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Valorisation of Cassava Wastewater as Substrate for Trichoderma virens

Production, Bio-control Agent Cocoa Black Pod Disease

Article in British Journal of Applied Science & Technology · November 2018

DOI: 10.9734/CJAST/2018/45488

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Current Journal of Applied Science and Technology

30(5): 1-8, 2018; Article no.CJAST.45488

ISSN: 2457-1024
(Past name: British Journal of Applied Science & Technology, Past ISSN: 2231-0843,
NLM ID: 101664541)

Valorisation of Cassava Wastewater as Substrate for

Trichoderma virens Production, Bio-control Agent
Cocoa Black Pod Disease
Pakora Gilles Alex1*, Amari Ler-N’Ogn Dadé Georges Elisée2,
Abo Kouabenan3, Silue Nakpalo2, Coulibaly Anne Edwige1 and Kone Daouda2,4
1
Laboratoire de Pharmacodynamie Biochimique, UFR Biosciences, Université Félix Houphouët-
Boigny d’Abidjan (UFHB), 22 BP 582 Abidjan 22, Côte d’Ivoire.
2
Laboratoire de Physiologie Végétale, UFR Biosciences, Université Félix Houphouët-Boigny d’Abidjan
(UFHB), 22 BP 582 Abidjan 22, Côte d’Ivoire.
3
Institut National Polytechnique Félix Houphouët-Boigny (INP-HB), Département de Formation et de
Recherche Agriculture et Ressources Animales (DFR-ARA) Laboratoire de Phytopathologie et de
Biologie Végétale B.P. 1313 Yamoussoukro, Côte-d'Ivoire.
4
Centre d’Excellence Africain sur les Changements Climatiques, la biodiversité et l’Agriculture
durable, Université Félix Houphouët-Boigny, 22 BP 582 Abidjan 22, Côte d’Ivoire.

Authors’ contributions

This work was carried out in collaboration between all authors. All authors read and approved the final
manuscript.

Article Information

DOI: 10.9734/CJAST/2018/45488
Editor(s):
(1) Dr. Jakub Kostecki, Professor, Faculty of Civil Engineering, Architecture and Environmental Engineering, University of
Zielona Góra, Poland.
Reviewers:
(1) P. Saravanakumari, Rathnavel Subramaniam College of Arts and Science, India.
(2) Anabelle Camarotti de Lima Batista, Federal University of Paraíba, Brazil.
Complete Peer review History: http://www.sciencedomain.org/review-history/27428

Received 11 September 2018

Original Research Article Accepted 21 November 2018
Published 27 November 2018

ABSTRACT

Aim: In Côte d'Ivoire, several food products are made from cassava. However, this production
generates effluents such as wastewater responsible for soil, as well as water and air pollution.
Meanwhile, cassava wastewater can be used as an inexpensive substrate for the production of
biopesticides, such as Trichoderma virens, a bio-control agent for black pod disease. This could
address both the problems of cassava wastewater treatment and the use of conventional synthetic
substrates for biopesticide production.
_____________________________________________________________________________________________________

*Corresponding author: E-mail: [email protected];

Pakora et al.; CJAST, 30(5): 1-8, 2018; Article no.CJAST.45488

Methodology: The experiments described here were conducted using the cassava wastewater,
without any supplement, to produce spores of T. virens by liquid state fermentation, solid state
fermentation on kaolin grains. The production of gliovirin in cassava wastewater was monitored by
LC / MS analysis.
8
Results: The maximum production (≈ 1.7 x 10 spores / mL) was achieved at dilution 1/4 of the
cassava wastewater while at dilutions 1 and 1/2 the concentrations were 6.3 x 106 and 7.2 x 106
spores / mL, respectively. Spores concentration increased when cassava wastewater was highly
diluted. The cultures of T. virens on kaolin supplemented with the cassava wastewater recorded a
6
concentration of 1.13 x 10 spores / g of kaolin. The presence of gliovirin was detected by LC/MS
analysis of solid state fermentation of T. virens in cassava wastewater.

Keywords: Cassava wastewater; Trichoderma virens; fermentation; spore production; gliovirin.

