BIOREMEDIATION OFBIOREMEDIATION OF PETROLEUM AND...

Chapter 3

BIOREMEDIATION OFBIOREMEDIATION OFBIOREMEDIATION OFBIOREMEDIATION OF PETROLEUM AND OTHER PETROLEUM AND OTHER PETROLEUM AND OTHER PETROLEUM AND OTHER

HYDROCARBONHYDROCARBONHYDROCARBONHYDROCARBON CONTAMINATED SOIL CONTAMINATED SOIL CONTAMINATED SOIL CONTAMINATED SOIL USINGUSINGUSINGUSING

EARTHWORMSEARTHWORMSEARTHWORMSEARTHWORMS

4. 3.1. Introduction

Bioremediation is the biological remediation of contaminated sites/soil/water using

mostly microorganisms. The bioremediation techniques have the benefit of high

treatment efficiency, low cost, in-situ and ex-site application and compatibility with other

techniques (Alexander, 1999; Hong et al., 2005). Polycyclic aromatic hydrocarbon (PAHs

are major) recalcitrant components in oil contaminated soil and are known to be

carcinogenic to human and other living organisms. Physical, chemical and biological

methods can be used for both environmental and economic reasons (Kim et al., 2001).

However the biological processes will have no negative impact on the environment as

they are natural processes. The biological processes rely on the natural ability of

microorganism to carry out the mineralization of organic chemicals, leading ultimately to

the formation of CO2, water and biomass (Cunha and Liete, 2000).

Strategies to accelerate the biological breakdown of hydrocarbons in soil include

stimulation of indigenous microorganisms by optimizing the nutrient and oxygen supply

Part IV

258 Studies on the potentials of Earthworm species as Bioremediating and Biological control agents

and the temperature and pH conditions (biostimulation) and through inoculation of an

enriched mixed microbial consortium into the soil (bioaugmentation). The use of

enormous amount of petroleum products contributes to environmental pollution. Mainly

spill of hydrocarbon occurs either due to the leakage of storage tanks or dumping of

waste petroleum products in the environment. The elevated loading of petroleum

hydrocarbon in soil causes a significant decline in soil quality reflecting negatively on the

soil use (Wonga et al., 2004). Biodegradation by natural population of microorganisms or

in-situ attenuation is a primary mechanism by which petroleum hydrocarbon could be

eliminated from contaminated site. Biostimulation, a process in which intrinsic

hydrocarbon degrading bacteria are stimulated with appropriate nutrient supplements to

ensure that the microbial growth is sustained. Addition of biosurfactants increases the

availability of long chain hydrocarbons to microbes and renders them more accessible to

microbial enzyme systems for utilization (Banat et al., 2000).The remediation of

contaminated soil is therefore an essential process in the waste management by the

petroleum industry worldwide. A number of successful remediation techniques have

been developed and used at petroleum- contaminated sites. Physical and chemical

methods are relatively high energy processes that are usually expensive (Fingas, 2001).

However biological processes can be cost effective, utilizing living microorganisms to

transform contaminants in soil into none or less harmful byproducts. Composting has

several advantages over other technologies including relatively low operating costs;

simplicity of operation, design and relatively with better treatment efficiency.

4.3.2 Objectives of the present study

Reports on the bioremediation of oil-contaminated sites using microbes are

sporadic; however using earthworms in such studies are scarce in the literature. The

significance of the present study lies on the use of earthworms and associated

microorganisms together in remediating petroleum and other hydrocarbon-hence forth

referred as petroleum hydrocarbon in the rest of the text- contaminated soil.

In the present study the soil contaminated with petroleum hydrocarbon was

subjected to composting with and without the addition of microbial inoculum. Then the

resulting compost - referred as precompost the rest of the text- was used as the feed for

earthworms in vermicomposting. In this chapter the potential of two earthworm species-

Eisenia fetida and Eudrilus eugeniae- in bioremediating petroleum hydrocarbon

contaminated soil was studied.

Chapter 3 Bioremediation of

petroleum and other hydrocarbon contaminated soil using Earthworms

Studies on the potentials of Earthworm species as Bioremediating and Biological control agents 259

Objectives of the present study are listed below,

1. To bioremediate soil contaminated with petroleum hydrocarbon at two stages:

composting and vermicomposting.

2. To study the impact of various additives during composting on the ultimate

remediation of contaminated soil.

3. To assess the effectiveness of addition of microbial inoculum in contaminated soil

during composting.

4. To study the impact of additional feed provided during the vermicomposting of the

contaminated soil on remediation.

4.3.3 Materials and Methods

4.3.3.1. Substrates

a) Oil contaminated Soil

Soil samples were collected from three different sites of an automobile service

station near M G University campus, Kottayam. A composite sample was prepared and

used at each time for both experimental and analytical purposes.

b) Vermitea

Vermitea was prepared by percolating distilled water through a glass column of

500ml capacity packed with 250g vermicast. 500 ml of distilled water was allowed to

percolate in drop wise through packed bed of vermicast; the leachate was collected and

used as vermitea.

c) Enrichment Culture

The indigenous hydrocarbon degrading bacteria were isolated from petroleum

contaminated soil. 100g of soil collected from the service station was added to 100 ml

Minimal Salt Medium (MSM) and shaken overnight at 200 rpm. After shaking, the soil

was allowed to settle the supernatant was removed and distributed in four Erlenmeyer

flask of 100 ml capacity containing 100ml of fresh MSM, where diesel at a concentration

of 1% was used as the substrate. Then it was incubated in a shaker for four days at

200rpm. The culture was enriched by regular transfer of 10% (v/v) inoculum from these

Part IV


flasks into new flask containing fresh MSM with 1% diesel. 100 ml of the enriched culture

was centrifuged and the supernatant was used as inoculum (Yerushalmi et al., 2003).

