ARTICLE IN PRESS

0964-8305/$ - se

doi:10.1016/j.ib

�Correspondfax: +965 4847

E-mail addr

International Biodeterioration & Biodegradation 56 (2005) 1–7

www.elsevier.com/locate/ibiod

Inhibition of hydrocarbon bioremediation by lead in a crudeoil-contaminated soil

Esmaeil S. AL-Saleh�, Christian Obuekwe

Microbiology Program, Department of Biological Sciences, P.O. Box 5969, Safat 13060, Faculty of Science, Kuwait University, State of Kuwait

Abstract

Analyses of soil samples revealed that the level of lead (total or bioavailable) was three-fold greater in crude oil contaminated than

in uncontaminated Kuwaiti soils. Investigation of the possible inhibitory effect of lead on hydrocarbon degradation by the soil

microbiota showed that the number of hydrocarbon-degrading bacteria decreased with increased levels of lead nitrate added to soil

samples, whether oil polluted or not. At 1.0mg lead nitrate g�1 dry soil, the number of degraders of hexadecane, naphthalene and

crude oil declined by 14%, 23% and 53%, respectively. In a similar manner, the degradation and mineralization of different

hydrocarbons decreased with increased lead content in cultures, although the decreases were not significantly different (P40:05).The dehydrogenase activities of soil samples containing hydrocarbons as substrates also declined with an increase in the lead content

of soil samples.

r 2004 Elsevier Ltd. All rights reserved.

Keywords: Bioremediation; Hydrocarbons; Metal pollution; Mineralization

1. Introduction

Accidental and deliberate crude oil spills have been,and still continue to be, a significant source ofenvironmental pollution. Approx. 6� 107 barrels of oilwas spread over 2� 107m3 soil and 320 oil lakes werecreated across the desert during the first Gulf War inKuwait. Bioremediation of these oil lakes is thetechnology of choice, but this natural process isinfluenced by many factors, including the presence ofinhibitory compounds such as heavy metals. In fact,many sites that are contaminated with organic wastecontain high concentrations of heavy metals, which caninhibit the natural microbiota (Begley et al., 1996).Heavy metals at certain concentrations constrainmicrobial activity (Giller et al., 1998; Baath et al.,1998) and hence may create serious problems, especiallyfor authorities that depend on bioremediation as the

e front matter r 2004 Elsevier Ltd. All rights reserved.

iod.2004.11.003

ing author. Tel.: +965 4811188 x 5652;

054.

ess: keva5000@hotmail.com (E.S. AL-Saleh).

main strategy for pollution alleviation. In general,available technologies for metal remediation in soil areoften disruptive and costly, and usually total removal ofall metals is not feasible. Therefore, the decision toremediate hydrocarbon-contaminated sites pollutedwith metals should be based on risk assessment studieson the extent of inhibition which the metals exert onhydrocarbon bioremediation. Lead at certain concen-trations has been reported to be inhibitory to microbialactivity (Alloway, 1990; Hughes and Poole, 1989;Laskowski et al., 1994) and its presence in highconcentrations in hydrocarbon contaminated sites canprolong the bioremediation of these sites. Even atrelatively low concentrations, the inhibitory effect oflead has been shown to be accentuated in the presence ofother cations (Laskowski et al., 1994). In Kuwait, leadhas been detected in sediments collected from 21 coastalareas receiving industrial effluents. The concentrationsranged between 0.4 and 39mg kg�1 (Beg et al., 2001).However, no reports of studies are available concerningthe level of lead in hydrocarbon contaminated sites inKuwait and the possible inhibitory role of lead in

ARTICLE IN PRESSE.S. AL-Saleh, C. Obuekwe / International Biodeterioration & Biodegradation 56 (2005) 1–72

limiting bioremediation of such sites. Therefore, theprimary objective of this work was to determine theconcentration of lead in a hydrocarbon-contaminatedsite in the southern area of Kuwait and to determine thepotential inhibitory effects of lead on the biodegradationof polyaromatic and aliphatic hydrocarbons by theindigenous soil microbiota.

