School of Agricultural Sciences and Technology, Chinhoyi University of Technology, Private Bag 7724, Chinhoyi, Zimbabwe.
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Chrispen Murungweni
  • Chrispen Murungweni
  • Department of Animal Production and Technology, School of Agricultural Sciences and Technology, Chinhoyi University of Technology, Private Bag 7724, Chinhoyi, Zimbabwe.
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  •  Received: 17 October 2019
  •  Accepted: 20 December 2019
  •  Published: 31 January 2020

 ABSTRACT

The rapid expansion and reproduction of certain plant species represents one of the biggest problems in aquatic environments, ranging from eutrophication to the limited availability of water for human consumption. Among these plants is water hyacinth (Eichhornia crassipes), a herbaceous hydrophyte often branded the world’s worst aquatic weed due to its invasive aggression, negative impact on aquatic environments, and the cost usually associated with its management. Water hyacinth is a biomass, typically rich in lignocellulosic material and making it a potential raw material for the synthesis of products of industrial and domestic interest; e.g. edible fungi. Among the commonly known edible fungi is Lentinus edodes, a commercial mushroom whose versatile nature as a white rot fungus provides basis for the continued exploration of its biochemical processes during solid state fermentation on various lignocellulosic biomass as potential substrates. The fungus naturally feeds on lignocellulose by secreting various extracellular enzymes responsible for breaking down this organic polymer into simple soluble molecules that the hyphae can absorb and develop into mycelia. In this study, L. edodes was assessed for its ability to grow on water hyacinth and possibly utilizing it as a substrate. When cultured onto this noxious biomass followed by assessment by agar plate-based clearing assay and spectrophotometry, the fungus demonstrated its ability to secrete cellulases, xylanases, pectinases, peroxidases and laccases, thus showing its capabilities to physiologically utilize this hydrophyte as a substrate. If properly optimized, this approach can be remarkably used as a sustainable way to control water hyacinth in Zimbabwe.

 

Key words: Lentinus edodes, water hyacinth, lignocellulosic biomass, lignocellulolytic enzymes, cellulases, xylanases, pectinases, lignin peroxidases, laccases.


 INTRODUCTION

Water hyacinth or Eichhornia crassipes (Mart.) Solms-Laubach. is a tropical perennial aquatic plant belonging to the family Pontederiaceae (Crow et al., 2000; Penfound and Earle, 1948). It is a free-floating aquatic organism, originating from the Amazon River Basin in South America (Sornvoraweat and Kongkiattikajorn, 2010). The plant tolerates extremes in seasonal variations particularly in terms of flow velocity, nutrient availability, pH, temperature, water level fluctuations and toxic substances (Penfound and Earle, 1948). The hydrophyte also has an extensive dispersal capacity and an extremely fast growth rate (Gutierrez et al., 2001; Villamagna and Murphy, 2010) and duplication time of 7.4 days on average, by which a total of 144 ton/ha of dry matter can be accumulated in a year (Mwangi, 2013; Nigam and Singh, 2002).
 
Water hyacinth has emerged as a major weed, polluting water bodies in more than 50 countries in the tropical and sub-tropical regions, with profuse and permanent impacts (Mwangi, 2013). In Zimbabwe, this invasive exotic plant was first reported in watersheds as early as the 1940s, but had not yet posed any management problem (Magadza, 2003). The plant was introduced in the country initially, as an ornamental flower and then eventually spreading out to other water bodies uncontrollably (Chikwenhere, 1994).  By the mid-1960s, most of the major aquatic bodies in that country, including lake Chibero (the largest water body in Harare - the capital city) and Hunyani River (the main tributary of lake Chibero) were invaded (Magadza, 2003).
 
