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Catena 147 (2016) 25–31

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Selected microbial diversity of contaminated landfill soil of Peninsular


Malaysia and the behavior towards heavy metal exposure
B. Jayanthi a,⁎, C.U. Emenike a,b, P. Agamuthu a,b, Khanom Simarani a,b, Sharifah Mohamad c, S.H. Fauziah a,b
a
Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia
b
Centre for Research in Waste Management, Institute of Research Management & Monitoring, University of Malaya, 50603 Kuala Lumpur, Malaysia
c
Department of Chemistry, University of Malaya, 50603 Kuala Lumpur, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history: Microbes function normally and effectively when the site of action or immediate environment is intact and un-
Received 23 December 2015 polluted. Given that microbes can significantly improve the bio-geochemical cycling of toxic heavy metals or
Received in revised form 19 May 2016 the remediation of metal-contaminated environments, understanding the effect of microbe diversity on heavy
Accepted 23 June 2016
metal pollution caused by leachate seepage is imperative. This study focused on the difference in the distribution
Available online xxxx
of microbial species in non-sanitary landfill soil of Peninsular Malaysia (operational and non-operational status)
Keywords:
regarding the heavy metal tolerance of the bacterial species. Soil and leachate characterization identified the level
Bioremediation of pollutants in the landfill environment. Hence, microbial isolation and identification generated the microbial di-
Heavy metals versity of the contaminated landfill soils of Peninsular Malaysia. Exposure to Hg concentrations (5–20 ppm)
Landfills showed that all the organisms survived with a heavy growth pattern. All strains showed varied resistance to
Leachate the heavy metals. Pseudomonas mendocina demonstrated the highest resistance to metal exposure. Bacillus
Metals resistance pumilus was absolutely resistant to the heavy metals used in the study, except Ni. A comparison of isolates
from operational and non-operational landfill sites in Peninsular Malaysia suggests higher susceptibility to chro-
mium ions than to other highly toxic metals, especially lead and mercuric ions. The behavior/response of the iso-
lated microbes suggests suitability for enhanced bioremediation of heavy-metal-polluted environments.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction classified as toxic to the environment, even at minute concentrations.


Although different sources of heavy metals exist, metals also occur nat-
Microorganisms are integral components of the ecosystem. The urally in the environment as ores and few other complexes; hence, sev-
presence of microbes in atmospheric, terrestrial, and aquatic environ- eral metals play useful roles in the environment, particularly in plant
ments enhances different dimensions of metabolism and transforma- growth (Aydinalp and Marinova, 2009; Sing et al., 2011; Galal and
tions. This phenomenon explains the importance of microbes in Shehata, 2015). However, when heavy metals exist in excessively high
synthesis and degradation. Basically, microbes thrive more optimally concentrations, pollution occurs and can be catastrophic because of its
when the site of action or the immediate environment is intact than effect on microbes. Sometimes, metals distinctively perturb soil micro-
when the environment is contaminated. However, totally intact or un- bial biomass and activity and even reduce the composition and diversity
disturbed environments are gradually disappearing because of human of the microbial community of soil (Xu et al., 2015). Free radical nano-
interference. Anthropogenic activities that are guided by the insatiable particles of CuO exhibit toxicity towards bacteria (Gajjar et al., 2001).
societal desires are now a global concern, as most green environments Similarly, nanoparticles of TiO2 rupture cell wall/membrane of
have been negatively affected by man-made pollutants. The list of pol- Nitrosomonas europaea to induce cell permeability and mortality (Fang
lutants is long and has been updated every now and then by profession- et al., 2010). One source of heavy metal pollution is the leachate pro-
al bodies and organizations based on research findings. duced from landfill operation. The inevitable waste generation pattern,
However, the distribution of numerous pollutants in the environ- especially in developing countries, often leads to daily generation of
ment does not imply equal prevalence and influence. Several pollutants high volumes of leachate. Leachate is the liquid/fluid that flows out
are very toxic and dangerous to every form of life, whereas other pollut- from waste because of increased moisture levels from water penetra-
ants are considered negligible. Heavy metals are pollutants that are tion or degradation. The global characterization of leachate, especially
from municipal solid waste (MSW) landfills, has shown that it is highly
⁎ Corresponding author.
heterogeneous and often contains massive amounts of dissolved organ-
E-mail addresses: jayne_barath@yahoo.com, jayne.barath@siswa.um.edu.my ic matter, pesticides, xenobiotics, and heavy metals (Emenike et al.,
(B. Jayanthi). 2013, 2012; Fauziah et al., 2013; Kjeldsen et al., 2002). When any

http://dx.doi.org/10.1016/j.catena.2016.06.033
0341-8162/© 2016 Elsevier B.V. All rights reserved.
26 B. Jayanthi et al. / Catena 147 (2016) 25–31