2. Materials and methods

2.1. Soil samples and treatment

Soil samples were aseptically collected during the latesummer period, from the upper 10 cm of crude oilcontaminated and uncontaminated sites in AL-Douha,south east of Kuwait city, Kuwait. The general area ofAL-Douha was subject to crude oil contamination. Thesoil is a sandy loam with the following particledistribution: sand 74.4%, silt 14.2% and clay 11.0%.Each soil sample comprised 5–7 scoops of soil from a

1m2 area, pooled and mixed in a sterile zipped bag. Thissampling mode was adopted randomly in three siteswithin the contaminated and uncontaminated sites. Thesoil samples were sieved (o2mm), moistened to 30% ofthe maximum water-holding capacity, and incubatedundisturbed at 25 1C for one week. Based on preliminaryexperiments, this procedure was adjudged necessary tostabilize and restore the active indigenous microbiota ofthe soil samples.Before use for cultural purposes, the soil samples were

divided into several portions, which were separatelyamended to contain different concentrations of leadnitrate (0.1, .05, 1.0mg g�1 dry soil) and naphthalene(1mg g�1) or hexadecane (1.1mg g�1).

2.2. Total and bioavailable (soluble) lead in soil

The total lead content of the soil samples wereanalysed according to Rowell (1994). The methodinvolved digestion of dried soil samples, followed byanalysis of the digest by flame atomic absorptionspectrometry (FAAS) using a Varian AA-1475 FAAS.The same method (Rowell, 1994) was also applied in thedetermination of bioavailable (soluble) lead followingextraction in deionised water and digestion.

2.3. Enumeration of lead-tolerant bacteria in soil

The culturable hydrocarbon-degrading, lead-tolerantbacteria, and the total heterotrophic, lead-tolerantbacteria present in the soil samples were determined bya standard plate dilution method on Hutner’s minimalagar (Cohen-Bazire et al., 1957) containing hydrocar-bons and Luria-Bertani (LB) agar (Maniatis et al.,1989), respectively. Both the Hutner’s minimal and LB

agars were prepared containing different concentrationsof lead nitrate (0, 0.1, 0.5 and 1.0mgml�1 medium). A10-g portion of each soil sample was suspended in 40ml50mM phosphate buffer (pH 7.2), and vortexed for1min before diluting in the same buffer up to 10�6.Aliquots (0.1ml) were spread in triplicates on Hutner’sminimal agar and LB agar containing various concen-trations of lead nitrate. The Hutner’s minimal agarcontained crude oil, naphthalene or hexadecane (as solecarbon and energy source) supplied in vapour phase indesiccators. All plates were incubated at 30 1C for up to14 days.

2.4. Effect of lead nitrate on hydrocarbon degradation

The effects of different concentrations of lead (0, 0.1,0.5, 1.0mg g�1 dry soil) on the degradation ofhydrocarbons by the indigenous microbiota of the soilsamples were determined in cultures containing thevariously lead-amended soils and different hydrocarbonsubstrates. Each culture consisted of 100ml Hutner’sminimal liquid medium, contained in 250-ml screw-capbottles, supplemented with various levels of leadnitrate, 100 g of each soil sample, and naphthalene,hexadecane or crude oil supplied at the rate of 1mg g�1

dry soil. Hexadecane was supplied at the rate of1.1mg g�1 dry soil. All cultures were in triplicate andwere incubated at 30 1C for up to 14 days in NewBrunswick shaking incubator operating at 200 rpm. Thecontrol flasks contained autoclaved soil samples, andsoil samples without hydrocarbon substrates.

2.5. Recovery and analysis of residual hydrocarbons

The residual hydrocarbon present in each culturebottle after the growth of the soil microbiota wasrecovered by three extractions with 10ml dichloro-methane at 5 1C. Extracts were pooled and filteredthrough a Acrodisc CRPTFE filter (pore size 0.45 mm)to remove emulsions. Extracts then were analysedchromatographically in Varian 3400 chromatographicequipment fitted with a 30� 0.32mm DB5 capillarycolumn (J & W Scientific, Faison, USA) and equippedwith a flame-ionization detector. The column wasmaintained initially at 50 1C for 5min and increased atthe programmed rate of 7 1Cmin�1 to 270 1C. Injectorand detector temperatures were maintained at 290 1C.The carrier gas was nitrogen at a flow rate of 0.5 ml s�1.