4.3.2. Choice of earthworm

Two epigeic earthworm species Eudrilus eugeniae Kinberg, and Eisenia fetida

Savigny, were used in this study. They typically inhabit humus-laden upper layers of

garden earth and manure-pits. They have a higher frequency of reproduction and a

faster rate of growth to adulthood than most other species of earthworms. Further, as

they don’t burrow deep into the soil, the vermireactors need not contain a deep bed of

soil. This has the potential of contributing towards saving on reactor volume.

4.3.3. Analytical methods

a) Total Petroleum Hydrocarbon (TPH)

Total petroleum hydrocarbon was determined by spectrophotometric method, similar to

the method described by Adekunle ( 2011). TPH in soil samples were extracted using

hexane as solvent in a Soxhlet apparatus (Plate ) as described by Khan et al., (2005).

The extracted samples were purified by passing through a silica gel column (Plate ) as

per the method of Mishra et al., (2001). Then the column fractions were quantitatively

estimated for TPH using UV spectrophotometer at 254 nm. The concentration of TPH

was obtained from a calibration curve and related to sample weight.

4.3..3.4 Microbiological Analysis

a) Enumeration of heterotrophic bacteria

The number of colony forming heterotrophic bacteria in compost samples was

determined by using Trypton Glucose Yeast (TGY) extract medium. Compost samples

were serially diluted and appropriate dilution was plated on TGY plates using spread

plate technique. A control plate was also kept. All the plates were incubated at 300C for

48 hours (Jorgenson et al., 2000).

b) Enumeration of hydrocarbon degrading bacteria

Enumeration of hydrocarbon degrading bacteria was attempted on a mineral

medium-containing diesel as the sole carbon source. The mineral medium contained the

following in mg/L: KH2PO4 – 8.5; K2HPO4 – 21.7; Na2HPO4.2H2O – 33.4; NH4Cl – 0.5;




CaCl2.2H2O – 39.4; MgSO4.7H2O – 22.5; FeCl3.6H2O- 0.2; Cycloheximidine – 150;

agar – 1500.Diesel (200µL) was added to a small piece of filter paper which was placed

in the lid of the Petri dish. Control plates were prepared without any carbon source. All

the plates were incubated at 280C for four days (Jorgenson et al., 2000).

4.3.3.5 Experimental Methods

The experiments were conducted in two phases. In the first phase, contaminated

soil was subjected to aerobic composting – referred as precomposting in the rest of the

text. The products of the precompost were subsequently subjected to vermicomposting

in the second phase (Figure 4.3.1). However in between these two phases an additional

experiment known as avoidance test was also conducted in order to understand whether

all the precomposted soils are acceptable to the earthworms either as vermibed/feed. To

assess this an avoidance test was conducted with different categories of precomposted

soils and based on the result further vermicomposting of the precomposted soils (second

phase) was designed.

4.3.3.5. a) Precomposting

Two types of precomposting were carried out. In one type microbial enrichment

was done by adding a definite volume of microbial mixture. The other type of

precomposting was done without microbial enrichment. In order to study the effect of

different forms of nutrients on precomposting, a few additives such as cowdung and

vermitea were added in each of the types mentioned above before the start of

precomposting. Precomposting was also done without the additives, which serve as

controls (Plate V).

i) Precomposting with cowdung

The contaminated soil with petroleum hydrocarbon was mixed with cowdung in 6:

1 (soil: cowdung, w/w) ratio and set in plastic aerated bins. Six bins were prepared. In

three bins microbial enrichment culture (1ml/220g of contaminated soil) was added. The

other three bins were kept without the addition of microbial culture. The entire contents

were sprinkled with adequate quantity of water to generate average moisture content of ~

50% and were covered with thick plastic sheets. The temperature of the bin contents were

recorded periodically using a thermometer. After the initial setting, the compost boxes were

Part IV


left undisturbed. The aerobic process of composting started and the temperature of the

bin contents gradually increased from the initial ~ 31°C to ~40°C within 5-8 days after

start. During the second week the temperature began to fall then the plastic covers

were removed and the contents were mixed manually on every alternate days in order

to aerate the compost mixture. The covers were then replaced after mixing. Out of the

three bins started with microbial inoculums, one bin was operated for 15 days, the next

one was for 30 days and the last one was operated for 50 days.

ii) Precomposting with Vermitea

Six precomposted bins were prepared in the similar manner as mentioned in the

previous section. That is, all six bins had contaminated soil and cowdung in 6:1 (w/w)

ratio. Out of the six bins, three were inoculated and rest was un inoculated. In all bins

instead of water vermitea was used to generate adequate moisture content. All the bins

were subjected to Precomposting for the duration as mentioned in the previous section.

iii) Precomposting of contaminated soil only

Here the contaminated soil only was subjected to pre composting. Six bins were

prepared, three bins with the addition of microbial enrichment culture and the other three

bins without microbial enrichment. Moisture was maintained by the addition of water.

Composting was done as in the first case for 15 days, 30 days and 60 days.