Table 1 soils across Peninsular Malaysia in relation to the heavy metal tolerance
General conditions of the landfill sites. of the bacterial species.
Condition class Taman Beringin landfill Bukit Beruntung landfill

Landfill type Non-sanitary Non-sanitary


2. Materials and methods
(non-operational) (operational)
Period of landfilling 1991–2005 2001 - date 2.1. Soil and leachate samplings and characterizations
Age classification Stabilized Mature
Daily average of waste 1800–2000 1500
Municipal solid waste (MSW) landfill sites were selected for the
disposed (tonnage)
Waste type Household, commercial Household, commercial study based on their status and grade. The two selected landfills were
and industrial and industrial graded non-sanitary because membrane liners were not installed. One
Form of leachate treatment Physical and biological Biological of the landfills was “operational” because waste was dumped at the
Distance to river/stream 5 NA site. The other landfill had not received MSW for more than 10 years
(m)
Fate of generated landfill No facility No facility
and was considered “non-operational”. Table 1 reflects the general con-
gas ditions of the landfill sites. Hence, soil samples were excavated at 30 cm
Coordinates of sampling °
A 3 13′ 40.17 N A 3°42′49.21 N depth from each of Taman Beringin (TBL) (3° 13.78′N; 101° 39.72′E;
spots 101 39′43.48 E 101 54′55.87 E non-operational) and Bukit Beruntung (BBL) (3° 32.14′N;101° 25.80′E;
B 3 13′ 43.86 N B 3 42′49.81 N
operational) landfills in accordance with the 2014 ASTME–1197 stan-
101 39′51.74 E 101 54′53.35 E
C 3 13′37.91 N C 3 25′31.88 N dard guidelines for conducting terrestrial soil-core microcosm tests
101°39′51.74 E 101°32′48.92 E (Sprocati et al., 2011). The excavated samples were analyzed for pH
D 3°13′36.44 N using a multiprobe meter (YSI Professional Plus, USA), while the soil
101°39′46.72 E total nitrogen, total potassium, and total phosphorus were analyzed
NA – not available. by adopting ASTM E778-87, ASTM E96-94, and ASTM D5198-92
methods, respectively. Elemental concentrations of metals in the soil
were analyzed based on the USEPA 3050B guidelines except for mercury
landfill lacks proper liners and an adequate collection system for the (Hg), which was analyzed based on the USEPA 3052 method. All assess-
leachate generated from waste degradation, raw leachate will laterally ments were duly replicated (including different trials). Similarly, the
seep into soil compartments and contaminate the soil. Therefore, the in- raw leachate samples were collected from the environment and ana-
teractions and responses of microbes in the presence of leachate as a soil lyzed for parameters similar to the soil samples. Part of the leachate as-
contaminant are important. The responses of microbes to pollution may sessment included on-the-spot analysis of raw leachate collection for
vary from one environment to another or may vary among species be- several parameters, especially pH (HANNA HI 8424). Similarly, several
cause of the nature of pollutants and their varying concentrations. For other physico-chemical properties of the leachate samples determined
instance, microbial growth can be enhanced at low concentrations of in the laboratory were BOD5, COD, total N, P, K, and the metal distribu-
copper (Cu), but will be repressed at high concentrations. Meanwhile, tion. The assessment was conducted based on APHA (1998) standards.
low concentrations of cadmium (Cd) can cause severe toxicity Preliminary investigation and assessment of the landfill site, which in-
(Lucious et al., 2013; Wei et al., 2009; Karnachuk et al., 2003). Given clude soil testing, topographic outlay, and visual observation, deter-
that microbes can significantly improve the bio-geochemical cycling of mined the degree of heterogeneity and siting of the sampling spots.
toxic heavy metals or remediating metal-contaminated environments,
understanding the diversity of microbes during heavy metal pollution 2.2. Bacteria isolation and identification
caused by leachate seepage into soil is imperative. Similarly, increasing
evidence on the metal resistance among naturally resident microbes Bacterial species were isolated by mixing 1 g of soil sample with
found in the contaminated sites has become available (Lucious et al., 10 ml of normal saline water (0.9% NaCl) as stock. The mixture was
2013). However, studies on understanding similar resistance within shaken vigorously (3 h at 180 rpm) with the aid of a Lab-line 3521
landfill sites still remain limited. Considering the indiscriminate distri- orbit shaker, and the resulting suspension was subjected to 20 times se-
bution of non-sanitary landfills in Southeast Asia, including Malaysia, rial dilution. Dilutions (0.1 ml) were dispensed on freshly prepared nu-
contamination of soil with leachate is common, and the effect of con- trient agar under aseptic conditions (Kauppi et al., 2011). The inoculated
tamination on microbial diversity has not been thoroughly investigated. media plates and associated replicates were incubated at 37 °C for 24 h.
Therefore, this research focuses on the difference in microbial species Developed colonies were further sub-cultured to ensure the purity of
resident in non-sanitary landfill (operational and non-operational) samples prior to identification. Subsequently, the Biolog GEN III Micro-
plate protocol was used to test the isolated microbes according to
Bochner (1989a,1989b). An omni log reader was used to identify the
bacteria species contained in the microbial identification system
Table 2 software.
Characteristics of heavy metals used.
2.3. Heavy metal resistivity test
Mol.wt Atomic wt
S/N Metal Salt Product (g/mol) (g)
Isolated bacteria were aseptically re-grown by inoculating each spe-
1 Pb PbCl2 Merck 278.1 207.2
2 Mn MnSO4 Friendemann 169.02 54.93
cies into individual test tubes containing 5 ml of nutrient broth at 37 °C
Schmidt for 18–24 h. Each inoculum was later introduced into test tubes contain-
3 Fe FeSO4·7H2O HumbG Chemicals 278.02 55.85 ing 4.5 ml of normal saline water for standardization (NCCLS, 1993) to
4 Hg HgSO4 Bendosen 296.65 200.59 obtain 0.1 ABS (absorbance)/0.5 McFarland at 860 nm. However, the
5 Zn ZnSO4·7H2O AnalaR 287.55 65.38
final inoculums required for the heavy metal sensitivity assessment
6 Cu CuSO4 Bendosen 159.60 159.60
7 Cd CdCl2 Friendemann 228.85 112.41 were obtained by dispensing 0.1 ml of the resultant standard into corre-
Schmidt sponding test tubes containing 9.9 ml of normal saline water for each
8 Ni NiCl2·6H2O Bendosen 237.73 58.69 test organism to provide an approximate cell density of 5 × 105 CFU/ml.
9 Cr K2Cr2O7 HumbG Chemicals 294.19 103.8
Furthermore, the chemical characteristics of heavy metals used for
10 Al Al2(SO4)·16H2O Systerm 630.39 53.92
the resistivity test are in Table 2.
B. Jayanthi et al. / Catena 147 (2016) 25–31 27