Besides natural factors, human activities in most cases, also promote the spread of this weed by providing conducive conditions and environments for its proliferation and establishment (Gutierrez et al., 2001; Villamagna and Murphy, 2010). Run-offs from agricultural and industrial developments, pollution from septic and sewer systems and the other human-related practices, continuously increase the influx of organic and inorganic substances into water bodies (Chikwenhere, 1994). The fast growth rate of this weed and robustness of its seeds lead to various problems, which among others, include coverage of water ways, destruction of ecosystems through death of the aquatic life and eventually, the uncontrollable speeding up of eutrophication (Nigam and Singh, 2002). The usual humankind livelihood activities such as fishing and tourism have, to date, been severely constrained by the explosive infestations of this aquatic plant in various local and regional water bodies (Cilliers et al., 2003).
 
By this day, water hyacinth has been marked the world’s worst aquatic weed and has garnered increasingly a lot of international attention as an invasive species (Mwangi, 2013). The plant has been classified by the International Union for Conservation of Nature (IUCN) as one of the 100 top-most aggressive invasive species and one of the 10 top-most worst weeds in the world (Saha et al., 2014). Attempts to control the weed have resulted in very high marginal costs, which at times, were rather futile as they could only manage to temporarily reduce the weed but not completely eradicating it (Cilliers et al., 2003; Gutierrez et al., 2001). Some of the methods used e.g. chemical treatment, had very detrimental effects on aquatic life, further with the water being deemed unsafe for domestic and agricultural uses (Mwangi, 2013). According to Brown (2006), the economic impacts of this weed in several African countries, including Zimbabwe have been estimated to be between 20 and 50 million US dollars every year while across the whole continent of African, it is as much as US$100 million annually.
 
As a problematic weed but with an attractively high content of the lignocellulosic biomass, water hyacinth’s possible use in industries and commercial set-ups could potentially have significant benefits not only to the industries themselves but also to either or both the natural aquatic environments and/or local communities situated around such water-infested bodies. However, and like any other flowering land plant, water hyacinth is composed mainly of the biologically stable polymer - lignocellulose that is very resistant to either the physical, chemical, or enzymatic attacks (Dorado et al., 2001). Notably, white-rot fungi, which are a specialized group of microorganisms belonging to the unique class of Basidiomycetes, have been reported to be capable of attacking the lignocellulose fibre (Dorado et al., 2001; Jurado et al., 2011; Wang et al., 2019). These fungi secrete various extracellular enzymes and organic acids that breakdown fiber (Dorado et al., 2001; Jurado et al., 2011; Pandya and Albert, 2014). Among the enzymes are oxidases (laccases and peroxidases) that breakdown lignin (Wesenberg et al., 2003; Zirbes and Waldvogel, 2018), glucanases (exo- and endo-) that degrade cellulose (Kuhad et al., 2011; Legodi et al., 2019), xylanases that breakdown hemicelluloses (Punniavan, 2012), and pectinases that degrade pectins (Baldrian and Valášková, 2008; Collins et al., 2005).
 
A number of white rot fungi produce a whole cocktail of these enzymes while others produce only one or a few of them (Baldrian and Valášková, 2008; Maganhotto de Souza  Silva  et   al.,  2005).  Lentinus   edodes   (Berk. & 
Mont.) Pegler., (shiitake) and Pleurotus spp. (Jacq.: Fr.) Kumm., (oyster) comprise a group of white rot fungi that are edible (mushrooms) with important medicinal properties and biotechnological and environmental benefits (Cohen et al., 2002; Jia et al., 2019; Leatham, 1985; Reddy and D’Angelo, 1990; Thakur, 2018). If successfully grown on water hyacinth, these edible white rot fungi would yield mushrooms (Thakur, 2018), a whole cocktail of enzymes (Wang et al., 2019) and fine chemicals (Zirbes and Waldvogel, 2018), whose properties would be very essential and useful for the food and feed industries (Kiiskinen et al., 2004; Mikiashvili et al., 2006).
 