One control bin having only the contaminated soil without any additives including

water was operated for 60 days in order to check evaporative loss of hydrocarbon. Soil

samples from all precomposting bins including control bin was drawn and analyzed on

15th, 30th and 60th days for the TPH content. After pre composting the precomposted soil

from each category was subsequently subjected to an avoidance test prior to

vermicomposting.

9.3.5.b) Avoidance test

Two acrylic containers with one central chamber surrounded by five peripheral

chambers which are interconnected with each other through perforated plates were used

in this study (Plate VI). First container was used to conduct the avoidance test for the

earthworms against the contaminated soil which was precomposted (hence known as

precomposted soil-PS) with various additives filled in peripheral (test) chambers. The

second container was also used for the same purpose but to study the influence of




microbial inoculum. Originally each of the five chambers was connected to adjacent

chambers and to the central cylinder by five perforated plates. Ten worms of selected

species were placed into the soil free central chamber at the beginning of the test.

Because of their negative photo tactical reaction, the earthworms moved quickly into the

soil filled chambers. The compartment entered by each earthworm was recorded (t0).

The test chambers were filled with the precomposted soil in different combinations

namely contaminated soil with cowdung, contaminated soil with cowdung and vermitea,

contaminated soil with synthetic fertilizer and contaminated soil only and fertile garden

soil. The second container possessed all these combinations with the addition of

microbial enrichment culture (inoculum). The moisture content of the soil in all chambers

were maintained as ~60%. After all of the test organisms had migrated into the soil filled

chambers, the central chamber was closed. Thus, movement of earthworms was only

possible among the soil filled peripheral chambers within a test unit. To prevent worms

from escaping, the containers were covered with a lid with small holes in order to provide

sufficient air. The experimental set up was kept for 48hrs. Both the species of

earthworms E. fetida and E. eugeniae were subjected to this avoidance test separately.

9.3.5. c) Vermicomposting

Vermicomposting was conducted using the precomposted soil as bedding media as well

as feed for earthworms. Six categories of precomposts were used in this experiments,

viz (i) contaminated soil derived precompost (ii) CS with cowdung mixed in 6:1 ratio

and precomposted (iii) CS with cowdung and vermitea precompost. The remaining three

categories of precomposts were also of the same combinations but additionally

supplemented with an enriched microbial culture (inoculum)at the time of starting of

precomposting. In the usual vermireactor design (as indicated in chapter 3 of part II) the

top layer of garden soil was replaced with the precompost in the present study.

Four vermireactors were prepared for each pre-composted soil. In one reactor the

top layer consisting garden soil was replaced by pre-composted soil which served a dual

purpose of soil bed as well as feed for the worms. The second reactor was also

maintained similar to first reactor except an additional feed of cowdung (37.5g) dry wt.

was also provided.

All these vermireactors were inoculated with 10 adult healthy worms of selected

species. These animals were randomly picked from cultures maintained in the laboratory,

Part IV


with cowdung as feed. Other two reactors were prepared in the same manner as that of

the first and second and were kept as control reactors without the introduction of worms.

Same procedure was followed for all other pre composted soils.

The average moisture content of the vermirectors was maintained at 60 ± 5% by

periodic sprinkling of adequate quantities of water. These vermireactors (plastic vessels

with vermibed and worms) were provided with a thin nylon mesh covering and a metal

mesh (5mm) covering in order to avoid the entry of insects and rodents.

Figure 4.3.1: Design of experiment for vermicomposting of precompost derived from

contaminated soil

CS + CD With and without inoculum

CS With and

without inoculum

CS+CD+VT With and

without inoculum

Oil contaminated soil

Precomposted soil

V E R M I C O M P O S T I N G

Precomposting of the contaminated soil done with three combinations




4.3.3.6 Vermireactor operations

The vermireactors - after the bed preparation - were inoculated with 10 healthy

adult earthworms of chosen species. All the quantities of feed mass were adjusted in

terms of dry weights (taken after oven drying at 105°C to constant weight). The

earthworm biomass is reported as live weight, taken after rinsing the adhering

materials from the worms and carefully blotting them dry (Gajalakshmi et al., 2001).

From the reactors, after 15 days, the castings were harvested and the mass of

castings were recorded. After recording the vermicast harvest the vermireactors were

operated continuously without any additional feed except the reactors which were

started with additional feed cowdung at the rate of 37.5g(dry weight) of cowdung as

feed again with same quantity of fresh feed, as used while the reactors were started

initially as stated above. On 30th day the castings and the earthworms were removed

from the reactors and placed in separate containers for quantification, after collecting

sufficient soil samples for TPH analysis the rest of the reactor contents were discarded.

The juveniles, if any are generated in the previous run, were separated, counted and

then transferred to the culture bed. After recording the weight, the 10 worms with which

the reactors were originally started, were reintroduced into culture bed.

Part IV


Plate IV : Precomposting Bins

Plate V : Avoidance test chambers




Plate VI : Soxhlet apparatus and Silica gel column

Part IV


Plate VII a) : Colonies of Hydrocarbon degrading bacteria in contaminated soil

Plate VII b) : Colonies of Hydrocarbon degrading bacteria in contaminated soil

amended with Cowdung and vermitea




Plate VIII : Vermicomposting units of bioremediation experiment

4.4.4. RESULTS

4.4..4.1 Characteristics of the contaminated soil and other raw materials used

The characteristics of contaminated soil and other materials used in this study are

shown in the table 9.1. The contaminated soil had an average Total Petroleum

Hydrocarbon (TPH) concentration of 43750±2500mg/kg. Contaminated soil has organic

carbon concentration 6.123% and C: N ratio of 27.09. The soil was neutral in its pH. The

cowdung used in this study had 37.44% and 1.9% of organic carbon and Kjeldhal

Nitrogen respectively. C: N ratio of the cowdung was 19.7.