2.6. Effect of lead nitrate on hydrocarbon mineralization

in soil

The effects of lead nitrate (0, 0.1, 0.5 and 1.0mg g�1

soil) on hydrocarbon mineralization by the indigenousmicrobiota in soil samples were determined in a Micro-Oxymax respirometer. (Columbus Instruments). The

ARTICLE IN PRESSE.S. AL-Saleh, C. Obuekwe / International Biodeterioration & Biodegradation 56 (2005) 1–7 3

reaction vessels contained 40 g soil, the water content ofwhich was adjusted to 30%. Soil samples were amendedwith naphthalene or hexadecane (1mg and 1.1mg g�1

soil, respectively) and the desired metal concentration.The reaction vessels were incubated in a shaking waterbath at 30 1C. The control constituted vessels inoculatedwith autoclaved soil samples, and soil samples withoutadded hydrocarbons.

2.7. Effect of lead nitrate on the dehydrogenase activity

of soil

The method of Alef and Nannipieri (1995) was usedto determine the effect of lead nitrate on the dehydro-genase activity of the soil samples containing naphtha-lene or hexadecane as substrate. Each assay tube was a200-ml screw-cap centrifuge tube containing 5 g soilamended with 0, 0.1, 0.5, 1.0mg lead nitrate, and 1mgnaphthalene or 1.1mg hexadecane, all g�1 soil) samples.The assay tube also contained 5ml triphenyltetrazolium

Table 1

Total and dissolved concentrations of lead in hydrocarbon contami-

nated and uncontaminated soils

Soil sample Lead

Total

(mg kg�1 soil)Dissolved

(mg l�1)

Hydrocarbon contaminated 43007300 30.572

Uncontaminated 1200780 970.6

Each value is the mean of triplicate analyses of three soil

samples7S.D.

Table 2

Occurrence of hexadecane-degrading lead-tolerant bacteria (cfu g�1 soil) in h

Soil sample Lead nitrate (mgml�1)

0 0.1

Hydrocarbon contaminated 2.0� 10570.15 1.9�

Uncontaminated 0.78� 10570.68 0.70�

Each value is the mean of triplicate analyses of three soil samples7S.D.

Table 3

Occurrence of naphthalene-degrading lead-tolerant bacteria (cfu g�1 soil) in

Soil sample Lead nitrate (mgml�1)

0 0.1

Hydrocarbon contaminated 4.0� 10370.30 3.6�

Uncontaminated 1.0� 10370.07 0.88�

Each value is the mean of triplicate analyses of three soil samples7S.D.

chloride (TTC) solution prepared by dissolving 0.1 gTTC in 100ml 100mM Tris HCl buffer (pH 7.6). Thesealed tubes were incubated at 30 1C in a NewBrunswick shaking incubator operating at 200 rpm for24 h. The controls were assay systems containingautoclaved soil samples and tubes without addedhydrocarbons. Following incubation, 40ml acetonewas added to each tube and the tubes agitated in thedark for the extraction of the reduction product,triphenyl formazan (TPF). The extract was membrane-filtered (0.45 mm) and TPF measured at 546 nm in aGenesis 5 spectrophotometer.

3. Results

The concentrations (total and bioavailable) of lead inhydrocarbon contaminated and uncontaminated soilsamples from the AL-Douha area of Kuwait (Table 1)varied widely but were significantly higher (Po0.05) inhydrocarbon contaminated samples than in uncontami-nated samples. When the number of microorganismsthat were able to grow on different hydrocarbonsubstrates in the presence of different levels of leadwere determined (Tables 2–4), generally, whetherthe substrate was hexadecane (Table 2), naphthalene(Table 3) or crude oil (Table 4), the number oforganisms present in the soil samples that were able togrow on each substrate declined with increasingamounts of lead in the growth media. This decline inmicrobial numbers that were able to grow on thedifferent hydrocarbon substrates was more pronouncedamong the organisms able to grow on crude oil (50%decline) than on naphthalene (25–27%) or hexadecane

ydrocarbon-contaminated and-uncontaminated soils

0.5 1.0

10570.15 1.8� 10570.14 1.7� 10570.14

10570.54 0.65� 10570.48 0.60� 10570.50

hydrocarbon-contaminated and-uncontaminated soils

0.5 1.0

10370.27 3.4� 10370.26 3.0� 10370.22

10370.07 0.80� 10370.06 0.74� 10370.05

ARTICLE IN PRESS

Table 4

Occurrence of crude oil-degrading lead-tolerant bacteria (cfu g�1 soil) in hydrocarbon-contaminated and -uncontaminated soil