In this reported work, L. edodes was tested for its practical capability to grow on water hyacinth growing locally in Zimbabwe and its potentials to utilize it as a substrate. The study was designed on the backdrop that if this edible white rot fungus could successfully utilize water hyacinth as a substrate, then this whole approach could then provide a viable strategy for sustainable management (Jia et al., 2019; Thakur, 2018) of this problematic weed in local aquatic environments of the country. On the other hand, the same approach would also provide prospects for the possible conversion of a low-value indigenous lignocellulosic rich waste into products of high commercial value such as mushrooms, enzymes and fine chemicals (Buswell et al., 1993; Thakur, 2018; Villamagna and Murphy, 2010; Wang et al., 2019; Zirbes and Waldvogel, 2018).
 


 MATERIALS AND METHODS

Source of the test fungus 
 
The L. edodes strain used in this study as the test fungus was purchased from Sylvan Africa (PTY) Ltd., RSA, in form of a partially-dried spawn, maintained at 4°C.
 
Viability assessment of the test fungus
 
Growth viability of the purchased test fungus was tested and ascertained as already detailed elsewhere (Sibanda et al., 2019).
 
Source of substrate and substrate preparation
 
The water hyacinth and liver seed grass biomasses used in this study as the test and control substrates were obtained from Zimbabwe (Permit number: P0079761; Appendix; Figure A1) and South Africa respectively. The biomasses were dried and prepared for experimental work as outlined before (Sibanda et al., 2019). 
 
Cultivation of fungi and production of enzymes
 
The culture cultivation of L. edodes on the two prepared biomasses and the  subsequent  preparation  of  crude  enzyme  extracts  were undertaken as previously detailed (Sibanda et al., 2019). 
 
Assaying for lignocellulolytic activities
 
L. edodes’ probable ability to secrete various lignocellulolytic enzymes when cultured on water hyacinth as a potential substrate (and liverseed grass as a control substrate) was assessed via the agar plate-based clearing assay and spectrophotometric methods (Miller, 1959; Pointing, 1999; Sibanda et al., 2019a; Téllez-téllez et al., 2013).
 
Resolution and analysis of the secreted total protein content in the crude enzyme extract
 
Total protein content secreted by L. edodes in the crude enzyme extract during its growth on water hyacinth was resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), according to a previously established method (Laemmli, 1970). In addition, the subsequent zymographic analyses of the resolved protein bands for cellulolytic and xylanolytic activities were carried out in accordance with the method of Téllez-téllez et al. (2013) and Pointing (1999). For more detail, refer to Sibanda et al. (2019a).
 
Reaction kinetics of the cellulolytic and xylanolytic protein fractions in the crude enzyme extract
 
Reaction kinetics of the SDS-PAGE resolved cellulolytic and xylanolytic protein fractions in the crude enzyme extract were assessed and determined via the Hanes-Woolf plot and in accordance with the established method of Kwezi et al. (2011) and Meier et al. (2010). For further detail, refer to Sibanda et al. (2019a).
 
Statistical analysis
 
All data from enzyme assaying in this work are means of triplicate assays (n = 3) subjected to analysis of variance (ANOVA) (Super-Anova, Statsgraphics Version 7; Statsgraphics Corp., The Plains, VI, USA). Wherever ANOVA revealed significant differences between treatments, means were separated by post hoc Student–Newman–Keuls (SNK) multiple range test (p < 0.05).


 RESULTS

L. edodes has a good viability status to grow on water hyacinth as a potential substrate
 
When the viability status of L. edodes, as a test fungus for the study, was tested through spawn culturing, it became apparent that this white rot fungus could both viably and significantly grow on either the two provided commercial grade substrates (carboxymethylcellulose and birchwood xylan) (Figure 1a) or the test experimental substrate (water hyacinth) (Figure 1b). On water hyacinth, L. edodes produced hyphal growths that could be visually observed on the substrate biomass, thus demonstrating its probable capabilities to physiologically utilize the tested experimental biomass as a substrate.
 