Part IV


Table 4.4.1: Characteristics of the raw contaminated soil and other materials used

Parameters Substrates(raw materials)

Cowdung Vermitea Contaminated soil

Organic Carbon (%) 37.44 - 6.123

Total Kjeldhal Nitrogen (%) 1.90 0.014 0.0226

Available phosphorous (%) 0.029 1.69 2×10-4

C/N 19.70 - 27.09

pH - - 7.05

Conductivity - - 0.226ms/ppt

TDS - - 0.095

TPH - - 43750mg/kg

4.3.4.2. Avoidance test

Avoidance test was conducted in two boxes. In the first box all the test chambers

were filled with the contaminated soil subjected to precomposting- mentioned as

precomposted soil (PS) in the rest of the text- in different combinations namely

precomposted soil with cowdung (CD), precomposted soil (PS) with vermicompost tea

(VT), precomposted soil with inorganic (synthetic) fertilizer, precomposted soil and fertile

garden soil. After 48 hours all Eudrilus eugeniae worms were present in the chamber

having PS+ CD. The other substrates were found to be avoided by the earthworms in the

first box with E. eugeniae. The second box contained the same combinations with the

addition of enrichment culture (inoculum-I). In this case, the combination of PS+ I was

preferred by 10% worms, while 50% preference was observed in PS +CD+ I and 40 %

preference was observed in PS+CD+VT+ I (Table 4.3.2). In Eisenia fetida inoculated

avoidance test boxes, in the first category test (without inocula supplementation) 90%

preferred cowdung amended soil and only 10% preferred CD+ Vermitea amended soil.

In the second box with E. fetida 10%, 70% and 20% preference were noted in PS+, CD+

and CD+VT compartments respectively. From the observations of avoidance test the

combination of PS with CD and inoculum was found to be the best substrate for

earthworm. Both worm species in both the cases ie with and without addition of inoculum

avoided fertilizer amended soil. So further studies were conducted based on these

observations, i e, fertilizer amended contaminated soil was not used in further studies.




Table 4.3.2: Substrate preference (% of total number of worms, n= 10 worms) as exhibited in the

avoidance test by Eudrilus eugeniae and Eisenia fetida after 48 hrs (t 48).

Substrate Eudrilus eugeniae Eisenia fetida

Without inoculum

(Box I)

With inoculum

(Box II)

Without

inoculum

(Box I)

With inoculum

(Box II)

PS 0 10 0 10

PS +CD 100 50 90 70

PS+CD+VT 0 40 10 20

PS+F 0 0 0 0

PS+FS 0 0 0 0

4.3.4.3. Nutrient status of precompost

The nutrient status and C: N ratio of the contaminated soil after precomposting with

various combination is presented in table 4.3.3. The concentration of available

phosphorous was found to be very less in the case of precompost obtained using

contaminated soil with or without inoculum. High amount of available nutrients were

found in the precompost obtained using the combination of contaminated soil with

cowdung and inoculum. A wide range of C/N ratio was noted with the different kinds of

precomposts, the minimum C/N ratio of 8.1 was noticed with the precompost obtained

from a combination of PS + CD + VT (Table 4.3.3). A decrease in C: N ratio was noted

during the precomposting phase in all combinations of contaminated soil.

Part IV


Table 4.3.3: The nutrient status in terms of N, P, K and C/N ratio of the samples during precomposting

Nutrient parameter

Time interval

Category

PS PS + I PS+CD PS+CD + I

PS+CD +VT

PS+CD+ VT + I

Organic Carbon (%)

0 15 30 60

4.72 2.82 2.01 3.05

5.54 3.40 2.80 3.15

5.48 4.20 3.27 4.10

10.74 8.40 3.81 4.40

7.02 4.46 3.16 4.21

7.80 5.90 5.20 5.61

Total Nitrogen (%)

0 15 30 60

0.23 0.154 0.099 0.15

0.24 0.156 0.136 0.153

0.52 0.442 0.35 0.403

0.97 0.89 0.413 0.424

0.64 0.527 0.390 0.41

0.706 0.56 0.51 0.54

C/N ratio

0 15 30 50

20.52 18.32 20.30 20.33

23.08 21.79 20.59 20.59

10.54 9.50 9.30 10.17

11.07 9.44 9.22 10.38

10.96 9.40 8.10 10.27

11.05 10.54 10.20 10.38

Available Phosphorus (%)

0 15 30 60

0.00007

0.00006

0.00004.

0.00054

0.00008

0.00007

0.00005

0.00006

1.5 0.8 0.53 0.65

2.0 1.0 0.55 0.65

1.5 0.9 0.55 0.65

2.4 0.95 0.7 0.65

Available Potassium (%)

0 15 30 50

0.0125 0.01 0.008 0.01

0.028 0.01 0.009 0.01

0.035 0.031 0.028 0.030

0.042 0.031 0.031 0.032

0.035 0.033 0.032 0.034

0.043 0.032 0.028 0.031

4.3.4.4. Enumeration of total heterotrophic and hydrocarbon degrading bacteria

The data on cfu/g (colony forming units) counts of total heterotrophic

microorganisms and hydrocarbon degrading bacteria in the precomposted samples was

shown in tables 9.4 and 9.5. The combination of precomposted soil (PS) with cowdung

(CD) exhibited maximum number of hydrocarbon degraders than the combination having

PS, CD and Vermitea (VT) together and PS alone. The addition of inoculum to the

substrate resulted in increase of both heterotrophic and hydrocarbon degrading bacteria

in the precomposting samples (Plate VIII a, b). Gradual increase in both total




heterotrophic and hydrocarbon degraders were noted in all different combinations up to

30th day, afterwards a decline in population was noted.

