Effects of cement components and contaminants on dehydrogenase activity … dataset on... ·...

October 2012

Laboratory Report on Dehydrogenase Assay Experiments in Soil-Cement Systems

1

Effects of cement components and

contaminants on dehydrogenase activity

in a mixed contaminated soi l

Reginald B. Kogbara1,2

1Department of Engineering, University of Cambridge, Trumpington Street, Cambridge CB2 1PZ, UK.

2Present Address: Mechanical Engineering Program, Texas A&M University at Qatar,

P.O. Box 23874, Education City, Doha, Qatar.

Email: [email protected]

Summary

Microbiological findings from investigations aimed at encouraging biodegradation of organics

within cement-stabilized contaminated soils revealed that magnesium phosphate cement could

resuscitate and sustain microbial activity in a heavily polluted soil. Microbial activity in soil

contaminated with lead nitrate, zinc chloride and 2-chlorobenzoic acid and treated with

magnesium phosphate cement formulations was monitored using dehydrogenase assay. Low

levels of the contaminants stimulated soil dehydrogenase activity. There was a total inhibition of

soil dehydrogenase activity at co-contamination levels above 1500 mg/kg. Magnesium oxide is

identified as a stimulant, triple super phosphate an inhibitor, of dehydrogenase activity. The

implications of these findings would be useful in improving the robustness of biological clean-up

Some of the findings in this report have been published as part of a wider study in the paper,

Kogbara et al. (2011), Water Air Soil Pollut. 216: 411–427, DOI: 10.1007/s11270-010-0541-7.

mailto:[email protected]

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of polluted sites, as well as in fertiliser management practices for agricultural productivity.

Introduction

Co-contamination with organics and heavy metals is a common feature of polluted soils. This

has led to concerted efforts to develop remediation techniques that could deal with a wide range

of contamination. One such technique is the incorporation of a biodegradative mechanism

within a soil treated with stabilization/solidification (S/S) binders. Stabilization/solidification

basically involves chemical fixation and physical encapsulation of contaminants by the addition

of binders, mainly Portland cement-(PC) based (Kogbara et al., 2011a; Kogbara et al., 2012). It

is effective and widely used for treatment of heavy metals but not very effective with organics.

Details of the techniques and processes of the technology can be found elsewhere (Conner and

Hoeffner, 1998).

Successful incorporation of a biodegradative mechanism within stabilized/solidified soils

requires a cementititious system with pH lower than that of PC (12 – 13) since the high alkalinity

of PC is unsuitable for biological activity. This led to research into magnesium phosphate

cements (MPCs). MPCs like other phosphate based cements are formed at room temperature by

rapid acid-base reaction between dead burned magnesia and an acid phosphate source. A

comprehensive d e s c r ip t io n o f t he cem en t has b een docum ented ( Wagh, 2 0 0 4 ). A

m a j o r advantage of MPCs beneficial to biological activity is its potential to form mixes with

different pH ranges, depending on the magnesia and phosphate contents. The MPCs

employed in this study had triple super phosphate (TSP, supplied by RS Minerals, UK) as acid

phosphate source and dead burned magnesia (MgO, Richard Baker Harrison, UK) as base

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source. TSP is a common fertilizer; it is composed mainly of calcium dihydrogen phosphate,

and also contains some gypsum (Iyengar and Al-Tabbaa, 2008). The chemical constituents of

dead burned magnesia have been reported (Harbottle and Al-Tabbaa 2008; Kogbara et al.,

2011b).

Harbottle and Al-Tabbaa (2006) investigated enhancing microbial activity in cementitious

systems using different additives like compost, nutrients, oxygen releasing compounds, water

retaining compounds and carbon sources. The study demonstrated the effectiveness of compost

in this regard as it provides a nutrient and microbe source as well as enhances the porosity of the

final S/S treated soil. Compost has also been found to enhance biodegradation of

recalcitrant hydrocarbons in hazardous wastes (Ayotamuno et al., 2010). The above

cited works set the stage for the present study which sought to encourage microbial activity

within a co-contaminated soil amended with compost and treated with magnesium phosphate

cement formulations. It is thought that successful maintenance of microbial life within an S/S

system would bring about a sustainable remediation technique where heavy metals are stabilized

and organics biodegraded over time. Thus, it was the aim of this study to investigate the

possibility of encouraging microbial activity in a co-contaminated soil with relatively high

concentration of contaminants, treated with an S/S binder.