 
 
 
L. edodes has the ability to secrete lignocellulolytic enzymes when cultured on water hyacinth
 
Using the agar plate-based clearing assay method, it was noticed that the L. edodes could viably secrete a whole cocktail of the lignocellulolytic enzymes during its growth on water hyacinth as a potential substrate (Figure 2). In this particular assaying method, lignocellulolytic enzymes secreted by microbes, breakdown and solubilize complex lignocellulosic polysaccharides in the media to generate zones of clearance on the solidified agar plates that can be easily visualized as clear halos after addition of particular dyes and clearing with specific de-staining solutions (Sibanda et al., 2019a; Téllez-téllez et al., 2013) (Figure 2a-e).
 
Validating the lignocellulolytic capacity of L. edodes when cultured on water hyacinth
 
Using spectrophotometry, it was validated that the L. edodes could actually produce a whole cocktail of the lignocellulolytic enzymes when grown on water hyacinth as a substrate (Figure 3). In this advanced scientific technique, a 3,5-dinitrosalicylic acid detection system for reducing sugars is employed. It results in the generation of coloured compounds that are measurable by various analytical equipment and easily convertible  into  rates  of enzyme activity (Miller, 1959; Pointing, 1999); for instance, and in this case, ~ 1.2650, 2.0625, 2.4375, 0.9375, 2.0225 µmole/sec/ml for lignin peroxidases, laccases, cellulases, pectinases and xylanases, respectively (Figure 3).
 
Resolution and activity assaying of the various protein fractions in the crude enzyme extract
 
When SDS-PAGE was used to resolve the various enzymatic protein fractions secreted by L. edodes during its growth on water hyacinth  for probable further analysis, it emerged that only fractions of the molecular weight sizes of around 50-70 kDa and 20-25 kDa could be visually observed (Figure 4a). This kind of resolution thus suggested cellulases and xylanases respectively. Notably, a further analysis of the same SDS-PAGE gel by zymography then firmly confirmed that such ~50-70 kDa and ~20-25 kDa protein fractions were indeed carboxymethylcellulose-degrading (Figure 4b) and birchwood xylan-degrading proteins (Figure 4c) respectively.
 
Kinetic assaying of the cellulolytic and xylanolytic activities in the crude enzyme extract
 
After determining that, when cultured on water hyacinth, L. edodes mostly produces cellulolytic and xylanolytic proteins as its major components, the kinetic rates of these two most secreted protein components were then assessed  and   ascertained   via   the  Hanes-Woolf  plot method (Figure 5). This was undertaken in order to relate activities of these two major enzymatic protein components to their counterparts in other known organisms and/or related microbial systems. Based on this approach and as is shown in Figure 5, a Km value of 0.247 mM and a Vmax value of 2 177.88 µmol/sec for the cellulolytic proteins were obtained (Figure 5a) while a Km value of 0.147 mM and a Vmax value of 1 208.33 µmol/sec for the xylanolytic proteins were obtained (Figure 5b).
 
 
 


 DISCUSSION

Water hyacinth or E. crassipes (Mart.) Solms-Laubach. originates from Brazil (Crow et al., 2000; Penfound and Earle, 1948)and has by this day spread to almost all tropical and sub-tropical nations such as Zimbabwe (Parsons et al., 2001), where it is considered as one of the world’s most deadliest invasive aquatic plants (Mwangi,  2013).  The   plant   is   perennial   and   mostly wide-spread on freshwater wetlands of most tropical and sub-tropical areas, particularly in stagnant water (MWBP/RSCP, 2006). The hydrophyte multiplies very rapidly, forming dense mats (Gopal and Goel, 1993) that normally interfere with existing waterways, ruin aquatic life and create suitable conditions for breeding of parasitic vectors and the outbreak of their related diseases (Kushwaha, 2012). Water hyacinth is also known to have various notable ecological and socio-economic issues, which among others, include suppression of the local aquatic biodiversity, obstruction of river flows which may aggravate flooding and promote siltation, interference with water utilization for activities such as recreation, tourism and/or irrigation, and increased rates of evapotranspiration from water storages (Chikwenhere, 1994; Cilliers et  al.,  2003). Its  infestation  also  poses  a potential health risk in that the plant has been implicated in the creation of breeding habitats for mosquitos and/or their larvae that can cause malaria as well as other water disease-carrying vectors like bilharzia snails (Villamagna and Murphy, 2010).
 