Table 3
Soil characterization.a, b

Test parameter Test method Taman Beringin landfill Bukit Beruntung landfill

Meana Rangeb Meana Rangeb

pH 7.57 7.3–8.1 7.09 7.04–7.20


Total N (%) ASTM E778-87 0.62 0.25–0.8 0.46 0.35–0.49
Total K (ppm) ASTM E926-94 396.9 0.046–418 935.5 55.2–980
Total P (ppm) ASTM D5198-92 568 0.1–608 1858 10.5–2170
Ar (ppm) USEPA 3050 B 0.1 b0.01–0.1 0.38 0.1–0.38
Ca (ppm) USEPA 3050 B 1608 47–2019 8614 63.4–9281
Fe (ppm) USEPA 3050 B 5209 134.6–5209 5367 43.2–848
Mn (ppm) USEPA 3050 B 470.5 b0.01–520.5 174.8 2.42–928
Mg (ppm) USEPA 3050 B 127.2 52.2–160.2 618.8 35.2–723.8
Na (ppm) USEPA 3050 B 4.54 0.95–29.7 269.9 100.7–315
Cu (ppm) USEPA 3050 B 20.4 b0.01–22.8 371.3 1.88–392
Zn (ppm) USEPA 3050 B 15.1 0.54–18.4 547.5 3.57–602
Pb (ppm) USEPA 3050 B 3.67 b0.001–3.67 79.9 0.49–90.4
Cd (ppm) USEPA 3050 B b0.01 b0.01 2.64 0.19–3.36
Hg (ppm) USEPA 3052 b0.02 b0.02 b0.002 b0.002
Cr (ppm) USEPA 3050 B 7.27 b0.01–8.54 11.4 0.57–17.4
Ni (ppm) USEPA 3050 B 4.38 b0.01–6.22 10.21 0.67–14.01
Al (ppm) USEPA 3050 B 2060 5.47–2130 10,087 0.47–12,261
a
Mean values (n = 3).
b
Range values (n = 5).

Therefore, the metal tolerance for each bacterial isolate was deter- between resistance and inhibition zone diameter. A p value below
mined by agar diffusion. The standard suspension of each organism 0.05 was regarded as statistically significant.
(5 × 105 CFU/ml) was used to seed each sterile plate, which contained
20 ml of nutrient agar. Pre-diffusion was allowed before a core borer 3. Results and discussion
was used to make 6 mm diameter wells (4) on the seeded plates. Four
concentrations (5, 10, 15, and 20 ppm) of each metal were prepared. Based on landfill status, the studied landfill soils were found to be
Metals (70 μl) were dispensed into corresponding wells. Hence, each percolated by raw leachate. Results of the characterized soil samples in-
plate accommodated four concentrations of a specified heavy metal dicated the presence of heavy metals and other associated components
and was allowed to stand for 1 h for pre-diffusion. Plates were then in- (Table 3). Notably, both landfill soil cores contained some metal concen-
cubated at 37 °C for 24 h. The minimum inhibitory concentrations (MIC) trations at levels that exceeded both local standards and international
of the heavy metals on the microbes were determined based on ob- standards (DOE, 2009; Aweng et al., 2011).
served growth pattern. Diameters of the corresponding clear zones However, the concentrations of the studied pollutants differed
that characterized the concentrations of the heavy metals that showed markedly between both landfill sites; concentrations from the Bukit
no visible growth were measured to determine the inhibition zone di- Beruntung landfill (BBL) were mostly higher than those of the Taman
ameter (IZD). Beringin landfill (TBL). This result can be associated with the active sta-
