1UST College of Science

Department of Biological Sciences

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Nickel Tolerance and Bioaccumulation in Various Fungal Isolates

from Contaminated Soil

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An Undergraduate Thesis

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Submitted to the

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Department of Biological Sciences

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College of Science

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University of Santo Tomas

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in partial fulfillment for the requirements of

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Bachelor of Science in Biology

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by

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4 Bio 5 Group 2

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Gianpaolo N. Dailisan

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Lux M. Hardin

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Reggiana Mae R. Lanuza

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Jasper Jo A. Sabalburo

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March 2015

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3UST College of Science

Department of Biological Sciences

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Abstract

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36Introduction.
37Methods.
38Results.
39Conclusion.
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41Keywords:
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5UST College of Science

Department of Biological Sciences

63Introduction
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Land pollution means degradation or destruction of earths surface

65and soil, directly or indirectly as a result of human activities (Conserve

66Energy Future, 2009). Soil pollution is one form of land pollution that
67damages and contaminates the soil. Industrial waste is one of the most
68important sources of contamination in the surface environment. Numerous
69studies of environmental contamination due to industrial wastage activities
70have been undertaken to further the understanding of the impacts of
71heavy metals and metalloids in soils, plants, animals and humans
72(Gunwal, Singh and Mago, 2014).
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Many heavy metals exist in minute amounts in natural agricultural

74soil. However, when their amounts exceed a certain level due to pollutants
75brought from outside, soil contamination occur and agricultural products
76become contaminated (Arao et. al., 2010). Nickel is a nutritionally
77essential trace metal for at least several animal species, microorganisms
78and plants, and therefore either deficiency or toxicity symptoms can occur
79when, respectively, too little or too much Ni is taken up. Nickel is generally
80distributed uniformly through the soil profile but typically accumulates at
81the surface from deposition by industrial and agricultural activities (Cempel
82and Nikel, 2005). Nickel (II) is a more toxic and carcinogenic metal when
83compared with Nickel (IV) (Congeevaram, 2007).
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According to the European Economic Community, the typical

85agricultural soil maximum limit for nickel of dry matter soil sample with pH
866, ranges from 30-75 mg/L. Also, the typical agricultural sewage sludge
87maximum limit for nickel dry matter, ranges from 300-400 mg/kg (World
88Health Organization, 1991).
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Recently microbes like bacteria, fungus, and algae have been

90effectively used as adsorbing agents for the removal of heavy metals

91(Muoz, 2006). Among the microorganisms, fungi are very important for
92bioremediation due to their mycelial nature and well documented ability to
93accumulate metals of all kinds (Gadd, 1993). Some generalizations have

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Department of Biological Sciences

94stated that fungi are more resistant to heavy metals than bacteria. This is
95due to the observation that there is a relative abundance of fungi in highly
96metal-stressed

soils

(McGrath,

2001).

longterm

exposure

of

97microorganism to high metal concentration develops immunity in them

98(Shazia, et al., 2013). Different species of Aspergillus, Pseudomonas,
99Sporophyticus, Bacillus, Phanerochaete, etc., have been reported as
100efficient chromium and nickel reducers (Yan and Viraraghavan, 2003).
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The research aims to Isolate and identify fungi from nickel-

102contaminated soil. It also aims to determine the tolerance of the fungal

103isolates to high concentrations of nickel and lastly, to determine the
104efficiency of the isolated fungi in reducing nickel in under different
105conditions.
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107Materials and Methods
108Sample collection. Heavy metal soil samples were collected at areas near
109nickel mining sites at Brgy. Lipay, Sta. Cruz, Zambales. Each soil sample
110was placed in sealed bags and was properly labeled. Samples were
111stored in a chilled container until further examinations. 100g of each soil
112sample were subjected to AAS (F.A.S.T. Laboratory) to determine their
113nickel concentration.
114Isolation of fungi. One gram of each soil sample was serially diluted from
11510-3 10-4 and spread-plated to strength Sabouraud Dextrose Agar
116(SDA) in triplicates. Plates were incubated for 3-5 days at room
117temperature for fungal growth. Fungal colonies on SDA plates were then
118selected and re-inoculated on full strength SDA plates using agar block
119method and incubated for 3-5 days at room temperature. The identification
120of fungal isolates was based on their morphological characteristics
121(colonial formation, color, shape, and hyphae).
122Nickel tolerance test. Fungal spores were inoculated in a set of full
123strength SDA plates in duplicates amended with Ni(II) (anhydrous NiSO 4)
124concentrations from 100-900 mg/L. Control samples were prepared by