Table 4.3. 4: Enumeration of Heterotrophic Bacteria from the Precomposted Samples Sample Monitoring days

0th day 15th day 30th day 50th day PS 1.4×106 2.6×107 2.0×108 2.1×107

PS + I 2.1×106 3.0×107 2.9×108 2.4×107 PS+CD 2.3 ×106 2.5×107 3.4×108 3.1×107 PS+CD + I 3.4×106 4.4×107 4.5×108 4.0×107

PS+CD+VT 3.0×106 3.8×107 4.2×108 3.4×107

PS+CD+VT + I 4.0×106 4.2×107 4.9×108 4.0×107

Table 4.3.5: Enumeration of Hydrocarbon Degrading Bacteria from the Precomposted Samples

Sample Monitoring days 0th day 15th day 30th day 50th day

PS 6.0×104 1.4×106 1.0×107 1.2×105

PS + I 1.0×105 2.0×106 1.6×107 1.9×105

PS+CD 1.9×105 3.0×106 2.8×107 3.6×105

PS+CD + I 3.6×105 4.0×106 3.9×107 4.5×105

PS+CD+VT 2.2×105 3.2×106 3.0×107 2.8×105

PS+CD+VT + I 3.0×105 3.5×106 3.3×107 3.2×105

4.3.4.5. Total Petroleum Hydrocarbon (TPH) concentration in Vermicomposted

samples

Generation of calibration curve.

From mass-volume-density relationship, a known volume (1.4 ml) corresponding to 1 g

of the gasoline was transferred to 1000-ml volumetric flask, completely dissolved in 100

ml n-hexane and made up to mark using the same solvent to give 1000 mg/L stock

solution. Absorbance of each standard (100, 200, 300, 400, 500, 600, 700 and 800

mg/L), prepared from the stock, was read on the UV spectrophotometer. By plotting

absorbance against concentration, a calibration graph (Figure 4.3.3). The TPH in soil

extract, obtained from the calibration curve, was then related to soil sample weight and

Part IV


expressed as mg/kg. The biodegradation efficiency for TPH in soil sample was

calculated by relating the difference between the initial and final concentrations of soil

TPH to the initial concentration and multiplying the resulting quotient by a factor of 100.

Figure 4.3.2: Calibration graph for the determination of total petroleum hydrocarbon

using UV spectrometry

The calibration graph used for the determination of total petroleum hydrocarbon in this

study was presented in the figure 4.3.2. The total petroleum hydrocarbon in the

contaminated soil was an average of 43750 mg/kg. Gradual reduction in TPH

concentration was noted during precomposting phase itself (Table 4.3.6). During

precomposting phase contaminated soil is mixed with additives such as cowdung and

cowdung along with vermitea (VT). Addition of additives such as cowdung and cowdung

along with vermitea has resulted in better removal of total petroleum hydrocarbon from

the contaminated soil during the initial period of precomposting itself (Table 4.3.6).

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

100 mg/L 200 mg/L 300 mg/L 400 mg/L 500 mg/L 600 mg/L 700 mg/L 800 mg/L

Abs

orba

nce

Concentration mg/L




Table 4.3.6 : Total petroleum hydrocarbon concentration of Precompost

Precompost Soil TPH mg/kg

% reduction over initial

concentration

15 days 30 days 60days 15 days 30 days 60days

PS 42750 39050 36900 2.29 10.74 15.66

PS+I 38500 34000 31375 12.00 22.29 28.29

CD+PS 36250 28750 25750 17.14 34.29 41.14

CD+PS+I 31125 25500 22500 28.86 41.71 48.57

CD+PS+VT 35500 29100 25250 18.86 33.49 42.29

CD+PS+VT+I 33125 26000 25375 24.29 40.57 42.00

TPH concentration in the vermicomposted soil

The percentage removal of Total Petroleum Hydrocarbon (TPH) in the vermicomposted

soil with earthworm Eudrilus eugeniae is presented in summary Table 4.3.7. The

contaminated soil precomposted with inoculum (Enrichment culture) irrespective of the

amendments when fed in the vermireactors has shown a better removal of TPH than the

precomposted PS without the inoculum (Enrichment culture). Maximum removal of TPH

was obtained with 60 day precomposted samples.

Part IV


Table 4.3.7: Percentage removal of TPH from the contaminated soil subjected to precomposting and vermicomposting with E. eugeniae

Precompost

TPH* mg/kg

% Reduction

TPH *mg/kg

% Reduction

TPH mg/kg

% reduction

Precompost

TPH* mg/kg

% Reduction

TPH* mg/kg

% Reduction

TPH mg/kg

% Reduction

PS 33275 23.94 20125 54 12750 70.86 PS+I 32000 26.86 19625 55.14 11750 73.14

PS+ Feed 31000 29.14 18750 57.14 11875 72.86 PS+I +Feed

30875 29.43 18500 57.71 10250 76.57

PS (with CD) 19750 54.86 9125 79.14 4875 88.86 PS (with CD)+I

10500 76 4375 90 4625 89.43

PS (with CD)+Feed

18750 57.14 8500 80.57 4375 90

PS (with CD)+Feed+I

9125 79.14 3500 92 2875 93.43

PS (with CD and VT)

18625 57.43 5625 87.14 3625 91.71

PS (with CD and VT) + I

15250 65.14 5000 88.57 3125 92.86

PS (with CD and VT)+Feed

17625 59.71 5250 88 3000 93.14

PS (with CD and VT)+ Feed+I

15625 64.29 4875 88.86 3000 93.14

The combination with which the contaminated soil was subjected to precomposting is given in parenthesis. For instance, if it is given as PS (with CD and VT)it means the PS was obtained after subjecting the contaminated soil for precomposting in combination with CD and VT *TPH content remaining in the soil of the vermireactors after 30 days of vermicomposting (only average values of the concentration are given).