However, and despite its various negative impacts as a notorious weed, water hyacinth has several other potential benefits to humankind, which include its use as a protein supplement in animal feeds (Mako et al., 2011; Yagi et al., 2019), for water purification, as fibreboard or fertilizer and in paper and biogas production (Lindsey and Hirt, 1999). The fact that this plant has very high levels of protein content (especially in its leaves) (Virabalin et al., 1993) accompanied by its rapid growth (Gopal, 1987), has essentially made it very suitable for this hydrophyte to be commonly  used as fodder (Yagi et al., 2019) for the various kinds of livestock such as cows (Rashid and Iqbal, 2012), sheep (Abdalla et al., 1987), goats (Dada et al., 2002) and pigs (Men et al., 2006), and domesticated birds such as ducks (Jianbo et al., 2008) as well as the aquatic fishes like tilapia fingerlings (El-Sayed, 2003). Moreover, the harvesting process of this hydrophyte for use as fodder is quite simple and straightforward as it can be done manually on a small scale level and without requiring any new harvesting techniques to be introduced (Gunnarsson and Petersen, 2007). In the middle-income earning countries such as Vietnam and Thailand, where poor quality rice straw is mainly the major source of fibre, water hyacinth has proved as an excellent alternative. Furthermore, in tropical African countries like Tanzania, this aquatic plant has since been proven to be very good substrate for large-scale production of either the exotic mushroom, Pleurotus ostreatus (ÇaÄŸlarırmak, 2007)or its indigenous counterpart, P. flabellatus (Kivaisi et al., 2003). The use of this plant (for example as livestock feed or for production of mushrooms) for the general benefit of mankind is typically considered as an effective method of its mechanical control in most nations (Murugesan et al., 2006).
 
Apparently, in an African tropical country like Zimbabwe, none of these efforts have ever been reported and/or made. As a result, this present study was therefore, undertaken to assess if the edible fungus L. edodes could perhaps grow successfully on water hyacinth native to the waters of that country and utilizing it as a substrate. A successful use of this problematic plant as a substrate for L. edodes could probably serve as a sustainable and cost-effective way of controlling it in the Zimbabwean local aquatic ecosystems while at the same time, generating protein-rich foods for the surrounding communities and perhaps, also production of commercial enzymes and/or fine chemicals for the local and/or national industries.
 
The L. edodes strain used in this study is an exotic mushroom that was commercially acquired from a local supplier Sylvan Africa (PTY) Ltd., (Pretoria, South Africa) in form of a partially-dried spawn. However, before this fungal strain could be used in the planned study, its growth viability was first checked and ascertained through culturing on two different substrates of commercial grade. As is shown in Figure 1a, the test fungus could both viably and vigorously grow on either carboxymethylcellulose or birchwood xylan. When the L. edodes was then cultured on water hyacinth, followed by assessment of its ability to grow on this test substrate, biomass colonization was relatively good with hyphal almost completely covering the whole substrate (Figure 1b). This could be as a result of the L. edodes secreting the various lignocellulolytic enzymes that then enabled it to grow and colonize the provided substrate. Generally, white rot fungi like L. edodes are known to be  capable  of secreting oxidases (laccases and peroxidases) that degrade lignin (Wesenberg et al., 2003; Zirbes and Waldvogel, 2018), glucanases (exo- and endo-) that breakdown cellulose (Kuhad et al., 2011; Legodi et al., 2019), xylanases that degrade hemicelluloses (Punniavan, 2012), and pectinases that breakdown pectins (Collins et al., 2005).
 