1. INTRODUCTION aeruginosa [1]; and enzymes as cellulase by

Trichoderma asperellum [7]; or cellulase and
Cassava is one of the six main agricultural xylanase by Trichoderma reesei [8]. The present
products including wheat, rice, corn, potatoes work carried out on the soil under cocoa farm in
and oatmeal flour. It is a major source of dietary Côte d'Ivoire successfully isolated several strains
energy for more than 500 million people. This of Trichoderma sp, of which three (Trichoderma
tuber represents an industrial interest for its virens, Trichoderma asperellum and Trichoderma
starch richness [1]. In Côte d'Ivoire, the national harzianum) showed very high inhibitory powers
production of cassava is about 5 millions of tons against Phytophthora palmivora, the agent of
per year; its consumption comes above that of cocoa black pods disease [9]. Chemical studies
rice [2]. The cassava processing produces many implemented on these three strains helped to
highly valuable derivatives, the most important of purify and identify several active molecules,
which is a slightly acidic fermented semolina including gliovirin, a compound responsible for
called "Attiéké". This homemade food, generally the inhibition of the mycelial growth of several
produced by women or women's associations, is species of Phytophthora palmivora and
increasingly on demand around the world. Thus Phytophthora megakarya [10-11].
industries began to settle with aim to meet this
great need for "Attiéké". However, the production The main objective of this study is to promote the
of this food as well as and many other foods cassava wastewater, available in large amount,
obtained after several cycles of dewatering of by using it as an inexpensive raw material to the
fermented cassava paste, generates large preparation of culture medium for the mass
amounts of starch-riched acidic wastewater. This production of spores and active molecules of T.
untreated wastewater is directly discharged onto virens, the agent for the bio-control of cocoa
the processing sites, sometimes into black pod disease. To this regards, fermentations
watercourses, which causes unsanitary of this microorganism were performed to
conditions like emission of gas, pollution of water determine the potential of the CW as ingredient
and soil and, degradation of the immediate of microbiological culture medium. Thus, two
environment [3]. These wastewaters can be used approaches have been developped for spore
as raw materials for fermentation in order to production: (i) liquid-state fermentation and
massively produce spores and metabolites of (ii) solid-state fermentation on kaolin grains, and
microorganisms of agricultural and biotechnology one for gliovirin production (iii) solid-state
interest. Indeed, due to both constraints of low fermentation.
spore yield and very high costs of conventional
synthetic media used for the production of 2. MATERIALS AND METHODS
biopesticides [4], many studies were undertaken
on new residues as raw materials. Successful 2.1 Cassava Wastewater
use of sewage sludge compost was reported [5],
as well as wastewater from starch industries [4] The cassava wastewater was collected in
for bio-control agent production. Besides, Abidjan (South, Côte d'Ivoire) thanks to a group
biological waste and cassava wastewater were of women producers of "Attiéké". These women
tested as a substrate for the production of used to process an average of 200 kg of fresh
molecules as volatile compounds by Geotrichum cassava per week for 100 kg of Attiéké and for
fragrans [6]; or rhamnolipids by Pseudomonas the same amount of untreated wastewater

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Pakora et al.; CJAST, 30(5): 1-8, 2018;; Article no.CJAST.45488
no.

discharged on site. A volume of 5 L of this fresh 2.3 liquid-state Fermentation

cassava wastewater is collected, sent to the
laboratory and stored at 4°C to minimise any Cultures media were obtained from CW after
growth of microorganisms. The characteristics of dilution (1, 1/2 and 1/4) with distilled water. The
the CW are the following: pH 4.3 ± 0.1; ratio C:N pH of these media was adjusted to 5.6 ± 0.2
≈ 34.25
25 and the following metals (Table 1). before autoclave sterilisation. The fermentations
were carried out in Erlenmeyer flasks (1L)
Table 1. Metal characteristics of cassava containing 400 mL of culture
ture medium (Table 2).
wastewater After inoculation with 1 mL of the T. virens spore
suspension (105 spores / mL) obtained as
Parameter Concentration (µM)
describe above, the flasks were incubated in a
Cd 0.24
rotary shaker at 26°C ± 1 and 130 rpm for 120
Cr 0.07
hours. For each incubation, three samples (2 mL)
Mn 1.6
were withdraw aseptically after 72 hours and 120
Fe 40.1
hours of incubation and then stored at 4°C for
Ni 1.7
subsequent analyses.
Cu 2.9
Zn 20.9
Table 2. Method of dilution of cassava
2.2 Fungal Material wastewater

