Environment and Natural Resources Journal 2018; 16(1): 39-57

Heavy Metal Tolerance and Removal Capacity of Trichoderma species

Isolated from Mine Tailings in Itogon, Benguet
Myra Tansengco*, Judith Tejano, Fe Coronado, Carmel Gacho and
Joven Barcelo
Environment and Biotechnology Division, Industrial Technology Development Institute, Department of Science and Technology,
Bicutan, Taguig, Metro Manila, Philippines

ARTICLE INFO ABSTRACT

Received: 6 Sep 2017 Waste from mining industries contains various heavy metals that can pollute the
Received in revised: environment. Bioremediation using efficient fungi can help in eliminating these
27 Oct 2017 heavy metal contaminants. This study focused on the isolation, identification, and
Accepted: 7 Nov 2017 characterization of heavy metal-resistant fungi from mine tailings in Itogon,
Published online: Benguet. Isolation of fungi was done by serial dilution and spread plate techniques
27 Nov 2017 on potato dextrose agar (PDA) with an individual heavy metal, i.e. chromium (Cr),
DOI: 10.14456/ennrj.2018.5 copper (Cu), lead (Pb), zinc (Zn), and nickel (Ni). Of the 29 fungal isolates, four
species were selected and molecularly identified as Trichoderma virens, T.
Keywords: harzianum, T. saturnisporum, and T. gamsii. Growth tolerance on PDA with
Heavy metal/ Fungi/ increasing concentrations (200-1000 ppm) of an individual heavy metal indicated
Trichoderma/ Itogon/ the following trend: T. virens > T. harzianum > T. gamsii > T. saturnisporum.
Benguet/ Mining/ Mine Growth test indicates that all Trichoderma isolates can tolerate high levels of Cr
tailings and Pb, however tolerance to Cu, Zn, and Ni was species specific. Shakeflask
culture using T. virens showed high lead removal (91-96%) over broad pH range
* Corresponding author: while and at neutral pH, T. virens had 70% and 63% reductions for Cu and Cr,
E-mail: respectively. Results of this study highlights the potential of Trichoderma isolates
[email protected] for biological wastewater treatment in mining industries.

1. INTRODUCTION environment (Anahid et al., 2011; Dixit et al., 2015).

Mining activities contribute to various It is important to examine the mechanism involved
environmental problems including deforestation, loss in controlling the growth and activity of microbes in
of biodiversity, and contamination of soil and water polluted area, their metabolism process, and how
bodies. In the Philippines, the law that governs all they respond to environmental changes. Micro-
mining operations including measures for envi- organisms tend to tolerate heavy metal-contaminated
ronmental protection, is indicated in the Philippine environments through various mechanisms such as
Mining Act of 1995 (DENR, 1995). Despite the biosorption, bioaccumulation, biotransformation, and
promulgation of this law, several cases of envi- biomineralization (Ayangbenro and Babalola, 2017).
ronmental damage caused by big mining firms have Fungi are efficient biological agents for
been reported. Some problems in the implementation remediating pollutants for their capability to produce
of the law and in enforcement of regulations are the various extracellular enzymes, less sensitivity to
lack of government personnel with technical expertise variations in nutrients, aeration, pH, and tempera-
and limited budget for monitoring mining activities ture, and for being appropriate for large-scale
(Raymundo, 2014). production (Mustapha and Halimoon, 2015).
Bioremediation using microorganisms has Moreover, filamentous fungi are more preferred for
been considered as an innovative, safe, eco-friendly bioremediation because of their capacity to remove
and cost-effective method for environmental clean- concentrated heavy metals from liquid substrates
up (Ayangbenro and Babalola, 2017; Dixit et al., (Siddiquee et al., 2015). The proposed mechanism
2015; Siddiquee et al., 2015). Bioremediation for heavy metal tolerance of fungi can be extra-
approaches for toxic inorganic compounds such as cellular sequestration to avoid metal entry, or
heavy metals depend on the active metabolizing intracellular physical sequestration to reduce the
capacities of microorganisms, type of metals, and metal burden in the cytosol (Anahid et al., 2011).
40 Tansengco M et al. / Environment and Natural Resources Journal 2018; 16(1): 39-57

Extracellular sequestration can be done through Wastewater samples were collected from six
chelation wherein organic molecules, not part of the mine tailing sites in Itogon, Benguet (Figure 1).
cell wall, are excreted by the fungi to chelate the Identification of collection sites and actual sampling
metal ions. Another mode of extracellular system is were in coordination with Benguet Corp., and DOST-
through biosorption or cell-wall binding due to the CAR (Cordillera Administrative Region). The
presence of anionic structures in the cell surface sampling sites were located in four barangays in
(Bellion et al., 2006; Maghsoodi et al., 2007). Itogon; namely Virac, Ucab, Poblacion, and Loacan.
Intracellular sequestration may involve metal The pH of water samples was determined onsite using
transport proteins by extruding toxic metal ions out portable pH meter (Lovibond, IQ140 IQ Scientific
of the cell or by allowing metal sequestration into Instruments, USA). Water samples were collected and
vacuolar components (Bellion et al., 2006; Abdul et preserved according to standard method (APHA,
al., 2007). 1992). Heavy metal concentration in water samples
Incubation temperature and pH are important was determined using a flame atomic absorption
factors to consider in microbe-based heavy metal spectrophotometer (PerkinElmer, PinAAcle 500,
removal. The pH value of an aqueous medium affects USA). Metal concentrations in water samples were
microbial surface charge and the degree of ionization compared with the Philippine effluent standards
of its functional groups (Galli et al., 2003). Lower pH (DAO, 2016).
results to protonation of the binding sites, which
reduces or inhibits the binding of metal ions (Kapoor 2.2 Isolation and screening of heavy metal-
et al., 1999). Microbial species have an optimum tolerant fungi
temperature for growth and therefore incubation Isolation of fungi was done by serial dilution
temperature affects microbial biomass yield and and spread plate techniques (Seeley et al., 1991).
heavy metal removal capacity (Gadd, 1990; Chen and Potato dextrose agar (PDA) (HIMEDIA Lab. Pvt.
Yiacaumi, 1997; Ali and Hashem, 2007). Ltd., India) media with 20 ppm of different heavy
Potential heavy metal-tolerant microorganisms metals (Cr, Cu, Pb, Ni, Zn) were used for the
can be isolated from contaminated sites. There are isolation of fungi by serial dilution technique. Heavy
several abandoned mine sites in Itogon, Benguet and metal salts of potassium dichromate (K2Cr2O7),
remediation of existing mine tailings is a major copper (II) sulphate (CuSO4·H2O), lead (II) nitrate
challenge to the city government. Mine tailings (Pb(NO3)2), nickel sulphate (NiSO4), and zinc
contain various heavy metals and other toxic sulphate (ZnSO4·7H2O) were utilized as source of
pollutants that can damage the environment and cause heavy metals. PDA alone was used to determine the
serious health problems. At present, there are no total fungal population. Serially diluted samples
reported studies on the isolation of potential fungal were inoculated on PDA with and without heavy
species from mine tailings in Itogon, Benguet. This metals by spread plate method. Plates were
study focused on the isolation, identification, and incubated at 30oC and colonies that grow on agar
characterization of indigenous heavy metal-resistant plates were counted after four days. Morphologically
fungi from the premier mining town in Itogon, different colonies were sub-cultured and purified by
Benguet. The effects of temperature and pH on the streak plate technique (Seeley et al., 1991).
growth of fungal isolates were determined. Heavy metal-tolerant fungal isolates were
Moreover, the removal efficiency of fungal isolates in selected based on their growth on PDA plates with
various heavy metals were examined and compared at mixture of five different heavy metals at 20 ppm
different initial pH. The isolated heavy metal-tolerant each. Agar disk from each pure fungal isolate was
fungi can be useful for wastewater treatment in inoculated on PDA with the heavy metal mixture and
mining industries and in biological remediation of incubated at 30oC. Mycelial radial growth was
other contaminated sites. recorded after four days of incubation using a digital
caliper (METROLOGY, Jingstone, Taiwan). Means
2. METHODOLOGY of radial growth were computed from triplicate agar
2.1 Collection and characterization of water plates.
samples
 Tansengco M et al. / Environment and Natural Resources Journal 2018; 16(1): 39-57 41

