Catena
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.
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
Table 3
Soil characterization.a, b
Test parameter Test method Taman Beringin landfill Bukit Beruntung landfill
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
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 +− ++ ++ ++ ++ ++
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.
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)
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
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
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