Data are expressed as means of the three (3) replicates. Comparison tus (operational) of the BBL, wherein the fresh waste deposited on daily
of metal resistance among isolated microbes was analyzed using a one- basis could enhance metal leaching into the soil compartment. The
way ANOVA followed by a linearity plot to evaluate the correlation metal concentration found in the TBL may imply that extractable/mo-
bile metals ions have been immobilized over time and may exist as com-
plexes. A similar result was found when raw leachate samples of both
Table 4 landfills were analyzed (Table 4). With both showing concentrations
Characteristics of raw leachate samples from the landfills. that were above discharge limits (Emenike et al., 2012; EQA 1974,
Test Taman Beringin Bukit Beruntung 2007), the BBL nonetheless revealed higher metal concentrations than
parameter Test method landfilla landfilla those found in the TBL. This difference can be attributed to age of the
pH 7.57 ± 0.8 7.09 ± 0.63 landfill as well, as the BBL is considered to be mature and the TBL is al-
BOD (mg/L) APHA 5210 B 127 ± 45 259 ± 37 ready stabilized. Levels of soluble metal in active landfills are often
COD (mg/L) APHA 5220 482 ± 103 985 ± 185 higher than what is obtained from non-active/older landfills
Total N (%) ASTM E778–87 0.25 ± 0.08 0.32 ± 0.05
(Lagerkvist, 2003; Yusof et al., 2009; McBean et al., 1995; Calli et al.,
Total K (ppm) ASTM E926–94 11.6 ± 2.1 40.4 ± 6.04
Total P (ppm) ASTM 0.1 24.3 ± 0.7 2005). Such solubilization is enhanced by low pH that results in the pro-
D5198–92 duction of acid during the acidogenic phase. However, both landfills are
Ar (ppm) USEPA 3050 B b0.01 0.21 considered to be at the acidogenic phase because the pH values were 6.8
Ca (ppm) USEPA 3050 B 242.1 ± 42 91.2 ± 11.6 and 7.1, respectively, for the TBL and the BBL. The upper limit of the
Fe (ppm) USEPA 3050 B 134.6 ± 16 60 ± 18.2
Mn (ppm) USEPA 3050 B 3.1 ± 0.32 5.1 ± 0.5
acidogenic phase is 4.5–7.5 (Kjeldsen et al., 2002).
Mg (ppm) USEPA 3050 B 52.2 ± 8.7 96.6 ± 16 Furthermore, the landfill soils were characterized for possible bacte-
Na (ppm) USEPA 3050 B 29.7 ± 5.1 242.1 ± 22.8 ria distribution based on isolation and identification. Table 5 contains
Cu (ppm) USEPA 3050 B 0.5 ± 0.1 2.62 ± 0.8
Zn (ppm) USEPA 3050 B 24.3 ± 3 236 ± 11.8
Pb (ppm) USEPA 3050 B b0.01 1.12 ± 0.04 Table 5
Cd (ppm) USEPA 3050 B 0.4 ± 0.1 0.4 ± 0.1 Isolated bacteria species from the landfill soils.
Hg (ppm) USEPA 3052 0.03 0.04
Cr (ppm) USEPA 3050 B 6.2 ± 1.4 17.3 ± 1.9 S/N Taman Beringin landfill Bukit Beruntung landfill
Ni (ppm) USEPA 3050 B 0.85 ± 0.1 12 ± 4.4 1 Aeromonas caviae DNA group 4 Bacillus pumilus
Al (ppm) USEPA 3050 B 5.47 ± 1.2 13.1 ± 3.2 2 Pseudomonas alcaligenes Ochrobactrum intermedium
a
Mean values (n = 3). 3 Pseudomonas mendocina Stenotrophomonas acidaminiphila
28 B. Jayanthi et al. / Catena 147 (2016) 25–31