In the experiments described, a model silty sand soil was prepared using Fraction-D Leighton

Buzzard sand (D50 ~150-300µm, David Ball Ltd., UK) and silt (silica flour, D50< 150µm, David Ball

Ltd, UK). Eco-compost (sieved past 2mm; Scotsdales Ltd, Cambridge, UK) was added as organic

matter to provide a microbial inoculum as well as a source of nutrients and to enhance the porosity of

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the soil/cement system. A model soil was used in place of real site soil so that the exact chemical

composition of the soil would be known and any effect on microbial assay test result by soil elements

isolated. The soil was composed of 70% sand, 20% silt and 10% compost. It had 15% moisture

content, dry weight. The effect of increased compost content on microbial activity was examined by

using a soil composed of 60% sand, 15% silt and 25% compost. The soil was contaminated with 2-

chlorobenzoic acid, lead nitrate and zinc chloride as organic and metallic contaminants and treated

with magnesium phosphate cement formulations.

Microbial activity was monitored using dehydrogenase assay following the combined methods of

Casida et al. (1964) and Harbottle and Al-Tabbaa (2008). The assay is indicative of the total

metabolic activity of soil microorganisms and has been used in several works (Weaver et al.,

1994). It has been shown to be very sensitive for evaluating the toxicity of anthropogenic

pollutants in soil (Gong et al., 1997). The test involved 6g of soil or crushed soil-cement and 3.5

ml of 0.75% 2,3,5-Triphenyltetrazolium chloride (TTC) solution containing 50mM Trizma

hydrochloride. Incubation was done for 24 hours at 37 C. Extraction of 2,3,5-

triphenyltetrazolium formazan (TPF—a water-insoluble red dye) was done with ethanol and the

concentration measured with a UV/VIS Spectrophotometer at a wavelength of 485 nm.

Results and discussion

Contamination of the soil with 3000 mg/kg each of lead nitrate, zinc chloride and 2-

chlorobenzoic acid caused a total inhibition of dehydrogenase activity, but the uncontaminated

soil with 10% and 25% compost had 3.582 µg TPF/ml and 6.270 µg TPF/ml respectively. Soil

contamination with 3000 mg/kg of only 2CBA and simultaneous addition of 3000 mg/kg of all

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three contaminants to compost only, further demonstrated that dehydrogenase activity was

inhibited by the high concentration of contaminants (Table 1, section A). Previous studies have

reported significant inhibition of soil microbial activity by heavy metals. Marzadori et al. (1996)

reported significant decrease in dehydrogenase activity with the addition of 5000 mg/kg lead,

while Salminen et al. (2001) reported that microbial biomass and activity were clearly reduced at

a zinc concentration of 1000 mg/kg. Compared with single pollutants, combined pollution is

obviously much more complex. Results obtained from combined pollution are generally more

difficult to interpret because of large number of factors and interactions between factors (Gong et

al., 1997). This led to investigations on the concentrations of the contaminants that can be

tolerated.

Five levels of co-contamination were arbitrarily chosen to investigate the range of concentration

at which dehydrogenase activity can occur in the soil. Section B of Table 1 shows that at levels

of contamination up to 187.5 mg/kg there was a stimulation of soil dehydrogenase activity as the

amount of TPF produced was more than that of the uncontaminated soil. Significant stimulation

of dehydrogenase activity has previously been observed on contaminated soil with poplar but

was thought to be influenced by the covering plant (Gong et al., 1997). The data shows that at

lower contaminant concentrations the contaminants may be useful to soil microbes as nutrients

thereby stimulating microbial activity. This is similar to the findings of previous studies where

microbial numbers increased in the aftermath of hydrocarbon contamination (Ayotamuno et al.,

2006; Kogbara 2008). However, at 750 mg/kg co-contamination, dehydrogenase activity was

reduced. At co-contamination levels ≥1,500 mg/kg there was a total inhibition of microbial

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activity.