(a) (b) (c)

Figure 1. Map showing the collection sites of water samples from mine tailings in Itogon, Benguet. (a) Map of the
Philippines with Benguet highlighted, (b) Map of Benguet with Itogon highlighted, and (c) Collection sites in Itogon,
Benguet

2.3 Molecular identification of selected fungal extension of 68oC for 3 min. The PCR products were
isolates resolved in 1.2% agarose gel (Vivantis, USA). After
Genomic DNA sequencing of selected fungal electrophoresis, PCR fragments were excised and
isolates was done at the Philippine Genome Center, purified using Zymoclean Gel DNA Recovery Kit
University of the Philippines, Diliman, Quezon City. (Zymo Research, USA). Purified amplicons were
Pure fungal cultures that were grown on PDA plates sequenced on ABI 3730xl DNA Analyzer (Applied
for four days served as DNA isolation source. Biosystem, Hitachi) using primers ITS1 and ITS4.
Fungal mycelia (100 mg wet weight) from each DNA sequences were aligned using Clustal W
culture were scraped and used for DNA extraction Multiple Alignment application in the BioEdit
following the protocol of ZR Fungal/Bacterial DNA Sequence Alignment Editor ver7.2.5 (Hall, 1999).
MiniPrep (Zymo Research, USA). The isolated The sequences were compared to the GenBank
genomic DNA was used as a template for PCR database using the nucleotide BLAST program
(polymerase chain reaction) amplification of the (Altschul et al., 1990).
ITS1 - 5.8S - ITS2 (internal transcribed spacer 1,
5.8S, and internal transcribed spacer 2) region of the 2.4 Characterization of fungal isolates
ribosomal RNA gene (Asis and Siddiquee, 2016). 2.4.1 Growth on potato dextrose agar (PDA)
Universal fungal primers used were ITS1 (5’- Selected fungal isolates were cultured on PDA
TCTGTAGGTGAACCTGCGG-3’) and ITS4 (5’- medium for morphological characterization. Agar
TCCTCCGCTTATTGATATGC-3’) as forward and disk from each isolate was placed at the center of
reverse primers, respectively (White et al., 1990). PDA and cultures were incubated at 30oC. Mycelial
Primers were synthesized by Integrated DNA growth and sporulation were monitored and recorded
Technologies (Singapore) through a local agent. from triplicate plates.
Amplification was done on a thermal cycler (BioRad
T100, USA) with an initial denaturation step of 95oC 2.4.2 Effect of pH and temperature
for 1 min, followed by 30 cycles of 95oC for 30 sec, PDA medium was prepared with varying pH
60oC for 30 sec, and 68oC for 1 min, and a final levels, i.e. 5, 6, 7, 8, and 9. The pH level of each
42 Tansengco M et al. / Environment and Natural Resources Journal 2018; 16(1): 39-57