Table 6
Heavy metals resistance test.

Metal Conc.
(ppm) Taman Beringin landfill microbes Bukit Beruntung landfill microbes

Aeromonas caviae DNA group Pseudomonas Pseudomonas Bacillus Ochrobactrum Stenotrophomonas


4 alcaligenes mendocina pumilus intermedium acidaminiphilia

Pb 5 ++ ++ ++ ++ ++ ++
10 ++ ++ ++ ++ ++ ++
15 ++ +− ++ ++ ++ ++
20 +− +− ++ ++ +− ++
Mn 5 ++ ++ ++ ++ ++ ++
10 ++ ++ ++ ++ ++ ++
15 ++ ++ ++ ++ +− ++
20 ++ ++ ++ ++ +− ++
Fe 5 ++ ++ ++ ++ ++ ++
10 ++ ++ ++ ++ ++ ++
15 ++ ++ ++ ++ ++ +−
20 ++ +− ++ +− ++ +−
Hg 5 ++ ++ ++ ++ ++ ++
10 ++ ++ ++ ++ ++ ++
15 ++ ++ ++ ++ ++ ++
20 ++ ++ ++ ++ ++ ++
Zn 5 ++ ++ ++ ++ ++ ++
10 ++ ++ ++ ++ ++ ++
15 ++ ++ ++ ++ ++ ++
20 +− +− ++ ++ ++ ++
Cu 5 ++ ++ ++ ++ ++ ++
10 ++ ++ ++ ++ ++ ++
15 ++ ++ ++ ++ ++ ++
20 +− ++ ++ ++ ++ ++
Cd 5 ++ ++ ++ ++ +− ++
10 +− +− +− ++ +− ++
15 +− +− +− ++ +− ++
20 +− +− +− ++ +− ++
Ni 5 ++ ++ ++ − ++ ++
10 ++ ++ ++ − ++ ++
15 ++ ++ ++ − ++ +−
20 +− +− ++ − ++ +−
Cr 5 ++ +− ++ ++ +− ++
10 ++ +− +− ++ +− +−
15 +− +− +− ++ +− +−
20 +− +− +− +− +− +−
Al 5 ++ ++ ++ ++ ++ ++
10 ++ ++ ++ ++ ++ ++
15 ++ ++ ++ ++ ++ ++
20 +− ++ ++ ++ ++ ++