Table 1. Dehydrogenase activity measurements on soil

Section Treatment Dehydrogenase activity

(µg TPF/ml)

A Model soil with 10% compost, contaminated with

3000 mg/kg each of 2CBA, Pb(NO3)2 and ZnCl2

0.000

Uncontaminated model soil with 10% compost content 3.582 + 1.476

Uncontaminated model soil with 25% compost content 6.270 + 0.138

Compost only, with 3000 mg/kg co-contamination 0.000

Model soil with 10% compost, contaminated with

3000 mg/kg of only 2CBA

0.000

B Simultaneous addition of 2CBA,

Pb(NO3)2 and ZnCl2 to model soil

with 10% compost content at the

following concentrations

0 mg/kg 3.582 + 1.476

93.75 mg/kg 7.602 + 1.080

187.5 mg/kg 5.724 + 1.230

750 mg/kg 3.000 + 0.792

1,500 mg/kg 0.000

2,250 mg/kg 0.000

Results represent mean + standard deviation of three replicates

After the contaminated soil was treated with two MPC formulations, it was observed that one

formulation resuscitated dehydrogenase activity while the other did not. The two cement

formulations used had the base materials mixed in the ratio, TSP:MgO = 8:1 for the lower pH

end (~6.5) and TSP:MgO = 1:2 for the higher pH end (~10). The soil:cement ratio was 2:1 for

both formulations. De-ionised water was used to produce the wet grout in which the

cement:water ratio was 2:1. Contaminated soil samples treated with TSP:MgO = 1:2 showed

evidence of microbial activity after 14 days of treatment but those treated with TSP:MgO = 8:1

did not. To investigate this, dehydrogenase activity test was carried out on crushed soil-cement

samples without any contamination, five days after treatment, and it was observed that samples

October 2012


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treated with TSP:MgO=1:2 had evidence of dehydrogenase activity (3.408 µg TPF/ml) while

those treated with TSP:MgO=8:1 did not show any trace of dehydrogenase activity (Table 2). At

this stage, it was hypothesized that the influence of pH noted by Mahmoud and Ghaly (2004),

whereby high pH environments (9.5 < pH < 13) can lead to non-enzymatic production of TPF in

the TTC dehydrogenase assay may be responsible for the dehydrogenase activity observed in

samples with the higher pH. However, tests on the cement constituents only, showed that this

was not the case as the cement formulations at pH extremes, 6.5 and 10 exhibited abiotic

dehydrogenase activities of 2.562 and 3.300 µg TPF/ml respectively (Table 2).

Further tests were carried out on the base materials, MgO and TSP, and the results demonstrated

that the abiotic dehydrogenase activity observed was due to the effect of dead burned magnesia.

The material induces the same coloration (pink to reddish) ascribed to the action of

dehydrogenase enzymes in the assay. With TSP alone there was no trace of abiotic

dehydrogenase activity which shows that the abiotic dehydrogenase activity observed in the

mixtures of cement constituents only was due to MgO stimulation. Although the mixture of

cement constituents only with TSP:MgO=8:1 exhibited abiotic dehydrogenase activity,

surprisingly when it was used for S/S treatment of the contaminated soil there was no trace of

dehydrogenase activity. The said cement formulation was projected to be favorable for microbial

activity due to its near neutral pH.

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Table 2. Dehydrogenase activity measurements on soil-cement components

Treatment Dehydrogenase activity

(µg TPF/ml)

Uncontaminated soil with 10% compost content treated

with TSP:MgO = 8:1 cement (after 5 days)

Uncontaminated soil with 10% compost content treated

with TSP:MgO = 1:2 cement (after 5days)

0.000

3.408 + 0.318

Cement constituents only, TSP:MgO = 8:1 (pH~6.5) 2.562 + 0.000 (abiotic)

Cement constituents only, TSP:MgO = 1:2 (pH~10) 3.300 + 0.018 (abiotic)

Triple Super Phosphate (TSP) only 0.000

Dead burned magnesia (MgO) only 2.526 + 0.054 (abiotic)