Chapter 3 Bioremediation of petroleum and other hydrocarbon

contaminated soil using Earthworms


Table 4.3.8: Percentage removal of TPH from the contaminated soil subjected to precomposting and vermicomposting with E .fetida Reactors without microbial inoculum

TPH* mg/kg

% reduction

TPH * mg/kg

% Reduction

TPH* mg/kg

% Reduction

With microbial inoculum

TPH* mg/kg

% Reduction

TPH * mg/kg

% Reduction

TPH* mg/kg

% Reduction

PS 37000 15.43 26750 38.86 12000 72.57 PS+I 35375 19.14 20600 52.91 9625 78

PS+Feed 33350 23.77 24125 44.86 8000 81.71 PS+Feed 31250 28.57 19500 55.43 8750 80 PS (with CD) 14500 66.86 12500 71.43 2000 95.43

PS (with CD)+I 11875 72.86 8750 80 2625 94

PS (with CD)+Feed 12500 71.43 9500 78.29 2625 94

PS (with CD)+Feed+I 11750 73.14 8000 81.71 2500 94.29

PS (with CD and VT) 16125 63.14 11750 73.14 3500 92

PS (with CD and VT)+ I 11875 72.86 8750 80 2625 94

PS (with CD and VT)+Feed 15875 63.71 10375 76.29 3125 92.86

PS (with CDand VT)+Feed+I 11750 73.14 8000 81.71 2500 94.29

The combination with which the contaminated soil was subjected to precomposting is given in parenthesis. For instance, if it is given as PS(with CD and VT)it means the PS was obtained after subjecting the contaminated soil for precomposting in combination with CD and VT.

*TPH content remaining in the soil of the vermireactors after 30 days of vermicomposting (only average values of the concentration are given).

Part IV


Table 4.3.8 give the % removal of Total Petroleum Hydrocarbon (TPH) in the Eisenia fetida

inoculated reactors for three different vermicomposting periods. Here also the contaminated

soil precomposted with enrichment culture (inoculum) irrespective of the amendments when

fed in the vermireactors has shown a better removal of TPH than the precomposted PS

without the inoculum (Enrichment culture) during the 15day precompost. Maximum removal

of TPH was obtained with 60 day precomposted samples in all three combinations such as

contaminated soil, contaminated soil with cowdung amendments and vermitea addition. Also

contaminated soil when amended with different additives such as cowdung and cowdung

along with vermitea showed increased percentage removal than contaminated soil alone.

Table 4.3.9 shows the concentration of TPH in the soil samples of control, reactors with and

without feed. The control reactors were operated without earthworms while the contents

were same as test reactors. As shown in tables better removal of TPH was found in PS (with

CD and inoculums) soil with additional feed.

Table 4.3.9: Percentage removal of TPH in the soil samples of control reactors( ie., without

earthworms) with and without additional cowdung as feed

Category 30d 60d

With feed Without feed With feed Without feed

PS+I 22.58 22.61 31.29 30.81

PS 11.6 12.8 21.46 20.12

PS(with CD)+I 42.96 42.8 53.14 49.81

PS(with CD) 34.94 34.59 39.44 38.14

PS(with CD and

VT) +I 42.8 41.2 46.24 45.11

PS(with CD and

VT) 34.9 33.8 45.59 44.03




Concentration of TPH in the vermicast

Concentration of TPH present in the worm cast of 60 day precomposted samples

was shown in table 4.3.10. Vermicast obtained through the vermicomposting of

contaminated soil – which was precomposted with the combination of CD+I- with an

additional feed of cowdung have shown the least quantity of TPH. Maximum level of TPH

(24.12) was observed with casts obtained from the reactors fed with PS (precomposted

alone without any additives. Table 4.3.11 shows the evaporative loss of TPH at different

time intervals. The loss of TPH due to mere evaporation from the contaminated soil (PS)

was assessed by operating a set of reactors in which PS alone was taken without any

amendments in order to avoid the influence of microbial action. Only 2.03% loss was

occurred after 50 days by evaporation (Table 4.3.11).

Table 4.3.10: Concentration(mg/kg) of TPH present in the worm cast obtained after 30 days of

vermicomposting with the 60 day precomposted soil as feed.

Category Eudrilus eugeniae Eisenia fetida

With feed Without feed With feed Without feed

PS+I 8063 (18.43) 9564 (21.86) 6344 (14.5) 7219 (16.5)

PS 9247 (21.14) 10553 (24.12) 6771 (14.79) 9595 (21.93)

PS(with CD)+I 2000 (4.57) 2415 (5.52) 967 (2.21) 1094 (2.5)

PS(with CD) 2000 (5.00) 2686 (6.14) 3.5 (1531) 1719 (3.93)

PS(with CD and

VT) +I 814 (1.86) 936 (2.14)

1.07 (468) 656 (1.5)

PS(with CD and

VT) 1251 (2.86) 3.29(1439)

1.64 (718) 1094 (2.5)

Values in the parenthesis indicate percentage of TPH remaining in the cat with respect to the initial TPH of the

contaminated soil.