−: No growth; +−: mild growth with some inhibition; ++ heavy growth.

the list of selected six bacteria species isolated from each of the contam- concentrations were pronounced, the overall growth of the microbes
inated soils. Selection of the bacteria species was based on preliminary declined as metal concentrations decreased. This trend was in contrast
impact assessment on total bacteria diversity that demonstrated toler- to the situation in the control (0.0 ppm of metals), where absolute
ance to metal-induced soil. growth of all the bacteria species was observed. At 5 ppm of the metals
The landfill soils have diverse genera of bacteria that are mostly (except Ni) concentrations, absolute/strong growth of all the microbes
Gram-negative organisms, except for Bacillus pumilus, which is Gram- was observed, except for B. pumilus (at Ni concentrations). Both mild
positive. Table 6 shows the results of the heavy metal resistance test. Al- and heavy growth occurred across 10–20 ppm of the metals. Microbes
though the growth across the microbial diversity and metal from both landfills showed absolute resistance to Mn, except for

Table 7
Minimal inhibitory concentrations of heavy metals on the isolates.

Metal Taman Beringin landfill microbes Bukit Beruntung landfill microbes

Aeromonas caviae DNA group 4 Pseudomonas alcaligenes Pseudomonas mendocina Bacillus pumilus Ochrobactrum intermedium Stenotrophomonas acidaminiphilia
Conc(ppm) Conc(ppm) Conc(ppm) Conc(ppm) Conc(ppm) Conc(ppm)

Pb 20 20 N20 N20 N20 N20


Mn N20 N20 N20 N20 N20 N20
Fe N20 20 N20 20 N20 20
Hg N20 N20 N20 N20 N20 N20
Zn 20 20 N20 N20 N20 N20
Cu 20 N20 N20 N20 N20 N20
Cd 10 10 10 N20 5 N20
Ni 20 20 N20 b5 N20 15
Cr 15 5 10 20 5 10
Al 20 N20 N20 N20 N20 N20
B. Jayanthi et al. / Catena 147 (2016) 25–31 29

Fig. 1. Measured inhibition zone diameter (IZD) of A. caviae DNA group 4 exposed to Fig. 3. Measured inhibition zone diameter (IZD) of P. mendocina exposed to different
different metals. metals.