Soil sample Lead nitrate (mgml�1)

0 0.1 0.5 1.0

Hydrocarbon contaminated 3.4� 10570.25 3� 10570.22 2.4� 10570.18 1.8� 10570.14

Uncontaminated 1.2� 10570.09 1.1� 10570.08 0.71� 10570.52 0.63� 10570.47

Each value is the mean of triplicate analyses of three soil samples7S.D.

0

10

20

30

40

50

60

Hexadecane Naphthalene Crude oil

% d

ecre

ase

in b

acte

rial

cou

nts

Contaminated soil Uncontaminated soil

Fig. 1. Percent decrease in the counts of hydrocarbon-degrading

bacteria in soil sample amended with lead nitrate (1mg g�1 soil). Each

value is the mean7S.D. of triplicate analyses of three soil samples.

0

0.2

0.4

0.6

0.8

1

1.2

0.1 0.5 1

Lead nitrate (mg g-1 soil)

Hyd

roca

rbon

deg

rade

d (m

g)

Naphthalene Hexadecane

Fig. 2. Effect of lead nitrate on the degradation of hydrocarbons in the

uncontaminated soil. Each value is the mean7S.D. of triplicate

analyses of three soil samples.

E.S. AL-Saleh, C. Obuekwe / International Biodeterioration & Biodegradation 56 (2005) 1–74

(15–22%), depending on whether the soil was crude oil-contaminated or not (Fig. 1).Examination of the ability of the soil microbiota to

degrade hydrocarbons in media containing differentconcentrations of lead (Fig. 2) revealed a generaldecrease in the amount of hydrocarbons degraded withincreased levels of lead present in the medium. However,there was no marked difference in this inhibitory effectof lead on the degradation of individual hydrocarbonsubstrates by the indigenous microbiota of the con-taminated and uncontaminated soil samples. The effectof lead on the mineralization of hexadecane andnaphthalene (Tables 5 and 6) was evident from theamount of carbon dioxide evolved from these substratesdecreasing with an increase in the concentration of leadin the medium. Surprisingly, the data showed thataddition of lead elicited a greater inhibitory effect onmineralization of the substrates by the indigenousmicrobiota in the contaminated soil than by thecorresponding microbiota of uncontaminated soil. Thisinhibitory effect on mineralization of naphthalene andhexadecane increased linearly with an increase in leadconcentration (Fig. 3).As shown in Table 7, the dehydrogenase activities of

the soil samples with hydrocarbon substrates alsodeclined with an increase in lead concentration.

4. Discussion

The analysis of soil samples from crude oil con-taminated and uncontaminated areas of AL-Douha,Kuwait showed that the lead content (total andbioavailable) was significantly higher (Po0.05) in theformer, a situation that points to the crude oil as thesource of the metal contaminants. It is well known thatcrude oil contains a wide variety and levels of heavymetals associated with the humic or asphaltene fractionsof crude oils (Tissot and Wette, 1978). The levels of leaddetermined in the soil samples are within the rangereported for coastal sediments contaminated by indus-trial effluents (Beg et al., 2001), and comparable to levelsof contamination in soils in the vicinity of metalsmelting plants (Laskowski et al., 1994). Although thetotal lead content was higher, it varied markedlybetween samples taken from the area. This variationwould reflect the heterogeneity of soils in this zoneresulting from dumping of contaminated soils fromdifferent sources following a wide-scale oil pollution as aresult of invasion by Iraqi forces. The apparently lowlevels of soluble lead observed in these soil sampleswould suggest a low risk of lead toxicity to soil

ARTICLE IN PRESS

Table 6

Effect of lead nitrate on the mineralization (mgCO2 g�1 soil) of naphthalene in hydrocarbon-contaminated and -uncontaminated soils

Soil sample Lead nitrate (mg g�1 soil)

0 0.1 0.5 1.0

Hydrocarbon contaminated 29.472.2 29.1172.2 28.2272.0 26.8072.0

Uncontaminated 38.5373.0 38.1473.0 36.9872.6 35.1272.7

Each value is the mean of triplicate analysis of three soil samples7S.D.