Naturally, some white rot fungi produce the whole cocktail of lignocellulolytic enzymes while others produce only one or a few of them (Baldrian and Valášková, 2008; Maganhotto de Souza Silva et al., 2005; Wang et al., 2019). Therefore, in order to ascertain if the L. edodes was capable of secreting the whole cocktail of the white rot fungal enzymes or part of it when growing on water hyacinth, its crude extracellular extract was tested for the various lignocellulolytic enzyme activities via the agar plate-based clearing assay method (Figure 2) and spectrophotometry (Figure 3). Under the agar plate-based clearing assay method, the Congo red assay showed zones of clearance in diameters of over 2.74 cm for cellulases (Figure 2a) and 2.53 cm for xylanases (Figure 2b), demonstrating ability of the excreted enzyme extract to breakdown carboxymethylcellulose and birchwood xylan respectively. These revealed zones of clearance were not that much different from the ones generated by plant endophytes, P. ostreatus and some filamentous fungi from termite mounds on the same commercial substrates, which were >2 cm (Eichlerová et al., 2012; Sibanda et al., 2019a). Such capabilities may be gained due to the adaptation abilities of fungi to their habitats, which are a whole set of lignocellulosic materials (Yopi et al., 2014). A related trend of clearance was also observed for the pectinases (Figure 2c), lignin peroxidases (Figure 2d) and laccases (Figure 2e), signifying ability of the excreted enzyme extract to hydrolyze polygalacturonic acid, veratryl alcohol, and guaiacol respectively.
 
The same results as is reported above were also revealed by spectrophotometry (Figure 3), a method that is alternative to the agar plate-based clearing assay but being rather more sensitive. Collectively, these findings therefore, showed that the L. edodes is capable of secreting the whole cocktail of the white rot fungal enzymes when grown on water hyacinth, and thus able to utilize this notorious weed as an alternative substrate. Lentinus spp. have previously been reported to have a unique ability to produce xylanases (Bhagobaty et al., 2007), carboxymethylcellulases, β-glucosidases, β–xylosidases, and extracellular lignocellulolytic enzymes, including laccases, pectinases and lignin peroxidases (Elisashvili et al., 2015; Jia et al., 2019; Wang et al., 2019).
 
When the various fractions of the total protein content secreted by the L. edodes during its growth on water hyacinth   were    resolved    by    SDS-PAGE  for   further analysis, it emerged that the dominant protein fractions produced were most likely cellulases (~50-70 kDa) and xylanases (~20-25 kDa) (Figure 4a). Cellulases are multi-enzyme complexes composed of various protein components with endoglucanase, exoglucanase and β-glucosidase activities that normally operate synergistically (Legodi et al., 2019; Liming and Xueliang, 2004; Stajić et al., 2006). Of these protein components, cellobiohydrolase I (52.2 kDa) and cellobiohydrolase II (47.2 kDa) are the predominant ones (>90%) while endoglucanases and hemicellulases represent less than 10% (Da Vinha et al., 2011). On the other hand, xylanases are single polypeptide chain proteins with a molecular weight size of around 21 kDa (as judged by SDS-PAGE) and a pI value of 4.5 (Bray and Klarke, 1995; Zirbes and Waldvogel, 2018). Unlike cellulases, xylanases are not glycosylated (Bray and Klarke, 1995).
 