.

Part IV


Table 4.3.11: Assessment of TPH loss due to mere evaporation from the reactors operated

without any amendments and without precomposting

Monitoring days 0th day 15th day 30th day 50th day

Soil TPH

mg/kg 43750 43417.5 43194.38 42861.88

% Reduction over initial

concentration -- 0.76 1.27 2.03

4.4.6. Earthworm survival, Biomass and Young ones production

Tables 4.3.12-4.3.14 show the worm biomass, survival, mortality and young ones

produced during the vermicomposting of various precomposted samples. Maximum mortality

of worms were observed with reactors in which PS- precomposted for 30 and 50 days with

inoculum and no supplement of CD or VT as feed.(Table 4.3.12-4.3.14).




Table 4.3.12: Worm biomass, juveniles produced, mortality and survivability of earthworms in the reactors operated with 15

day precomposted soil as feed

Experimental

Reactors

Eudrilus eugeniae Eisenia fetida

Biomass

Adu

lt

surv

ived

Mor

talit

y

You

ng o

nes

Biomass

Adu

lt

surv

ived

Mor

talit

y

You

ng o

nes

Initi

al

Fin

al

Cha

nge

in

biom

ass

Initi

al

Fin

al

Cha

nge

in

biom

ass

PS + I 5.621 2.496 -3.125 6 4 - 2.176 0.645 -1.531 4 6 -

PS + I with feed 6.559 7.465 0.906 10 Nil 16 1.731 2.029 0.298 7 3 2

PS 9.572 7.808 -1.764 6 4 - 1.828 1.175 -0.653 5 5 -

PS with feed 10.455 9.625 -0.83 7 3 13 2.148 2.748 0.6 6 4 3

PS (with CD + I) 8.054 9.013 0.959 9 1 21 1.829 2.26 0.431 9 1 6

PS (with CD + I)+feed 10.158 14.078 3.92 10 Nil 67 1.981 2.985 1.004 9 1 12

PS (with CD) 9.489 8.600 -0.889 9 1 22 2.180 5.644 3.464 10 Nil 7

PS (with CD)+feed 5.893 5.202 -0.691 7 3 17 2.182 5.593 3.411 10 ,, 16

PS (with CD&VT + I 10.804 15.351 4.547 10 Nil 41 2.350 3.798 1.448 10 ,, 4

PS (with CD&VT +I with

feed 13.253 16.572 3.319 10 Nil 49 1.805 5.330 3.525 10 ,, 6

PS (with CD&VT ) 12.524 16.572 4.048 10 Nil 21 1.774 2.465 0.691 10 ,, 6

PS (with CD&VT +

feed

10.729 10.813 0.084 7 3 46 2.105 2.721 0.616 10 ,, 11

Part IV


Table 4.3.13: Worm biomass, juveniles produced, mortality and survivability of earthworms in the reactors operated with 30 day precomposted soil as feed Experimental Reactors

Eudrilus eugeniae Eisenia fetida Biomass

Adu

lt su

rviv

ed

Mor

talit

y

You

ng o

nes Biomass

Adu

lt su

rviv

ed

Mor

talit

y

You

ng o

nes

Initi

al

Fin

al

Cha

nge

in

biom

ass

Initi

al

Fin

al

Cha

nge

in

biom

ass

PS + I 7.410 6.213 -1.197 8 2 0 2.085 1.508 -0.577 6 4 0 PS + I with feed 9.179 7.610 -1.569 10 0 6 1.850 1.723 -0.127 10 Nil 6 PS 9.407 6.357 -3.05 6 4 0 1.928 1.093 -0.835 5 5 0 PS with feed 7.206 8.103 0.897 7 3 3 1.989 1.243 -0.746 6 4 9 PS (with CD + I) 9.335 12.473 3.138 9 1 20 1.537 1.873 0.336 8 2 33 PS (with CD + I)+feed 9.942 13.912 3.97 9 1 26 1.801 2.671 0.87 10 Nil 182 PS (with CD) 6.776 8.600 1.824 9 1 22 1.735 2.599 0.864 6 4 119 PS (with CD)+feed 6.080 9.168 3.088 9 1 67 1.930 3.348 1.418 10 Nil 146 PS (with CD&VT + I 7.238 8.789 1.551 10 0 41 1.766 2.936 1.17 10 Nil 44 PS (with CD&VT +I with feed 10.048 14.26 4.212 10 0 49 1.77 3.33 1.56 7 3 32 PS (with CD&VT ) 7.095 9.641 2.546 9 1 21 2.099 1.800 -0.299 9 1 57 PS (with CD&VT + feed

9.835 11.641 1.806 9 1 46 1.680 1.800 0.12 9 1 22




Table 4.3.14: Worm biomass, juveniles produced, mortality and survivability of earthworms in the reactors operated with 60 day precomposted soil as feed