0

4

8

12

16

20

0 0.2 0.4 0.6 0.8 1

Lead nitrate (mg g-1 soil)

Ext

ent o

f in

hibi

tion

(%)

Naphthalene Hexadecane

Fig. 3. Inhibition of hexadecane and naphthalene mineralization in

lead nitrate- amended uncontaminated soil.

Table 5

Effect of lead nitrate on the mineralization (mgCO2 g�1 soil) of hexadecane in hydrocarbon-contaminated and -uncontaminated soils

Soil sample Lead nitrate (mg g�1 soil)

0 0.1 0.5 1.0

Hydrocarbon contaminated 40.8073.0 38.9372.9 37.6972.7 34.7672.6

Uncontaminated 53.5273.7 49.9673.7 47.873.2 44.7673.2

Each value is the mean of triplicate analyses of three soil samples7S.D.

E.S. AL-Saleh, C. Obuekwe / International Biodeterioration & Biodegradation 56 (2005) 1–7 5

microbiota since it is commonly believed that extrac-table metal ions in soils are related to metal uptake and,therefore, toxicity to organisms (Logan and Chaney,1983). The current opinion is that extractable metals insoils are not an exact measure of the free ionicconcentrations available to organisms, since severalenvironmental factors are known to influence thebioavailability of metals in soil (Giller et al., 1998;Angle et al., 1993). Moreover, it has been reported(Laskowski et al., 1994) that even at moderately lowlevels of lead, interaction of the metal ion with othercations enhances its toxic effects. Thus, the apparentlylow level of extractable lead observed in this study maynot indicate the actual level available during the growthof the hydrocarbon degraders. For, example, it is knownthat hydrocarbon degradation is normally accompaniedby the acidification of the environment (Obuekwe andAL-Zarban, 1998) as a result of the presence of carbon

dioxide and organic acids as degradation products. ThepH is one of the most important factos in thesolubilization of metals in soils (Giller et al., 1998).Consequently, the available lead in the soil would beexpected to deviate (usually increase) with the solubili-zation of insoluble forms as a result of the acidity arisingfrom hydrocarbon degradation. Such solubilization,coupled with the prevailing high temperature and aridityat sampling time, would increase the stress imposed onthe natural microbiota of the environment, and might beresponsible for the low soil microbial counts (data notincluded) observed in our preliminary experiments. Itwas this low level of microbiota of the soils thatnecessitated the rehydration and pre-incubation of thesoil samples in order to re-establish and stabilize theindigenous microbial populations before the onset ofthis investigation.The results of this study show a general decline in the

number of soil bacteria that were able to grow on amedia containing increasing levels of lead. This declinewas, however, not significant between the different levelsof lead. Similar observations on biomass, numbers andactivities of microbes have been reported by severalauthors (Angle and Chaney, 1991; Angle et al., 1993;Bardgett and Saggar, 1994; Baath et al., 1998; Horswellet al., 2003). These effects derive from the selectivepressure exerted by the elevated heavy metal levels onsoil microorganisms such that only the culturable,tolerant and resistant species survive. The nonsignificantalterations in the number of bacteria resistant to higherlevels of heavy metals has been ascribed to theoccurrence of intrinsic resistance of soil bacteria toheavy metals (Angle et al., 1993).The growth of soil bacteria on Hutner’s minimal

medium containing hydrocarbons as sole carbon source

ARTICLE IN PRESS

Table 7

Effect of lead nitrate on dehydrogenase activity (mgTPFg�1 soil) of hydrocarbon-amended soil samples

Soil sample Substrate Lead nitrate (mgml�1)

0 0.1 0.5 1.0

Hydrocrbon contaminated Naphthalene 0.17070.01 0.15370.009 0.14470.008 0.12870.007

Hexadecane 0.37070.02 0.35170.017 0.33370.016 0.31570.016

Uncontaminated Naphthalene 0.15070.01 0.14970.009 0.14470.007 0.13570.007

Hexadecane 0.41570.03 0.38270.028 0.36570.027 0.34070.024

Each value is the mean of triplicate analyses of three soil samples7S.D.