Notably, when the same SDS-PAGE gel described above (Figure 4a) was further subjected to a zymogram analysis, results obtained then showed that the resolved ~50-70 kDa proteins were indeed responsible for the decomposition of carboxymethylcellulose (Figure 4b) while the ~20-25 kDa proteins were responsible for the degradation of birchwood xylan (Figure 4c), thus firmly affirming our initial claim in the SDS-PAGE analysis (Figure 4a) that the ~50-70 kDa proteins were cellulases while the ~20-25 kDa proteins were xylanases. Incidentally, our work also managed to reveal a number of carboxymethylcellulose-decomposing proteins, ranging from ~10-200 kDa (Figure 4b), concurring with the fact that cellulases are multi-enzyme complexes composed of various protein components such as endoglucanase I (46.0 kDa), II (42.2 kDa), IV (33.4 kDa), V (22.8 kDa) and VII (25.1 kDa); cellobiohydrolase I (52.2 kDa) and II (47.2 kDa); β-glucosidase I (75.3 kDa) and II (52.1 kDa); and β-glucosidase-1,4-glucanase (23.5 kDa) (Da Vinha et al., 2011; Legodi et al., 2019). This outcome is closely related to that of Elisashvili et al. (2015), who recorded carboxymethylcellulose-decomposing proteins of around 25, 50 and 100 kDa from three unnamed Indonesian endophytic fungi, isolated from medicinal plants (Yopi et al., 2014); and to that of Ncube et al. (2012), who reported molecular masses of 20-45 kDa for cellulases isolated from Aspergillus niger when Jatropha curcas seed cake was substrate (Ncube et al., 2012). Furthermore, Nayebyazdi et al. (2012) reported a range of cellulolytic proteins of the molecular weight size 25-50 kDa in Trichoderma reesei and Phanerochaete spp. (Nayebyazdi et al., 2012). Overall, other studies that have been undertaken and reviewed independently, also have reported the molecular masses of fungal cellulases to be as low as 12 kDa and up to 250 kDa (Kuhad et al., 2011; Li et al., 2011; Liming and Xueliang, 2004; Ritter et al., 2013; Vivekanandan et al., 2014; Zhang and Zhang, 2013). In  addition,   our   work  also  revealed  numerous birchwood xylan-hydrolyzing proteins of the molecular size range of ~20-40 kDa (Zirbes and Waldvogel, 2018) (Figure 4c), relating closely with findings of the other previously undertaken studies. For instance, some xylanolytic proteins of the molecular weight size ranges of 20-50 kDa, 18-52 kDa, 29 kDa, 19 kDa, and 45-70 kDa were reported in endophytes (Polizeli et al., 2005), A. aculeatus (Fujimoto et al., 1995), Hypocrea lixii (Sakthiselvan et al., 2014), A. fumigatus (Silva et al., 1999), and Neocallimastix frontalis (de Segura and Fevre, 1993) respectively.
 
After determining that, when grown on water hyacinth, L. edodes mostly secretes cellulases and xylanases as its main protein components, the kinetic rates of these two highly produced lignocellulolytic protein components were then assessed and ascertained via the Hanes-Woolf plot method (Figure 5) (Irving et al., 2011; Meier et al., 2010; Sibanda et al., 2019a). This was done in order to relate activities of these two major L. edodes enzymes to their counterparts in other known organisms and/or related microbial systems. For the cellulases, the Km value of 0.247 mM and Vmax of 2 177.880 µmol/sec were obtained (Figure 5a). These obtained kinetic values are in close agreement with those previously shown by other closely related cellulases (Sibanda et al., 2019a) and the other various recombinant cellulases isolated from other different organisms such as termites, filamentous fungi and protists, whose Km values ranged from 2.0 to 14.7 mM and Vmax values ranging from 0.84 to 1 667.00 µmol/sec (Table 1).
 
For the xylanases, the Km value of 0.147 mM and Vmax of 1 208.330 µmol/sec were obtained (Figure 5b). Once more, these values do concur with the reaction kinetic rates of the other closely related xylanases (Sibanda et al., 2019a) and the other numerous recombinant and non-recombinant xylanases isolated from other different microorganisms such as termite fungal symbionts and bacteria, whose Km values ranged from 3.920 to 6.960 mM and Vmax values ranging from 256.000 to 7 407.000 µmol/sec (Table 2).
 