T. virens strain (Fig. 1) was provided by the Dilution Water Added volume (mL)
National Center for Agricultural Research 1 CW 400
(CNRA) in Ivory Coast [10].. It was cultured on SDW 0
agar of potato dextrose (PDA) in a 10 mL screw 1/2 CW 200
tubes for a period of 7 days. By the end of which, SDW 200
the tubes containing the culture of T. virens were 1/4 CW 100
stored at 4°C. The spore suspension was SDW 300
prepared as follows. On a 7-daysdays-old mycelial CW: cassava wastewater; SDW: sterilised distilled
cultures, 10 mL of sterilised distilled water were water
added with a sterile pipet. The surface of these
cultures was scraped using a sterilised metal 2.4 Solid-state Fermentation from Kaolin
spatula. The solution obtained was homogenised
homogen Grains
in a test tube using a vortex to release spores in The kaolin bought on the market in Abidjan was
the water. This suspension was then filtered with pulverised with a grinder and sieved. A quantity
muslin to remove the mycelial fragments. A of 500 g of the powder obtained was mixed
spore suspension of 200 µL was taken and put in homogeneously with a mixer in 500 mL of the
the wells of a Malassez hematimeter followed by CW at pH 5.6 ± 0.2. Grains were made from the
a count of spores. This number was adjusted to paste obtained. These kaolin grains were placed
5
10 spores / mL per dilution with sterilised in a container for 24 hours at room temperature
distilled water. to be dried. At the end of 24 hours, the kaolin
grains are placed in petri dishes (50 g per petri
dish) and then sterilised. These petri dishes are
inoculated
ted 4 mL of a spore suspension of
T. virens at a concentration of 105 spores / mL.
This spore suspension was prepared in sterile
manioc waste water diluted 1/2. The dishes were
subsequently incubated at 26°C ± 1 for 10 days.
The tests were repeated 3 times.

2.5 Enumeration of Spores

Spore suspensions from the liquid state were
obtained after filtering the culture medium with
muslin so as to remove the mycelial fragments
and spore concentrations were determinated
Fig. 1. Trichoderma virens cultured after 7 according to the method described in 2.2. In the
days on PDA cases of solid state fermentation, spore