E.S. AL-Saleh, C. Obuekwe / International Biodeterioration & Biodegradation 56 (2005) 1–76

indicates their ability to utilize the substrates as the solecarbon and energy sources. Therefore, the observeddecline in the number of hydrocarbon-degraders uponthe addition of lead suggests a decrease in theutilization, and consequently bioremediation, of hydro-carbons in environments contaminated with lead. Thisdecline in the number of hydrocarbon-degraders ap-peared to be reflected in the amount of the hydrocarbonsubstrate degraded, which decreased with an increase inthe level of lead in culture. Although little information isavailable in the literature which has specifically ad-dressed the inhibition of hydrocarbon, nonhydrocarbonorganic matter degradation in soil is known to beinhibited by metal pollution (Said and Lewis, 1991;Bardgett and Saggar, 1994; Laskowski et al., 1994;Giller et al., 1998). The need to expend energy derivedfrom hydrocarbon utilization to counter the effects ofthe stressful environment (maintenance energy) arisingfrom lead addition would reduce the energy availablefor biomass synthesis, which would result in decline ingrowth and number with attendance effects. However, itis possible that the reduced level of hydrocarbondegradation observed with rising levels of lead was aresult of toxicity of lead, per se, on the degradationpathways, and not as a result of population decline.As observed in degradation, mineralization of the

hydrocarbon substrates was also increasingly inhibitedupon addition of higher levels of lead. Surprisingly, thisinhibitory effect was more pronounced in crude oilcontaminated soil samples which had a higher level oflead, than was the case with uncontaminated soilsamples. This observation appears contrary to manyprevious reports (Chander and Brookes, 1991a; Bremerand Kuikman, 1994; Dahlin and Witter, 1998; Wetteand Kanal, 1998) in which carbon dioxide evolutionfrom organic matter (nonhydrocarbon) was greater inmetal-stressed than non-stressed microorganisms. How-ever, the apparent anomaly in this study may beexplained by a possible shift in the soil microbiota, asa result of selective pressure exerted by metal contam-ination, to a population with an inherently lowercapacity for hydrocarbon mineralization. Such popula-tion and physiological shifts in soil microbiota are notuncommon (Baath et al., 1998; Giller et al., 1998;

Ekelund et al., 2003). Linear regression analysis showeda high positive correlation (R2 ¼ 0:99) between thedegree of inhibition of hydrocarbon mineralization andthe amount of lead added to cultures. The observedinhibitory effect of lead on dehydrogenase activity of thesoil samples is consistent with previous observations onheavy metal contamination in soils (Chander andBrookes, 1991b; Kelly and Tate, 1998; Kelly et al.,1999).

Acknowledgement

This project was funded by a Grant numberSO 04/00 from the Research Administration of KuwaitUniversity.

References

Alef, K., Nannipieri, P., 1995. Soil sampling, handling, storage and

analysis. In: Alef, K., Nannipieri, P. (Eds.), Methods in Applied

Soil Microbiology and Biochemistry. Academic Press, London,

pp. 49–115.

Alloway, B.J., 1990. Soil processes and the behavior of metals. In:

Alloway, B.J. (Ed.), Heavy metals in soils. Blackie and Son,

London, pp. 7–27.

Angle, J.S., Chaney, R.L., 1991. Heavy metal effects on soil

populations and heavy metal tolerance of Rhizobium meliloti,

nodulation and growth of alfalfa. Water, Air and Soil Pollution

57–58, 579–604.

Angle, J.S., Chaney, R.L., Rhee, D., 1993. Bacterial resistance to

heavy metals related to extractable and total metal concentrations

in soil and media. Soil Biology and Biochemistry 25, 1443–1446.

Baath, E., Diz-Ravina, M., Frostegard, A., Campell, C.D., 1998.

Effect of metal-rich sludge amendments on the soil microbial

community. Applied and Environmental Microbiology 64,

238–245.

Bardgett, R.D., Saggar, S., 1994. Effects of heavy metal contamination

on the short-term decomposition of labeled [14C] glucose in a

pasture soil. Soil Biology and Biochemistry 26, 727–733.