9UST College of Science

Department of Biological Sciences

125inoculating isolates in SDA plates without Ni(II). Plates were incubated for
126seven days at room temperature. Observations were made on the seventh
127day and the results were recorded.
128pH test. Fungal spores were inoculated in a set of full strength 100 ml
129SDB in flasks amended with 300-500 mg/L Ni(II) (anhydrous NiSO 4) under
130varying pH (pH 5.0 - 7.0). It was incubated under room temperature for
131seven days. Observations were made on the seventh day and the results
132were recorded. Mycelia were harvested and decanted from the
133supernatant. The supernatant was placed in bottles and was subjected to
134AAS (F.A.S.T. Laboratory) to determine their final nickel concentration.
135Results were statistically analyzed using One-way Anova with 95% level of
136confidence.
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138Results
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Among the four soil samples collected from areas near nickel

140mining sites at Brgy Lipay, Sta. Cruz, Zambales., Soil Sample 1 has the
141highest nickel concentration (Table 1). A total of 5 different fungal isolates,
142excluding the yeasts, were isolated from the four soil samples. One fungal
143isolate was identified as Paecilomyces sp. and the other four were
144identified as Aspergillus sp.
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146Table 1. Soil nickel concentration
147 Samples
Soil Sample 1 (Dry Reddish Soil,

148 Open Lot)

Soil Sample 2 (Brown Soil, Riverside)
Soil Sample 3 (Black-ish Brown, Wet,
Near the shore)
Soil Sample 4 (Brown, dry soil, Open
lot)

Nickel, mg/L
6, 997

In

6, 862
2,002
3, 039

149comparison with samples amended with nickel, control samples showed

150greater growth rate, measured in diameter. In Fig. 1, two of the five
151isolates tolerated only 100 mg L-1 Ni(II) (Aspergillus sp.3 and Aspergillus

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Department of Biological Sciences

152sp.4). Paecilomyces sp. tolerated 500 mg L-1 Ni(II), Aspergillus sp.1

153tolerated up to 900 mg L-1 Ni(II) and Aspergillus sp.2 tolerated up to 300
154mg L-1 Ni(II). These nickel concentrations were inferred to be the Minimum
155Inhibitory Concentration (MIC) of fungal isolates against Nickel. The fungal
156isolates tolerance to nickel was based on their growth rate after seven
157days of incubation.

Paecilomyces sp.
Aspergillus sp2.
Aspergillus sp3.
Aspergillus sp4.
Aspergillus sp1.

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159Fig 1. Growth rate in diameter under varying Nickel Concentration
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Based on the Nickel tolerance tests results, Paecilomyces sp.,

162Aspergillus sp.1 and Aspergillus sp.2 were more efficient in tolerating high
163concentrations of nickel in comparison with Aspergillus sp.3 and
164Aspergillus sp.4. The three fungal isolates were further tested for its nickel
165reduction activity under varying pH levels. The nickel concentration used
166for Paecilomyces sp. and Aspergillus sp. 1 was 500 mg L-1 Ni(II) and 300
167mg L-1 Ni(II) for Aspergillus sp.2.

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Paecilomyces sp.
Aspergillus sp1.
Aspergillus sp2.

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169Fig 2. Percentage Reduction of 500 mg L-1 Ni(II) (Paecilomyces sp., Aspergillus
170sp.1) and 300 mg L-1 Ni(II) (Aspergillus sp.2) at varying pH levels
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The statistical analysis showed that there was no significant

173difference (p > 0.05) among the nickel reduction activity of the fungal
174isolates when subjected to various pH levels (5-6 pH) at 300 mg L -1 Ni(II)
175and 500 mg L-1 Ni(II) incubated at room temperature despite the
176differences in pH (Fig. 2). However, among the pH levels used, pH 7
177showed the highest reduction activity.
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179Discussion
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The soil samples collected from areas near nickel mining sites at

181Brgy. Lipay, Sta. Cruz, Zambales contained high concentrations of nickel,

182thus, considered to be toxic. The accepted concentration of nickel in a
183typical agricultural soil ranges from 30-75 mg/L based on the World Health
184Organization (1991). Soil is a rich habitat of all major microorganisms

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Department of Biological Sciences

185(Shazia, et al., 2013). Fungi, as a bioaccumulator, have been efficient for

186the removal or reduction of heavy metals.
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188Nickel is a transition element with atomic number 28 and atomic
189weight 58.69. In nature, Ni is mostly present in the form of
190nickelous ion, Ni2+. The hydrated form as Ni (H2O)6 is the most
191common form of Ni found in the soil solution (Gunwal, Singh and
192Mago, 2014). Ni(II) is more toxic and carcinogenic metal when
193compared with Ni(IV) (Congeevaram et al., 2007).
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195Acknowledgements
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197References
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199Supplementary Materials
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