3
Pakora et al.; CJAST, 30(5): 1-8, 2018; Article no.CJAST.45488

suspensions were obtained by homogenising a this last dilution increased with the incubation
granule in 1 mL of distilled water. The time (Table 2) in contrast to broths fermented at
concentration of spores was estimated by the 1 and 1/2 dilution in which the number of
determining the average mass of a kaolin grain spores did not vary. The rapid growth observed
and given spores / g of kaolin. Three repetitions could be due to the fact that CW contains a
were made to perform the count. source of carbon, easily metabolised by this
fungus and a metal content necessary for its
2.6 Solid State Fermentation for Gliovirin growth. Several parameters can influence the
Production and LC/MS Analysis growth and spore production capacity of
microorganisms in a broth. But the most
Three petri dishes (9 cm) containing 20 mL of important are the C: N ratio, the source of the
sterilised solid culture medium prepared from carbon source and the glucose concentration
CW (20 g of agar per 1 liter of cassava [12]. CW ratio C:N of 34.25 could be responsible
wastewater) were inoculated with 500 μL of 6
for conidiation ≥ 10 spores / mL. Indeed, studies
spore suspension of T. virens at the on the variation of the C:N ratio made it possible
4
concentration of 10 spores / mL. Petri dishes to observe the decrease in the spore
were incubated at 26°C ± 1 in the dark for 10 concentration with the decrease in the C:N ratio.
days. Controls for the production of gliovirin by In these studies, the ratios at 16 and 21 yielded
T. virens were carried out on the PDA (20 mL per 6 8
concentrations of ≈10 and ≈10 spores / mL,
petri dish) inoculated and incubated as respectively. High levels of spores may be due to
previously described. The tests were made in cellulose, which is the main source of carbon in
triplicate. After 10 days of incubation, cultures cassava wastewater. These results are in
were extracted with ethyl acetate and the agreement with those of [13] which observed an
extracts obtained were dissolved in methanol to increase in the number of spores when
perform U-High Performance Liquid Trichoderma viride was grown on a substrate of
Chromatography (U-HPLC) coupled with mass cellulosic origin. The 1/2 dilution of CW did not
spectrometry. Gliovirin (1 mg/mL) isolated from a give a significant effect on T. virens spore
culture of T. virens [11] was also analysed by production, probably because of the high glucose
LC/MS. Analysis was performed on an MAXIS II concentration in the medium despite dilution.
ETD © using a C18 column (Acclaim polar Glucose is easily used by T. virens as a carbon
Advantage II PA2, ThermoScientific) equilibrated source. An increase in glucose in the culture
at 40°C. The mobile phase consisting of medium can not only delay, but also decrease
CH3CN:H2O (acidified with 0.1% formic acid) was the conidiation due to high vegetative growth [4].
used, starting at 5:95 for 2 minutes then In contrast, a low glucose concentration could
increasing to 50: for 7 minutes, then at 90:10 in favor the production of spores. Thus, the fact that
the space of 6 minutes, holding for 2 minutes, the maximum concentration was reached at a
finally returning to the starting conditions in 4 dilution of 1/4 could be due to a low
minutes. For the mass spectrometry, the voltage concentration of glucose at this dilution.
was Es = 3500 V, the MS range: 100-1300 m/z
and the acquisition speed of 2 Hz. For the cultures on kaolin supplemented with the
CW, growth of T. virens was observed on all
3. RESULTS AND DISCUSSION grains (Fig. 2). The count of spores showed a
concentration of 1.13 x 106 spores / g of kaolin.
3.1 Spore Production by Liquid State This concentration was substantially the same as
Fermentation and Solid State that obtained with the fermentation in the liquid
Fermentation on Kaolin Grains state at dilutions 1 and 1/2.
Experiments were conducted in CW without any T. virens was grown directly on kaolin grains
supplement. Under the 3 conditions of culture supplemented with CW. In contrast to several
(dilution 1, 1/2 and 1/4), the growth and the studies in which kaolin was used in formulations
production of spores by T. virens were observed. as a carrier, the present study revealed that
The results showed that after 120h of incubation, kaolin could serve as an inorganic carrier for
the fermented broths at dilutions 1 and 1/2 spore production [14-15]. For growth on kaolin
produced substantially the same concentration of grains, T. virens metabolised the carbon source
6
approximately 10 spores / mL while a maximum of CW and used the kaolin minerals. In addition
concentration of approximately 1.73 x 108 spores to being a support for the formulation, kaolin
/ mL had been reached in the fermented broth at could constitute a growth support for
1/4 dilution. In addition, the number of spores at biopesticides.

4
Pakora et al.; CJAST, 30(5): 1-8, 2018;; Article no.CJAST.45488
no.

Table 3. Spore concentration as a function of time and different dilutions of cassava

wastewater

Dilution of cassava Number of spores (spores / mL)

wastewater 72 h 120 h
1 1.8 x 106 ± 0.05 6.3 x 106 ± 1.09
6 6
1/2 2.8 x 10 ± 0.11 7.2 x 10 ± 0.87
6
1/4 1.6 x 10 ± 0.11 1.73 x 108 ± 0.24
± SD Three repetitions were made to perform the count

Fig. 2. Aspect of growth of T. virens on kaolin grains supplemented with cassava wastewater

3.2 Solid State Fermentation for Gliovirin and the production of gliovirin by T. virens on
Production and LC/MS Analysis PDA (Fig. 4B). ). However, the peak intensity of
gliovirin on CW agar medium (Fig (Fig. 4A) was not
Growth of Trichoderma virens was observed on as high as that of gliovirin on PDA medium. The
both PDA (Fig. 3A)) and CW agar medium (Fig. (Fig production of secondary metabolites by fungi is
3B). LC/MS analysis of these extracts revealed influenced by a set of stimuli among which, the
the production of gliovirin by T. virens grown on source of carbon and, nitrogen gen [16]. As a
the CW agar. This production was demonstrated diketopiperazin [17],, gliovirin needs a source of
by the presence of the molecular ion peak m/z nitrogen from amino acids. Yet, CWs are very
+
([M+H] 481.073) that corresponds to gliovirin in poor in amino acids [18].. This could explain the
comparison to the control of gliovirin (Fig.
(Fig 4C) low intensity of gliovirin in cassava wastewater.