2.5%, 5%, 10%, 20% and 30% of TSP in a mixture of

compost and TSP, without any contamination

0.000

Results represent mean + standard deviation of three replicates

The above findings led to the hypothesis that TSP might inhibit dehydrogenase activity. To test

the hypothesis, different amounts of TSP was added to uncontaminated compost such that the

amount of TSP in the mixture of compost and TSP ranged from 2.5% - 30% (Table 2). There

was no trace of dehydrogenase activity even with the lowest amount of TSP in the mixture. This

demonstrated that TSP is an inhibitor of dehydrogenase activity. This is surprising as the

literature has it that phosphates stimulate soil microbial activity. A number of substances that

could stimulate or inhibit dehydrogenase activity have been documented but neither MgO nor

TSP was listed (Weaver et al., 1994). Similar findings in this direction previously reported

include: magnesium stimulation of catalytic activity of horse liver aldehyde dehydrogenase

(Takahashi and Weiner, 1980), superphosphate reduction of bacterial populations (Kelly and

Henderson, 1978) and inhibitory effects of TSP on microbial respiration and substrate induced

respiration (Thirukkumaran and Parkinson, 2000).

October 2012


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Noteworthy is the fact that although TSP inhibits microbial activity, the cement constituents

with TSP:MgO = 8:1 and TSP:MgO =1:2 had evidence of abiotic dehydrogenase activity. This

was due to MgO stimulation as the amount of TPF produced in the former was less than that in

the latter. Moreover, it is likely that the reaction product of the mixture, Ca-Struvite could also

enhance the stimulation as reports by Takahashi and Weiner (1980) have it that catalytic activity

of horse liver aldehyde dehydrogenase was also activated by the presence of Ca2+

. The absence

of dehydrogenase activity in contaminated soil samples treated with TSP:MgO=8:1 cement

formulation could be ascribed to limiting amount of MgO in the whole mixture thus giving way

to inhibition by TSP.

These results suggests that the dehydrogenase activity observed in contaminated soil samples

treated with TSP:MgO =1:2 cement formulation had a biotic and an abiotic component. The

biotic component was evaluated by using three kinds of treatment: soil with 10% compost

contaminated with all three contaminants (3000 mg/kg each), same with only 2CBA, and soil

with 25% compost contaminated with all three contaminants. The dehydrogenase activity results

after 14 and 28 days are shown in Figure 1. The error bars represent the standard deviation of

three replicates. Much of the hydration of typical cements is achieved in 28 days hence the test

was carried out at the set periods. Overall, the results show that the amount of TPF produced was

generally higher than that associated with abiotic dehydrogenase activity. Moreover,

the presence of heavy metal contaminants and increased amounts of compost caused different

trends in microbial activity with time. Samples with 10% compost (C) and only 2CBA, and 25%

compost content with all contaminants had higher dehydrogenase activities than those with 10%

October 2012


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compost and all contaminants at 14 and 28 days. The results demonstrate that the application of

the cement formulation to a heavily polluted soil where microbial activity was inhibited could

resuscitate and sustain it for a period. Chemical fixation of contaminants thus reducing their

toxicity to soil microbes coupled with magnesium oxide stimulation of microbial activity may

well be the mechanisms responsible.

10% C, all contam. 10% C, 2CBA only 25% C, all contam.

Figure 1. Dehydrogenase activity measurements in TSP:MgO=1:2 soil-cement samples

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Conclusions

The findings of this study has shown that the application of magnesium phosphate cement

formulation with the appropriate magnesia content to a heavily polluted soil where microbial

activity is inhibited could resuscitate and sustain it for a period of time. This knowledge would

be useful in improving the robustness of S/S technology as it would facilitate the incorporation

of a biodegradation mechanism thus leading to a more sustainable remediation technology. A

useful implication of these findings is that magnesium oxide may be effective in stimulating

microbial activity in a heavily polluted soil where it is limiting. The inhibition of microbial

activity by triple super phosphate observed in the study informs the need to review fertilizer

management practices, especially its effect on the contribution of soil biota to agricultural

productivity.

Acknowledgements

The provision of financial support for this work by the Cambridge Commonwealth Trust is

gratefully acknowledged.

References

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phytoremediation treatment of petroleum sludge. Soil and Sediment Contamination, 19 (6), 686 –

695.

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