medium was adjusted with 1 M NaOH solution and isolate. Potato dextrose broth (HIMEDIA Lab. Pvt.
pH was checked by a benchtop pH meter (Orion 3 Ltd., India) was prepared based on manufacturer’s
Star, Thermo Electron Corp., USA). Agar media instructions. Stock solution of each heavy metal was
were autoclaved at 121oC, 15 psi for 15 minutes. added to the medium to obtain 100 ppm
Sterile PDA media were plated on 9-cm diameter concentration in a volume of 50 mL. The pH of
glass petri dishes. Agar disk from each fungal isolate culture medium was adjusted to 5, 6, 7, 8, and 9 by 1
was inoculated at the center of PDA plate and M NaOH solution. Agar disks (4 mm diameter, 3
cultures were incubated at 30oC. Mycelial radial pcs) from selected fungal culture were inoculated
growth was daily recorded for up to 7 days from into each heavy metal-containing medium
triplicate agar plates. (Zapotoczny et al., 2006). The cultures were
For examining fungal growth at varying cultivated on a benchtop shaker (Lab Companion
incubation temperatures, PDA medium was SK-600) at room temperature with rotation speed of
autoclaved and inoculated as mentioned above. 150 revolutions per minute (rpm) (Ali and Hashem,
Inoculated agar plates were incubated at different 2007). After 7 days, fungal biomass was filtered
temperatures, i.e. 25oC, 30oC, and 35oC. Mycelial (Whatman No. 42) and washed with deionized water
radial growth was recorded from triplicate agar plates. to remove growth medium (Mohsenzadeh and
Shahrokhi, 2014; Nongmaithem et al., 2016). The
2.5 Growth tolerance to increasing levels of heavy harvested biomass was oven-dried at 105oC for 16 h
metal and dry weight was obtained using analytical
2.5.1 Tolerance to mixed heavy metals balance (Zhimadzu Corp., Japan).
Agar disk from each fungal isolate was After harvesting the fungal biomass from
inoculated on PDA with heavy metal mixture (Cr, culture broth, the filtrate was used to determine
Cu, Pb, Ni, and Zn). The same heavy metal salts as residual heavy metals based on standard method
mentioned above were used in this experiment. (ASTM, 1993). For digestion, 25 mL of sample was
Different concentrations, i.e. 20, 40, 60, 80, and 100 placed in 100-mL capacity beaker and 5 mL
ppm, of mixed heavy metals were prepared in concentrated HCl was added. The beakers were
triplicate agar plates. PDA without heavy metal covered with watch glass and placed in hotplate at
served as the control. Inoculated plates were 100oC until the volume was reduced to about 3 - 5
incubated at 30oC and mycelial radial growth was mL. Digested samples were transferred to 50-mL
measured after 3 days and 7 days of incubation. capacity volumetric flask and diluted to 50 mL with
deionized water. Samples were then filtered
2.5.2 Tolerance to individual heavy metals (Whatman No. 42) and filtrates were analyzed for
Growth tolerance of selected fungal isolates to heavy metals using flame atomic absorption
higher concentrations of individual heavy metals was spectrophotometer (PerkinElmer, PinAAcle 500,
also determined on PDA plates. The heavy metals USA). Heavy metal content in the culture media was
tested were Cr, Cu, Pb, Ni, and Zn and compared from the initial concentration before
concentrations used were 200, 400, 600, 800, and fungal inoculation. Percent heavy metal removal was
1000 ppm. PDA without heavy metal served as the computed from triplicate samples (Shroff and
control. Inoculation was done as mentioned above Vaidya, 2011). Means and standard deviations were
with triplicate agar plates for each concentration. used in data comparison.
Cultures were incubated at 30oC and radial growth
was measured after 3 days and 7 days of incubation. 3. RESULTS AND DISCUSSION
3.1 Characteristics of water samples from mine
2.6 Batch heavy metal removal capacity by tailings
selected fungal isolate The characteristics of wastewater samples
The efficiency of selected fungal isolate to collected from mine tailings in Itogon, Benguet are
remove heavy metals in liquid solution was shown in Table 1. Water samples were light brown
examined by shakeflask culture. Heavy metal salts of to dark brown in color. Two water samples collected
K2Cr2O7, CuSO4·H2O, Pb(NO3)2, NiSO4, and from Balatoc, Virac (sample 1) and Ambalanga
ZnSO4·7H2O were tested separately in the test fungal River, Ucab (sample 2) showed lower pH value (6.3)
 Tansengco M et al. / Environment and Natural Resources Journal 2018; 16(1): 39-57 43

as compared to standard effluent pH level for class C tailing sites in Itogon, Benguet were contaminated
water (6.5 - 9). Water samples collected from sites 3 by various heavy metals with concentrations beyond
to 6 were nearly neutral. For heavy metal the standard effluent limit and this may cause serious
concentration, all samples exceeded the standard environmental and health problems. As experienced
allowable effluent levels for Cr, Cu, and Pb. Sample by the Benguet indigenous people, large-scale
from Balatoc, Brgy. Virac (site 1) also contains high mining pollutes and distracts agricultural economies
level of Zn. Ni concentrations in all sampling sites as well as displaces local communities and villages
are within the standard allowable limit. Result of (Cordillera Peoples Alliance, 2007).
heavy metal analysis indicates that the existing mine

Table 1. Characteristics of wastewater from mine tailings in Itogon, Benguet

Collection sitesa
Parameters Standardb
1 2 3 4 5 6
pH 6.26 6.33 6.65 6.78 6.96 6.84 6.5 - 9
Chromium, hexavalent
0.53 0.39 0.63 0.34 0.45 0.45 0.01
(Cr+6), mg/L
Copper (Cu), mg/L 1.37 0.24 0.62 0.56 2.70 0.38 0.02
Lead (Pb), mg/L 4.90 0.21 0.69 0.77 0.23 0.27 0.05
Nickel (Ni), mg/L 0.15 0.06 0.13 0.10 0.11 0.10 0.2
Zinc (Zn), mg/L 4.79 0.17 0.89 0.84 0.27 0.17 2.0
aCollection
sites: 1-Balatoc, Virac; 2-Ambalanga River, Ucab; 3-Tailing storage facility (TSF) 1, Ucab; 4-Convergence Ambalanga-
Acupan River, Poblacion; 5-TSF 2, Poblacion; and 6-Antamok 440, Loacan
bDENR Administrative Order 2016-08 for Class C water (Inland water) (DAO, 2016)