A B

Fig. 3. Trichoderma virens cultured after 10 days on PDA (A)and on CW agar medium (B)

5
Pakora et al.; CJAST, 30(5): 1-8, 2018; Article no.CJAST.45488

Intens.
x105 A

2.0

1.5

1.0

0.5

0.0
0 2 4 6 8 10 12 14 16 18 Time [min]
20180515_GILLES_GP_LES_50_1_RA1_01_12922.d: EIC 481.0730±0.005 +All MS
20180515_GILLES_GP_LES_50_2_RA2_01_12924.d: EIC 481.0730±0.005 +All MS
20180515_GILLES_GP_LES_50_3_RA3_01_12926.d: EIC 481.0730±0.005 +All MS

Intens.
x106 B

0
0 2 4 6 8 10 12 14 16 18 Time [min]
20180515_GILLES_GP_PDA_1_RB2_01_12940.d: EIC 481.0730±0.005 +All MS
20180515_GILLES_GP_PDA_2_RB3_01_12942.d: EIC 481.0730±0.005 +All MS
20180515_GILLES_GP_PDA_3_RB4_01_12944.d: EIC 481.0730±0.005 +All MS

6
Pakora et al.; CJAST, 30(5): 1-8, 2018; Article no.CJAST.45488

Intens. 20180515_GILLES_GP_GLIO_RC2_01_12956.d: +MS, 9.7min #1158

x106 1+
481.0734
C

2.5

2.0

1.5

1.0

1+
231.0143
1+
0.5 961.1399

0.0
200 400 600 800 1000 1200 m/z

Fig. 4. Spectra showing gliovirin Extraction Ion Chromatogram (EIC) from CW agar medium
(A), from PDA (B) and control gliovirin mass spectrum (C)

4. CONCLUSION wastewater as a substrate for the

simultaneous production of rhamnolipids
8
In this study the maximum concentration (≈10 and polyhydroxyalkanoates by
spores / mL) indicated that cassava wastewater Pseudomonas aeruginosa. J Ind Microbiol
could be directly used for T. virens conidia Biotechnol. 2009;36(8):1063-72.
production of in formulations. This work showed 2. EU. Cassava value chain in Ivory Coast.
that wastewater could be recycled by using it as Chain value analysis for development;
a very low cost substrate for cocoa black pods 2018.
disease biocontrol agent production. 3. Ohimain EI. Environmental impacts of
smallholder ethanol production from
ACKNOWLEDGEMENT cassava feedstock for the replacement of
kerosene household cooking fuel in
We acknowledge Fond Compétitif pour Nigeria. Energy Sources, Part A:
l’innovation Agricole Durable (FCIAD, Côte Recovery, Utilization, and Environmental
d’Ivoire) for providing funding, the women Effects. 2013;35(16):1560-5.
association for providing cassava wastewater, 4. Verma M, Brar SK, Tyagi RD, Surampalli
UMR 7245 MNH/CNRS for making LC/MS RY, Valéro JR. Starch industry wastewater
analysis facilities available. We particilarly thank as a substrate for antagonist, Trichoderma
INRA, Versailles, Paris, France. viride production. Bioresource Technology.
2007;98(11):2154-62.
COMPETING INTERESTS 5. Cotxarrera L, Trillas-Gay MI, Steinberg C,
Alabouvette C. Use of sewage sludge
Authors have declared that no competing compost and Trichoderma asperellum
interests exist. isolates to suppress Fusarium wilt of
tomato. Soil Biology and Biochemistry.
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© 2018 Pakora et al.; This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly cited.

Peer-review history:
The peer review history for this paper can be accessed here:
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