Begley, J., Croft, B., Swannell, P.J., 1996. Current research into the

bioremediation of contaminated land. Land Contamination and

Reclamation 4, 199–208.

Beg, M.U., Al-Muzaini, S., Saeed, T., Jacob, P.G., Beg, K.R.,

Al-Bahloul, M., Al-Matrouk, K., Al-Obaid, T., Kurian, A., 2001.

Chemical contamination and toxicity of sediment from a coastal

area receiving industrial effluents in Kuwait. Archives of Environ-

mental Contamination and Toxicology 41, 289–297.

ARTICLE IN PRESSE.S. AL-Saleh, C. Obuekwe / International Biodeterioration & Biodegradation 56 (2005) 1–7 7

Bremer, E., Kuikman, P., 1994. Microbial utilization of 14C/u/glucose

in soil is affected by the amount and time of glucose addition. Soil

Biology and Biochemistry 26, 511–517.

Chander, K., Brookes, P.C., 1991a. Microbial biomass dynamics

during the decomposition of glucose and maize in metal-

contaminated and uncontaminated soils. Soil Biology and Bio-

chemistry 23, 917–925.

Chander, K., Brookes, P.C., 1991b. Is the hydrogenase assay invalid as

a method to estimate microbial activity in copper-contaminated

soils. Soil Biology and Biochemistry 23, 909–915.

Cohen-Bazire, G., Sistrom, W.R., Stainer, R.Y., 1957. Kinetic studies

of pigment synthesis by non-sulfur purple bacteria. Journal of Cell

Component Physiology 49, 25–68.

Dahlin, S., Witter, E., 1998. Can the low microbial biomass C-to-

organic C ratios in an acid and a metal contaminated soil be

applied by difference in growth characteristics, substrate utilization

efficiency or maintenance requirements? Soil Biology and Bio-

chemistry 30, 633–641.

Ekelund, F., Olsson, S., Johansen, A., 2003. Changes in the succession

and diversity of protozoan and microbial populations in soil spiked

with a range of copper concentrations. Soil Biology and

Biochemistry 35, 1507–1516.

Giller, K.E., Witter, E., McGrath, S., 1998. Toxicity of heavy metals to

microorganisms and microbial processes in agricultural soils: A

review. Soil Biology and Biochemistry 30, 1389–1414.

Horswell, J., Speir, T.W., van Schaik, A.P., 2003. Bio-indicators to

assess impacts of heavy metals in land-applied sewage sludge. Soil

Biology and Biochemistry 35, 1501–1505.

Hughes, M.N., Poole, R.K., 1989. The functions of metals. In:

Hughes, M.N., Poole, R.K. (Eds.), Metals and Microorganisms.

Chapman & Hall, London, pp. 1–37.

Kelly, J.J., Tate, R.L., 1998. Use of BIOLOG for the analysis of

microbial communities from zinc contaminated soils. Journal of

Environmental Quality 27, 600–608.

Kelly, J.J., Haggblom, M., Tate, R.L., 1999. Effects of the land

application of sewage sludge on soil heavy metal concentrations

and soil microbial communities. Soil Biology and Biochemistry 31,

1467–1470.

Laskowski, R., Maryanski, M., Niklinska, M., 1994. Effect of heavy

metals and mineral nutrients on forest litter respiration rate.

Environmental Pollution 84, 97–102.

Maniatis, T., Sambrook, J., Fritsch, E.F. (Eds.), 1989. Molecular

Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory

Press, New York.

Rowell, D.L., 1994. Pesticidesandmetals. In: Rowell, D.L.

(Ed.), Soil science. Addison Wesley Longmann, London,

pp. 303–327.

Said, W.A., Lewis, D.L., 1991. Quantitative assessment of the effects

of metals on microbial degradation of organic chemicals. Applied

and Environmental Microbiology 57, 1498–1503.

Tissot, B.P., Wette, D.H., 1978. Petroleum Formation and Occur-

rence. A New Approach to Oil and Gas Exploration. Springer,

Berlin, pp. 538.

Wette, E., Kanal, A., 1998. Characterization of soil microbial biomass

in soils from a long-term field experiment with different levels of C

input. Applied and Soil Ecology 10, 37–49.