3.2 Isolation and screening of heavy metal- isolation source would be: Balatoc, Virac (site 1) >
resistant fungi Antamok 440, Loacan (site 6) > TSF2, Poblacion
Different fungi were observed on PDA plates (site 5) > Convergence Ambalanga-Acupan River,
with and without heavy metal supplementation. The Poblacion (site 4) > TSF1, Ucab (site 3) >
fungal population (Figure 2) on PDA medium alone Ambalanga River, Ucab (site 2). Based from water
range from 3.0 x 101 to 9.5 x 103 spores/mL. On analyses (Table 1), site 1 mostly contains the highest
PDA plates with heavy metal, high population of load of heavy metals compared to the other
chromium-tolerant fungi (1.1 x 103 to 5.4 x 105 collection sites. Various microorganisms might have
spores/mL) was observed in all water samples from adapted to this heavily polluted site thus several
mine tailings, with population exceeding those morphologically different fungi were isolated from
grown on PDA alone. A number of fungi also grew this site.
on PDA plates with Cu (1.5 x 101 to 4.0 x 103 Pure fungal isolates were further screened for
spores/mL), Pb (2.0 x 101 to 2.5 x 103 spores/mL), Ni their growth tolerance on medium containing a
(2.0 x 101 to 9.5 x 103 spores/mL), and Zn (1.0 x 101 mixture of five heavy metals (20 ppm each of Cu,
to 6.0 x 103 spores/mL). Highest population of heavy Cr, Ni, Pb, and Zn). As shown in Figure 3, about
metal-tolerant fungi was observed in water samples 50% of the fungal isolates grown on PDA-mixed
from Balatoc, Brgy. Virac (site 1) with 5.4 x 105 heavy metals had 10-20 mm mycelial radial growth
spores/mL for Cr, 4.0 x 103 spores/mL for Cu, 2.5 x while eight isolates had <10 mm radial growth. Only
103 spores/mL for Pb, 9.5 x 103 spores/mL for Ni, four species (isolate nos. 2, 3, 17 and 27) showed
and 6.0 x 103 spores/mL for Zn. full mycelial colonization after four days on PDA
A total of 29 fungal species were isolated with mixed heavy metals (Figure 3). Fungal isolate
from different heavy metal-containing plates. The nos. 2, 3 and 27 were obtained from the most
majority (14 species) of the isolated fungi came from polluted site (Balatoc, Virac). Isolate no. 17 came
the mine tailing in Balatoc, Brgy. Virac (site 1). In from site 5 (Tailing Storage Facility 2), which had
terms of number of fungal isolates, the order of the highest concentration of Cu among the studied
44 Tansengco M et al. / Environment and Natural Resources Journal 2018; 16(1): 39-57

areas. These four isolates were selected for the metals (McGrath et al., 2001). Several fungal
succeeding experiments and designated as fungal isolates, such as Trichoderma species, Aspergillus
isolates F1 to F4. Selection of microorganisms that niger, and Penicillium sp., have been reported that
can tolerate multiple metals is advantageous in can tolerate more than one type of metal (Kapoor et
bioremediation studies because in actual condition, al., 1999; Kredics et al., 2001; Errasquin and
soil or water samples are contaminated with different Vasquez, 2003; Ahmad et al., 2006).

1000000
PDA
PDA-Cr
Fungal population (spores/mL)

100000 PDA-Cu
PDA-Pb
PDA-Ni
10000
PDA-Zn

1000

100

10

1
1 2 3 4 5 6
Mine tailing sites
Figure 2. Fungal population on agar plates with and without heavy metal. Data were taken after four days incubation
(30oC+1) on potato dextrose agar (PDA, pH 5.1+0.2) with 20 ppm of particular heavy metal. Control, PDA alone.

50

45
Mycelial radial growth (mm)

40

35

30

25

20

15

10

0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
Fungal isolates (code number)

Figure 3. Mycelial growth of fungal isolates after four days on agar plates (Potato dextrose agar, pH 5.1+0.2) with mixed
heavy metals. Data are means+SD from triplicate determinations. PDA-mixed heavy metal plates contain 20 ppm each of
Cu, Cr, Ni, Pb, and Zn and incubated at 30oC+1.
 Tansengco M et al. / Environment and Natural Resources Journal 2018; 16(1): 39-57 45

3.3 Molecular identification of selected fungal biocontrol agent (Harman et al., 2004a; Harman,
isolates 2006; Contreras-Cornejo et al., 2009). Trichoderma
PCR amplification of the genomic DNA of species have been used in bioremediation of soil and
four selected fungal isolates using ITS1 and ITS4 water pollutants such as heavy metals, xenobiotics,
primers produced fragments between 489 to 570 bp toxins and other environmental contaminants
containing the partial sequence of the internal (Harman, 2006; Siddiquee et al., 2013; Tripathi et
transcribed spacer 1, complete sequence of the 5.8S al., 2013; Siddiquee et al., 2015; Nongmaithem et
ribosomal RNA gene, and the partial sequence of the al., 2016). Studies indicate that the genus
internal transcribed spacer 2. Comparison of Trichoderma has degradative enzymes and performs
sequences in GenBank via nucleotide BLAST search other bioremediation strategies to tolerate, detoxify
showed that the fungal isolates all belong to the or degrade heavy metals and other environmental
genus Trichoderma (Table 2). Sequences of isolates pollutants (Kredics et al., 2001; Harman et al.,
F1 and F4 showed 100% identity with T. virens and 2004b; Ezzi and Lynch, 2005; Tripathi et al., 2013).
T. gamsii, respectively. Sequences of isolates F2 and Bioaccumulation mechanism to single or multiple
F3 showed 99% identity with T. harzianum and T. metals was observed in T. atroviride (Errasquin and
saturnisporum, respectively. The obtained sequences Vasquez, 2003) and T. viride (Anand et al., 2006).
for the ITS1 - 5.8S - ITS2 fragment of the four Both bioaccumulation and biosorption capabilities
isolates were submitted to GenBank and were for Cu(II) was reported in the viable and non-viable
assigned accession numbers KY010354, KX999713, cells of Trichoderma isolate SP2F1 (Ting and
KY010355, and KY010356 for fungal isolates F1, Choong, 2009). Bioaccumulation and biovolatili-
F2, F3, and F4, respectively. zation properties for pentavalent arsenic (Zeng et al.,
Trichoderma species are common group of 2010) and arsenite (Feng et al., 2015) were
fungi in the rhizosphere with various applications in investigated using T. asperellum. Moreover, high
agriculture, industry, health, and environment phytoextraction efficiency was reported in T.
(Mukherjee et al., 2013). Identification up to species atroviride F6-treated mustard plants grown in Cd
level is important in Trichoderma because some and Ni contaminated soils (Cao et al., 2008). The
beneficial traits are species specific, and also for application of Trichoderma to various environmental
safety of the users (Mukherjee et al., 2013). Various pollutants is highly dependent on its metabolic
Trichoderma species have been studied for their diversity and it is important to first identify the
antibiotic production, capacity to improve plant species well suited for a particular contaminated area
growth and development, and efficiency as a (Ting and Choong, 2009; Tripathi et al., 2013).