 
Apparently, when comparing the kinetic ratios (i.e., Vmax/Km) of cellulases to those of xylanases within as a single organism, it emerged from our work that the L. edodes cellulases had a relatively higher ratio than that of the xylanases (Figure 5). Arguably, this scenario is not unusual because previously, a recombinant protein from Clostridium thermocellum, CtCel5E, that had a dual function as a cellulase and xylanase, displayed a Km value of 2.1 mM and a Vmax of 1 564 µmol/sec for the cellulase and a Km value of 4.6 mM and a Vmax of 883.5 µmol/sec for the xylanase (Yuan et al., 2015). Notably, all the kinetic values of the CtCel5E together with most of the proteins in Tables 1 and 2 were generally lower than those of our own crude enzyme extract in this study, probably  due  to three possible technical reasons. Firstly, most of the proteins in Tables 1 and 2, including CtCel5E were recombinant while proteins in our own extract were not. Secondly, the source of some of the proteins, including CtCel5E was bacterial or prokaryotic whilst that of our own was fungal or eukaryotic, of which fungi are naturally known to be superior producers of lignocellulolytic enzymes (Favaro et al., 2013; Ramanjaneyulu et al., 2015). Lastly and in the event that substrate concentration was a limiting factor in the study, the cellulose content of most lignocellulosic substrates is always higher than that of hemicellulose, e.g., wheat straw, rice straw, switch grass and sugarcane bagasse - all have around 35% cellulose and at most 25% hemicellulose content (Chen, 2014; Koshy and Nambisan, 2012; Shawky et al., 2011).
 
Overall, comparing water hyacinth to its control substrate (liverseed grass), it is apparent that the control substrate was always performing better throughout the study. However, it is rather worth to note that the control substrate naturally has a higher biomass composition compared to the experimental substrate; that is, 30% cellulose, 50% hemicellulose, 20% lignin and 1% pectin for liverseed grass (Howard et al., 2013; Saito et al., 2003)and 20% cellulose, 33% hemicelluloses, 10% lignin, and 1% pectin for water hyacinth (Avci et al., 2013). In addition, water hyacinth has always been reported to possess a very high adsorption  capacity  that makes it capable of taking up numerous nutrients, toxic chemicals and metal substances, which perhaps may inhibit enzyme activity on its biomass (Idrees et al., 2013; Moyo and Mapira, 2012; Reddy and D’Angelo, 1990; Saha et al., 2014; Tham, 2012; Usha et al., 2014). However, even though the general production of mushrooms has always been undertaken using liverseed grass, rice or wheat straw, the overall performance of water hyacinth in this study as a substrate (0.75-0.98 folds) strongly proposes it as a probable alternate.
 
Finally, by collectively summing up all findings of this study, it is conceivable that the water hyacinth native to Zimbabwe can be viably utilized as a substrate of L. edodes, which if properly optimized, the approach can then be used as a sustainable and cost-effective way (Jia et al., 2019; Thakur, 2018) of managing this problematic and noxious weed in the country. More so, the possible effective utilization of this aquatic weed as a substrate of L. edodes can also be tailor-made towards the production of protein-rich mushrooms (Thakur, 2018) for local communities and a whole cocktail of the white rot lignocellulolytic enzymes (Wang et al., 2019) as well as specific fine chemicals (Zirbes and Waldvogel, 2018) for various applications in the industry and/or commercial systems. In addition, the degradation of a highly lignocellulosic biomass like water hyacinth by the white rot fungus L. edodes (Wang et al., 2019) produces various fermentable carbohydrates with numerous potential industrial applications such as bio-fuel, food, brewery and winery, animal feed, textile and laundry, pulp and paper and agriculture, which when properly optimized, may encourage communities to harvest this noxious aquatic weed and ameliorate its unabated growth and expansion. In this regard therefore, our study hereby strongly recommends for a further optimization of its findings so that L. edodes can be viably utilized for the sustainable and cost-effective management of water hyacinth in Zimbabwe (and even in other tropical and sub-tropical countries, where the weed is endemic).


 CONFLICT OF INTERESTS

The authors declare no competing interests.


 ACKNOWLEDGMENTS

The project was funded by the National Research Foundation (NRF) of South Africa (Grant Number: CSUR93635) and Chinhoyi University of Technology, Zimbabwe. CM, CJZ, ABM and OR conceived the idea and designed the study; NS carried out the experiments; TBD, MMT and DTK assisted in the experimental work; SSM and LMKT contributed chemicals and consumable; OR funded the study, provided facilities for the study as well as supervising the experimental work. All authors contributed to the writing of the manuscript and approved the final version.



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