Ochrobactrum intermedium isolated from the BBL (it offered slight resis- various heavy metals towards all the isolated strains. The study did
tance based on the mild growth observed at 15 and 20 ppm of Mn con- not adopt the conventional approach of testing the heavy metals on
centrations). The study observed a similar situation when only the species until the absence of absolute growth; instead, it adopted
Stenotrophomonas acidaminiphilia from the BBL gave slight resistance the set (5–20 ppm) that accommodates the range typical of most envi-
to 15 and 20 ppm of Fe concentrations. Exposure to Hg concentrations ronments. High similarity was observed in the tolerance developed by
(5–20 ppm) showed that all the organisms survived with a heavy the strains towards the heavy metals. P. mendocina demonstrated the
growth pattern. All strains showed varied resistance to the heavy highest tolerance, which we attribute to its nature as Gram-negative
metals. The resistance of microbes to the metal distribution was very bacteria. B. pumilus also demonstrated high tolerance, but did not
significant for all the bacterial species. An overall order of resistance show much resistance to Cd exposure, which is in contrast to the higher
was obtained from the variance of the group means, F. The following se- Cd2+ accumulation potential of Gram-positive bacteria. Previous stud-
quence was observed: Ochrobactrum intermedium [F(p b 0.05) = ies reported that such accumulation leads to the expression of a CadA
119.2] N B. pumilus [F(p b 0.05) = 110.3] N Aeromonas caviae DNA resistance system, which is located on plasmids p1258 and related plas-
group 4 [F(p b 0.05) = 95.9] N Pseudomonas alcaligenes F(p b 0.05) = mids (Novick and Roth, 1968; Nies and Silver, 1995). The resistance is
89.9] N Stenotrophomonas acidaminiphilia [F(p b 0.05) = 63.7] N P. mediated by active ion efflux (Nies and Silver, 1995; Lucious et al.,
medocina [F(p b 0.05) = 46.1]. B. pumilus was absolutely resistant to 2013). Responses of the microbes to the heavy metals were not too het-
all heavy metals used in the study except for Ni. This result may reflect erogeneous, but the result is consistent with findings of Lucious et al.
the ability of the species to grow even when exposed to high concentra- (2013), who reported that both Gram-positive and Gram-negative bac-
tions of respective heavy metals. Such behavior is significant because teria can be resistant to heavy metals.
the organism may potentially have special inhibitory mechanisms to We assessed zones of inhibition to evaluate the bioremediation po-
most free metal ions that are often lethal to the organisms. Therefore, tential of the isolates. Inhibition zone diameter (IZD) of the bacteria spe-
the degree of microbial resistivity to metal concentrations was charac- cies were measured against the heavy metal concentrations during the
terized by the extent of growth on the agar similar to study by resistivity test (Figs. 1–6). The IZDs demonstrated the extent of the toxic
Mgbemena et al. (2012). Resistance mechanisms clearly do not provide effect of the heavy metals on the isolated organisms. About 67% of the
protection at extremely high concentrations of the extractable/free isolated bacteria showed highest IZD on exposure to Cr concentrations,
metal ions (Konopka et al., 1999; Mgbemena et al., 2012). while the remaining isolates recorded highest IZD when exposed to Cd
Minimal inhibitory concentration (MIC) of the heavy metal ions on and Mn concentrations. Figs. 1 and 2 recorded a maximum IZD of 1.1
the bacteria strains were also evaluated from the inoculated media and 1.2 cm for A. caviae DNA group 4 and P. alcaligenes, respectively, at
plates. This procedure sought to establish relative baseline levels of re- exposure to Cr concentrations. P. mendocina was mostly inhibited
sistance against metals to compare values determined with the other upon exposure to Cd concentration with a corresponding 1.1 cm IZD
bacteria species (Lucious et al., 2013). Table 7 represents the MIC of (Fig. 3). This result revealed that both landfill isolates had similar behav-
ior/response to heavy metals because B. pumilus and O. intermedium also

Fig. 2. Measured inhibition zone diameter (IZD) of P. alcaligenes exposed to different


metals. Fig. 4. Measured inhibition zone diameter (IZD) of B. pumilus exposed to different metals.
30 B. Jayanthi et al. / Catena 147 (2016) 25–31

findings established the bacterial species as chemolithotrophs. Howev-


er, a comparison of isolates from operational and non-operational land-
fill sites in Peninsular Malaysia did not show much variation. Instead, all
the bacterial species demonstrated higher susceptibility to chromium
ions than to more toxic metals, especially lead and mercuric ions. This
study established a correlation between the degree of bacteria–metal
tolerance and extent of growth inhibition. It deduced that reduced IZD
is a reflection of increases in tolerance because both conditions show
an inverse relationship during susceptibility assessment. The behavior/
response of the isolated microbes may suggest their suitability for en-
hanced bioremediation of heavy metal-polluted environments due to
tolerance and growth patterns within the metal-polluted system.

Acknowledgment
Fig. 5. Measured inhibition zone diameter (IZD) of O. intermedium exposed to different
metals.
We would like to appreciate the sponsorship from University of Ma-
laya Research Grant (RP011A-14SUS) and the Center of Research Grant
Management (PG070-2014B). Furthermore, the Ministry Of Higher Ed-
recorded highest IZD (1.1 and 1.9 cm, respectively) upon exposure to Cr
ucation Malaysia is acknowledged for providing scholarship
concentrations (Figs. 4 & 5). The highest IZD value recorded by S.
(MyBrain15 – MyPhD). We thank the management of Taman Beringin
acidaminiphilia was 1.9 cm on exposure to Mn concentrations (Fig. 6).
and Bukit Beruntung Landfills for support our research.
This result implied that the reduction in measured IZD after exposing
the isolates may reflect the high tolerance of such microbes to heavy
metal contamination. Results showed a correlation between metal re- References
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