Table 2. Identity of selected fungal isolates and the assigned NCBI accession numbers of corresponding sequences

Isolate code Species identified % Identity GenBank-assigned accession number

F1 Trichoderma virens 100 KY010354
F2 Trichoderma harzianum 99 KX999713
F3 Trichoderma saturnisporum 99 KY010355
F4 Trichoderma gamsii 100 KY010356

3.4 Characterization of Trichoderma isolates day 3, these three Trichoderma species showed full
3.4.1 Growth on PDA plates colonization of PDA plates.
Of the four Trichoderma species, T. satur- Growth morphology of Trichoderma isolates
nisporum had the fastest growth on PDA plates. Full on PDA plates from days 2 to 6 is shown on Figure
mycelial colonization (mean radial growth = 46+0.0 4. T. virens, at 2nd day on agar plate, had white
mm) was observed in T. saturnisporum at second mycelia with green to yellow green sporulation at the
day of incubation. T. virens of the same age had an center. At 3rd day, green sporulation spreads from the
average radial growth of 35+0.0 mm, T. harzianum center of agar plate with white mycelia at the edges.
had 37+0.96 mm, and T. gamsii had 29+0.50 mm. At At 4th day, green sporulation covers the whole agar
46 Tansengco M et al. / Environment and Natural Resources Journal 2018; 16(1): 39-57

plate and at 5th and 6th days, dark green sporulation four strains of Trichoderma asperellum (PR10,
appeared denser at the center and near the edges. PR11, PR12, and 659-7) can grow at pH 4.5 to 8.5
Bottom of PDA plate at days 2 and 3 had wrinkled with fastest growth rate at pH 4.5 to 6.5. In the seven
lines and appeared brownish, especially at the center. Trichoderma isolates of Singh et al. (2014), highest
At day 4, bottom of PDA showed ring of green biomass production was observed in pH ranging
coloration near the edges and this ring turned dark from 5.5 to 7.5. These isolates were T. harzianum, T.
brown at days 5 to 6. T. harzianum, at 2nd day on viride, T. longibrachiatum, T. atroviride, T. koningii,
PDA medium, had profuse white mycelia covering and T. virens. Kredics et al. (2004) reported that T.
almost 2/3 of the plate. At 3rd day, plate was fully harzianum strains T66 and T334, T. aureoviride
colonized with white mycelia and had ring of light T122, and T. viride T114 and T228 can grow in a
green sporulation near the edges. After which, this wide pH range (2.0 to 6.0) with optimal growth at
ring turned yellow green to green with white pH 4.0. Rinu et al. (2014) isolated an endophytic,
particles while the center had white mycelia. Bottom psychrotolerant, plant growth promoting, and
of agar plate was plain cream with wrinkled lines antagonistic T. gamsii strain (NFCCI 2177), which
near the center. T. saturnisporum, at 2nd day on PDA, exhibited growth at pH between 2.0 and 13.5 with
had thin light yellow mycelia that covered the entire optimum growth at pH 4 to 6. Our results confirmed
plate. At 3rd day, whole plate was full of whitish and previous reports that T. harzianum, T. virens, and T.
light yellow sporulation which turned yellow green gamsii can grow well over a wide pH range (5.0 to
to green particles at 4th day. Sporulation was green to 9.0). However, published studies on T.
dark green at 5th and 6th days of incubation. The saturnisporum and its growth response to varying
bottom of the agar plate appeared light yellow at day pH are very limited. It is deemed important to
2, and then turned bright yellow at days 3 to 4. After examine the pH tolerance of our Trichoderma
which, the bottom of the agar plate showed isolates below pH 5 and greater than pH 9 as many
diminishing bright yellow. As noted in the literature, Trichoderma strains can tolerate extreme pH levels
the yellowish pigmentation is often formed in that would be advantageous in their applications in
members of Longibrachiatum clade (Gams and stress environments.
Bissett, 1998; Samuels et al., 2012) where T.
saturnisporum belongs. T. gamsii, at 2nd day on PDA 3.4.3 Growth on PDA incubated at varying
medium, had thin white mycelia which covered temperatures
about 2/3 of agar plate. At days 3 and 4 of Of the four isolates, T. saturnisporum showed
incubation, the center of PDA had light green to the widest tolerance to varying temperatures, having
green sporulation while edges had white mycelia. At full colonization on PDA plates incubated at 25oC,
days 5 to 6, green to dark green sporulation covers 30oC, and 35oC (Figure 5). However, denser
the whole agar plate and appeared denser at the sporulation on PDA was observed when T.
center. The bottom of the PDA appeared white at saturnisporum was incubated at 25oC and 30oC
days 2 and 3, and turned cream color at days 4 to 6. compared to those grown at 35oC. Trichoderma
Swirling lines were visible starting at day 3. virens, T. harzianum, and T. gamsii showed full
mycelial colonization on PDA after 3 days of
3.4.2 Growth on PDA with varying pH incubation at 25oC and 30oC. However, incubation at
All isolates showed full mycelial colonization 35oC affected the growth of the three Trichoderma
of agar plates after 7 days incubation, irrespective of isolates (Figure 5). T. virens grown at 35oC showed
pH level. Results indicate that all four Trichoderma 63% and 44% mycelial growth inhibition after 3 and
isolates can grow well on solid medium, in this case 7 days incubation, respectively. In the same manner,
PDA, with pH 5 to 9. Among the environmental growth of T. harzianum at 35oC was 65% and 52%
parameters, pH is considered vital for biomass inhibited after 3 days and 7 days incubation,
production, conidiation, and other activities of respectively. Moreover, browning of mycelia was
Trichoderma (Kredics et al., 2004; Begoude et al., noted in T. harzianum after 7 days incubation at
2007; Singh et al., 2014). Previous studies examined 35oC as seen at the bottom of the agar plate. In the
the effect of varying pH to different Trichoderma case of T. gamsii, growth was totally inhibited at
species. In the study of Begoude et al. (2007), all 35oC which is evident after 3 days of incubation.
 Tansengco M et al. / Environment and Natural Resources Journal 2018; 16(1): 39-57 47

Figure 4. Growth of Trichoderma species on PDA (potato dextrose agar, pH 5.1+0.2) medium at days 2 to 6 incubated at
30oC+1. Top panels: top view on PDA. Bottom panels: back view of PDA plates.
48 Tansengco M et al. / Environment and Natural Resources Journal 2018; 16(1): 39-57

Based on fungal growth on agar medium, the optimum temperature between 25oC and 30oC
trend of Trichoderma isolates in temperature (Hadar et al., 1984; Mukherjee et al., 2013; Singh et
tolerance would be: T. saturnisporum > T. virens > al., 2014). However, members of the Longi-
T. harzianum > T. gamsii. Our result was in brachiatum clade, which include T. saturnisporum,
accordance with the report of Malathi and were noted to have the highest temperature optima
Doraisamy (2003), which shows that growth and (Danielson and Davey, 1973). This was observed in
activity of Trichoderma was affected by temperature our T. saturnisporum isolate which showed good
and their response varied with species. Reports also mycelial growth at 35oC.
indicated that most Trichoderma species have

Figure 5. Fungal growth on PDA (potato dextrose agar, pH 5.1+0.2) incubated at different temperatures (25oC, 30oC,
35oC) after 3 days (top panel) and 7 days (bottom panel). Data are means+SD from triplicate determinations.

3.5 Growth tolerance to increasing levels of heavy heavy metals up to 100 ppm. T. saturnisporum, at 3
metals days incubation, showed slight inhibition in 40 ppm
3.5.1 Tolerance to mixed heavy metals mixed heavy metals, 76% inhibition in 60 ppm
Figure 6 shows the mycelial radial growth of mixed metals, and total inhibition in 100 ppm heavy
fungal isolates grown on PDA with and without metal mixture. After 7 days, T. saturnisporum only
heavy metal mixture. After 3 days incubation, T. showed 30% growth inhibition at 60 ppm mixed
virens and T. harzianum showed resistance to mixed metals but still greatly inhibited at 80 ppm and 100
 Tansengco M et al. / Environment and Natural Resources Journal 2018; 16(1): 39-57 49

ppm heavy metal mixture. T. gamsii, after 3 days 800 and 1000 ppm Cr while T. virens had 30%
incubation, showed full resistance up to 60 ppm growth reduction at 1000 ppm Cr. After 7 days,
heavy metal mixture, slight inhibition (28%) in 80 however, full mycelial colonization was observed in
ppm, and 83% mycelial growth reduction in 100 all Trichoderma species.
ppm heavy metal mixture. T. gamsii, however, was Lead (Pb). Initially at day 3, growth of
able to recover after longer period as noted by full Trichoderma isolates was affected at 800 and 1000
colonization of all agar plates at 7 days incubation ppm Pb. At day 7, all isolates can tolerate up to
on PDA with mixed heavy metal. maximum Pb concentration.
Zinc (Zn). T. gamsii, T. virens, and T.
3.5.2 Tolerance to individual heavy metals harzianum can tolerate up to 1000 ppm Zn after 7
Figure 7 shows the growth response of four days incubation but their growth was initially
Trichoderma isolates to each heavy metal tested. inhibited with increasing Zn concentrations. T.
Chromium (Cr). All Trichoderma isolates saturnisporum was very sensitive to the presence of
were tolerant up to 1000 ppm Cr. After 3 days on Zn showing total growth inhibition starting at 200
PDA with Cr, T. gamsii showed light inhibition at ppm Zn.

Figure 6. Tolerance of Trichoderma isolates on agar medium (potato dextrose agar, pH 5.1+0.2) with mixed heavy metals
taken after 3 days (top panel) and 7 days (bottom panel) incubation at 30oC+1. Heavy metal mixture consisted of Cu, Cr,
Ni, Pb, and Zn. Data are means+SD from triplicate determinations.
50 Tansengco M et al. / Environment and Natural Resources Journal 2018; 16(1): 39-57

Nickel (Ni). Of the four isolates, T. gamsii and Trichoderma sp. PDR1-7 isolate was effective in
T. virens were the most tolerant to nickel but their reducing significant amounts of Pb and other heavy
growth was greatly reduced at 1000 ppm Ni after 7 metals in media with single or multiple metals. The
days incubation. T. harzianum can tolerate up to 800 study of Siddiquee et al. (2013) showed that T.
ppm Ni while T. saturnisporum can only grow up to virens strain T128 had the highest tolerance for Zn,
400 ppm Ni. Pb and Ni while T. harzianum strain T32 had the
Copper (Cu). T. harzianum and T. virens can highest growth for Cu in different concentrations. In
tolerate up to 1000 ppm Cu but their growth was the report of Hasanzadeh et al. (2012), heavy metals
reduced with increasing Cu levels. T. gamsii can Ag, Co, Cu, Fe and Hg could stop the mycelial
only tolerate up to 400 ppm Cu while T. growth of Nematophagus fungi (including T.
saturnisporum can only grow at 200 ppm Cu. harzianum and T. virens), but Pb, Mn, and Zn cannot
Based on fungal growth (after 3 and 7 days) to completely inhibit fungal growth. In our result, T.
each heavy metal, the order of tolerance by the virens and T. harzianum had good mycelial growth
Trichoderma isolates can be arranged as follows: in Cr, Pb, and Zn but reduced growth was observed
Chromium: T. harzianum = T. saturnisporum > T. in Ni (600 – 1000 ppm) and Cu (200 - 1000 ppm)
gamsii > T. virens; Lead: T. virens > T. gamsii > T. (Figure 7). This result indicates the poor adaptive
harzianum > T. saturnisporum; Zinc: T. gamsii > T. nature of these Trichoderma isolates to Ni and Cu.
virens > T. harzianum > T. saturnisporum; Nickel: Similar behavior was reported by Anahid et al.
T. gamsii > T. virens > T. harzianum > T. (2011) wherein Ni was considered as one of the most
saturnisporum; Copper: T. harzianum > T. virens > toxic heavy metals for the fungi Aspergillus niger, A.
T. gamsii > T. saturnisporum. foetidus, and Penicillium simplicissimum. Even at
Table 3 summarizes the final heavy metal low concentration, Ni could exert toxic effects like
level up to which each Trichoderma isolate could blockage of the functional groups of enzymes
tolerate. The best isolate is T. virens which showed (Anahid et al., 2011). On the other hand, Hajieghrari
tolerance to all five heavy metals up to 1000 ppm. T. (2010) reported that CuSO4 exhibited the strongest
harzianum can tolerate up to 1000 ppm mycelia inhibition in Trichoderma harzianum T969,
concentration of Cu, Zn, Cr, and Pb and up to 800 T. hamatum T614, and T. virens T525. An inhibitory
ppm Ni. T. gamsii can tolerate the maximum effect of CuSO4 on the growth of T. harzianum
concentration of Zn, Ni, Cr, and Pb while up to 400 Rifaii T1 was also obtained by Kucuk et al. (2008).
ppm of Cu. T. saturnisporum can tolerate up to 1000 Indeed, the response of Trichoderma species to
ppm Cr and Pb but showed the least tolerance to Ni, heavy metal exposure may differ depending on the
Cu and especially to Zn. This is the reason for the Trichoderma isolate and type of heavy metals
inhibited growth of T. saturnisporum at 40 to 100 (Hajieghrari, 2010).
ppm mixed heavy metals (Figure 6). Data on For T. gamsii, Kavita and Keharia (2012)
mycelial growth indicate that the heavy metals Cr reported the biosorption potential of its biomass in
and Pb were well tolerated by all Trichoderma removing chromium ions from acidic electroplating
isolates (Table 3 and Figure 7). However, tolerance industrial effluent. No published report was noted for
of Trichoderma isolates to Cu, Zn, and Ni was T. saturnisporum in relation to heavy metal removal
species specific. Based on growth test on agar plates efficiency. A review article by Siddiquee et al.
with higher levels of individual heavy metals (Table (2015) listed the Trichoderma species isolated and
3), the order of tolerance of Trichoderma isolates characterized including their heavy metal tolerance.
can be summarized as follows: T. virens > T. Results of the current study confirm the tolerance
harzianum > T. gamsii > T. saturnisporum. capability of T. virens and T. harzianum to high
A number of Trichoderma isolates, including concentrations of different heavy metals. Our data on
T. virens and T. harzianum, have been reported that heavy metal tolerance by T. gamsii and T.
are tolerant to various heavy metals. In the study of saturnisporum to individual and mixed metals (Cu,
Babu et al. (2014a), Trichoderma sp. PDR1-7 was Cr, Pb, Ni, Zn) may be the first report in the
isolated from Pb-contaminated mine tailing soil. The literature.
 Tansengco M et al. / Environment and Natural Resources Journal 2018; 16(1): 39-57 51

Cr (ppm) Day 3 Cr (ppm) Day 7

50 50
45 45

Mycelial radial growth (mm)

Mycelial radial growth (mm)

40 40
35 35
30 30
25 25
20 20
15 15
10 10
5 5
0 0
T. virens T. harzianum T. saturnisporum T. gamsii T. virens T. harzianum T. saturnisporum T. gamsii
Fungal isolates Fungal isolates

Pb (ppm) Day 3 Pb (ppm) Day 7

50 50
45 45
Mycelial radial growth (mm)

40 Mycelial radial growth (mm) 40

35 35
30 30
25 25
20 20
15 15
10 10
5 5
0 0
T. virens T. harzianum T. saturnisporum T. gamsii T. virens T. harzianum T. saturnisporum T. gamsii

Fungal isolates Fungal isolates

Zn (ppm) Day 3 Zn (ppm) Day 7

50 50
45 45
Mycelial radial growth (mm)
Mycelial radial growth (mm)

40 40
35 35
30 30
25 25
20 20
15 15
10 10
5 5
0 0
T. virens T. harzianum T. saturnisporum T. gamsii T. virens T. harzianum T. saturnisporum T. gamsii

Fungal isolates Fungal isolates

0 200 400 600 800 1000

Figure 7. Tolerance of Trichoderma isolates on agar medium (potato dextrose agar, pH 5.1+0.2) with individual heavy
metals taken after 3 days (left panel) and 7 days (right panel) incubation at 30 oC+1. Data are means+SD from triplicate
determinations.
52 Tansengco M et al. / Environment and Natural Resources Journal 2018; 16(1): 39-57

Ni (ppm) Day 3 Ni (ppm) Day 7

50 50
45 45

Mycelial radial growth (mm)

Mycelial radial growth (mm)

40 40
35 35
30 30
25 25
20 20
15 15
10 10
5 5
0 0
T. virens T. harzianum T. saturnisporum T. gamsii T. virens T. harzianum T. saturnisporum T. gamsii

Fungal isolates Fungal isolates

Cu (ppm) Day 3 Cu (ppm) Day 7

50 50
45 45
Mycelial radial growth (mm)
Mycelial radial growth (mm)

40 40
35 35
30 30
25 25
20 20
15 15
10 10
5 5
0 0
T. virens T. harzianum T. saturnisporum T. gamsii T. virens T. harzianum T. saturnisporum T. gamsii

Fungal isolates Fungal isolates

0 200 400 600 800 1000

Figure 7. Tolerance of Trichoderma isolates on agar medium (potato dextrose agar, pH 5.1+0.2) with individual heavy
metals taken after 3 days (left panel) and 7 days (right panel) incubation at 30 oC+1. Data are means+SD from triplicate
determinations. (cont.)

Table 3. Final heavy metal concentration up to which each fungal isolate could tolerate

Fungal isolates Heavy metals (ppm)

Cr Pb Zn Ni Cu
Trichoderma virens 1000 1000 1000 1000 1000
Trichoderma harzianum 1000 1000 1000 800 1000
Trichoderma saturnisporum 1000 1000 - 400 200
Trichoderma gamsii 1000 1000 1000 1000 400
Data were taken after 7 days incubation from triplicate determinations.

3.6 Heavy metal removal by the best fungal irrespective of initial pH of samples, with reduction
isolate, Trichoderma virens ranging from 91 to 96%. Using other heavy metals,
Figure 8 shows the heavy metal removal by T. T. virens preferred specific initial pH to achieve high
virens in liquid culture at varying initial pH. T. removal. Maximum metal reductions were 69-70%
virens was very efficient in lead (Pb) removal, for Cu at pH 6-7, 63% for Cr at pH 7, 48% for Zn at
 Tansengco M et al. / Environment and Natural Resources Journal 2018; 16(1): 39-57 53

pH 9, and 39% for Ni at pH 6. Results indicate that tolerated by T. virens and did not affect its growth.
the order of heavy metal removal of T. virens is as On the other hand, biomass of T. virens was lower in
follows: Pb > Cu > Cr > Zn > Ni. media with Cr and Cu compared to control.
Figure 9 shows the growth response of T. Maximum dry weight in cultures with Cr was 4.50
virens on liquid medium with individual heavy g/L (pH 7) while only 3.27 g/L for those with Cu
metals and at varying initial pH. Growth of T. virens (pH 7). These are equivalent to biomass reductions
in control medium (PDB alone) showed increasing of 22% for Cr and 44% for Cu compared to control
trend as pH level increases. An almost comparable samples of the same initial pH. Results indicate that
trend was observed in those grown in Zn- although T. virens can reduce Cr and Cu levels, these
supplemented medium. The highest fungal biomass metals exhibit inhibitory growth function to this
was recorded in liquid medium with Ni at pH 5 and fungus. This might reflect the slow adapting strategy
6 and then with Pb at pH 6 to 8. These results of T. virens to Cr- and Cu-contaminated culture
indicate that Zn, Ni, and Pb at 100 ppm are well media.

100
90
pH
80
Percent removal (%)

70 5

60 6

50 7

40 8

30 9

20
10
0
Cu Ni Zn Pb Cr
Heavy metal

Figure 8. Heavy metal removal of Trichoderma virens at varying initial pH (5, 6, 7, 8, 9). Heavy metal concentration was
100 ppm in 50 mL liquid medium (Potato dextrose broth). Data are means+SD from triplicate shakeflask cultures taken
after 7 days at 150 rpm agitation and incubated at room temperature (28-30oC).

Few studies were noted that focused on T. T. virens strain T128 grown in liquid medium
virens’ capacity for heavy metal removal. In the showed highest tolerance to Zn compared to Ni, Pb,
report of Babu et al. (2014b), T. virens PDR-28 was and Cu. Their report suggests that the metal uptake
isolated from rhizosphere soil and tested in mine by the Trichoderma species was due to physical
tailing soil remediation and maize biomass binding to cell surfaces as mycelia appeared partially
production. Their results showed that T. virens PDR- degraded and that cell wall has an important role in
28 efficiently removed heavy metals in the order of biosorption of toxic metals (Siddiquee et al., 2013).
Pb > Cd > As > Zn > Cu from liquid media The mechanisms of heavy metal removal by
containing 100 mg/L heavy metal (Babu et al., microorganisms differ depending on the species, its
2014b). This is in accordance with our report origin, and processing of biomass (Ali and Hashem,
wherein T. virens showed high removal efficiency in 2007). In fungi, heavy metal sequestration can be
Pb. However, our T. virens isolate showed least extracellular to avoid metal entry or intracellular to
removal for Ni while T. virens PDR-28 isolate least reduce the metal burden in the cytosol (Bellion et al.,
preferred Cu. In the report of Siddiquee et al. (2013), 2006; Anahid et al., 2011). For our T. virens isolate,
54 Tansengco M et al. / Environment and Natural Resources Journal 2018; 16(1): 39-57

its strategies for heavy metal removal may involve ions out of the cell (Hall, 2002). Another hypothesis
complex mechanisms. For Pb, Ni, and Zn reduction, is the anti-oxidative detoxification mechanisms that
T. virens might be using extracellular system through allow the fungus to counteract the reactive-oxygen
chelation or biosorption. However, for Cu and Cr species induced by certain metals (Bellion et al.,
removal, T. virens biomass was reduced in spite of 2006). Our T. virens isolate might develop tolerance
heavy metal reduction at various initial pH levels. In to the redox-active metals, Cu and Cr, which induced
this case, T. virens might be slow in adapting to Cu- reactive-oxygen species and in the process caused
and Cr-polluted culture media and this may reflect stress or damage to the cells. For future research, it is
its characteristic biological function. The possible important to determine the exact mechanisms used
mechanism for its Cu and Cr tolerance and removal by our T. virens isolate so as to identify its potential
may be intracellular sequestration that involves applications in actual heavy metal bioremediation.
metal transport proteins, which extrude toxic metal

6 PDB
Dry weight (g/L)

Cu
5 Cr

4 Zn
Ni
3 Pb

0
5 6 7 8 9
pH

Figure 9. Dry weight of Trichoderma virens cultivated in liquid medium with individual heavy metals (100 ppm) and at
varying initial pH (5, 6, 7, 8, 9). Data are means+SD from triplicate shakeflask cultures taken after 7 days at 150 rpm
agitation and incubated at room temperature (28-30oC).

4. CONCLUSIONS that T. virens had high lead removal irrespective of

Wastewater samples collected from mine initial pH level. T. virens is also efficient in Cu and
tailings in Itogon, Benguet are rich sources of heavy Cr removal at neutral pH. Reductions in Ni and Zn
metal-resistant fungi. Twenty-nine (29) fungal were preferred at pH 6 and 9, respectively. Results
species were isolated and four species were selected of the study proved the occurrence of heavy metal-
based on their tolerance to mixed heavy metals tolerant fungi in Itogon, Benguet mine tailings.
containing Cu, Cr, Pb, Ni, and Zn. Selected fungal Isolated Trichoderma species can be used in heavy
isolates all belong to the genus Trichoderma and metal remediation of polluted sites.
sequences were highly identical to T. virens, T.
harzianum, T. saturnisporum, and T. gamsii. Growth ACKNOWLEDGEMENTS
test on agar medium with increasing concentrations This research was funded by the National
of individual heavy metals showed that the Research Council of the Philippines-Department of
Trichoderma isolates can be ranked as follows: T. Science and Technology (NRCP-DOST). The
virens > T. harzianum > T. gamsii > T. satur- authors are very grateful to: DOST-CAR (Cordillera
nisporum. Heavy metal removal in liquid shakeflask Administrative Region) and Benguet Corp. for
culture using the best Trichoderma isolate showed guidance and assistance during wastewater
 Tansengco M et al. / Environment and Natural Resources Journal 2018; 16(1): 39-57 55

collection; Mr. Jose Ricky Beraye, Ms. Cynthia effects of temperature, pH, and aw on the growth rate
Borromeo, and Mr. Bernard Gutierrez of ITDI for of Trichoderma asperellum. Journal of Applied
the laboratory assistance. Microbiology 2007;103:845-54.
Bellion M, Courbot M, Jacob C, Blaudez D, Charlot M.
Extracellular and cellular mechanisms sustaining
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