Experimental

Reactors

Eudrilus eugeniae Eisenia fetida

Biomass

Adu

lt su

rviv

ed

Mor

talit

y

You

ng o

nes

Biomass

Adu

lt su

rviv

ed

Mor

talit

y

You

ng o

nes

Initi

al

Fin

al

Cha

nge

In

biom

ass

Initi

al

Fin

al

Cha

nge

In

Bio

mas

s

PS + I 12.438 6.94 -5.498 4 6 10 2.641 1.10 -1.541 6 4 6

PS + I with feed 10.472 7.88 -2.592 9 1 40 2.278 3.50 1.222 10 0 29

PS 12.397 7.56 -4.837 10 0 13 2.369 1.91 -0.459 6 4 4

PS with feed 9.597 11.75 2.153 10 0 59 2.543 3.41 0.867 10 0 24

PS (with CD + I) 11.66 10.651 -1.009 10 0 48 2.898 4.82 1.922 10 0 79

PS (with CD + I)+feed 9.745 11.75 2.005 10 0 70 3.696 3.94 0.244 10 0 84

PS (with CD) 9.363 10.017 0.654 10 0 45 2.739 2.7 -0.039 7 3 54

PS (with CD)+feed 10.886 12.66 1.774 10 0 64 2.763 3.14 0.377 10 0 62

PS (with CD&VT + I 10.784 9.2 -1.584 9 1 40 1.910 2.14 0.23 8 2 25

PS (with CD&VT +I with feed

11.050 12.42 1.37 9 1 85 1.930 2.84 0.91

10 0 44

PS (with CD&VT ) 13.768 10.895 -2.873 9 1 64 2.538 2.88 0.342 6 4 28

PS (with CD&VT + feed

13.786 17.6 3.814 10 0 91 1.983 3.02 1.037

10 0 44

Part IV


4.4.7. Nutrient Status of vermicast

Tables 4.3.15 shows the nutrient status and C/N ratio of worm cast obtained by

vermicomposting the 60 day precomposted samples with microbial inoculum. The cast

obtained from the vermireactors fed with contaminated soil – which was precomposted for

60 days with CD- along with additional feed of cowdung possessed more amount of

nutrients – especially N and P than the casts obtained from other vermireactors. Casts from

the same reactors have also exhibited with least C\N ratio (Table 4.3.15).

Table 4.3.15: The nutrient status in terms of N, P, and K of the worm cast of 60 day precomposted sample with microbial inoculums

Ear

thw

orm

Nutrient parameter

Experimental reactors PS+I + FEED

PS +I

PS( with CD )+I+ feed

PS(CD + I)

PS( withCD&VT + I )+ FEED

PS(with CD+VT) + I

Eud

rilus

eug

enia

e

Total N (%) 0.610 0.216 0.770 0.47 0.441 0.347

Available P (%) 0.024 0.002 0.0046 0.0025 0.0035 0.0026

Available K (%) 0.065 0.060 0.165 0.150 0.20 0.175

Organic carbon (%) 13.065 5.85 13.455 8.385 15.015 6.63

C/N ratio 21.42 27.08 17.47 17.84 34.05 19.11

Eis

enia

fetid

a

Total N (%) 0.65 0.231 0.774 0.49 0.45 0.351

Available P (%) 0.025 0.0022 0.0046 0.0028 0.0035 0.0036

Available K (%) 0.061 0.05 0.172 0.15 0.22 0.17

Organic carbon (%) 7.01 6.09 14.723 9.23 12.89 4.46

C/N ratio 10.78 26.36 19.02 18.84 28.64 12.71



Studies on the potentials of Earthworm species as Bioremediating and Biological control Agents 285

4.4.5. DISCUSSION

Nutrient status of precompost

The concentrations of nutrients were found to be decreasing during the

precomposting (Table 4.3.3). The nutrient decrease seems mainly to be caused by its

incorporation into the microbial biomass and by its immobilization onto soil matrices as

reported by Schinner et al.,(1996). The organic carbon values also decreasing during the

precomposting (Table 4.3.3). These results agree with those reported by Riffaldi et al.,

(2005) in a study on the soil biological activities in monitoring the bioremediation of diesel oil

contaminated soil.

Microbiological analysis

The results show that the number of heterotrophic bacteria in the compost piles

having the additional inoculum was found to be higher than those without inoculum.

Microbial counts were found to be increasing during the initial stage of composting and

remain more or less constant towards the final stage. Many authors have reported an initial

increase in the number of oil degrading microorganisms during bioremediation of soils

correlated with biodegradation and soil biological activities (Atlas and Bartha, 1992). An

initial increase in cell number during the first 33 days was reported by Schinner et al.,(1996).

The results pertaining to the assessment of hydrocarbon utilizers in various

precomposted soils indicate a higher number of hydrocarbon utilizers in the soil samples

precomposted with CD and inoculum. The higher number of hydrocarbon degraders present

in this precomposted soil (with CD+I) has also led to the better removal of TPH after

vermicomposting when compared to PS+I. The increase in hydrocarbon utilizers coincided

with a high biodegradation activity, demonstrating the quick adaptation of the indigenous soil

microorganisms to the new substrates introduced by oil contamination.

Nevertheless, changes in bacterial numbers might be indicative of a simulative

biodegradation process, but do not necessarily represent an accurate measurement of the

actual biodegradation (VanderWaarde et al., 1995). The quantification of biological activities

is often used to interpret the intensity of microbial metabolism in soil (Schinner et al., 1996).

Part IV

286 Studies on the potentials of Earthworm species as Bioremediating and Biological control Agents

Percentage removal of TPH

There is a positive impact of the addition of inoculum in the removal of TPH. The

combinations having the enrichment culture shows better removal of TPH than without

enrichment culture in both earthworm species studied.

The mixing of CD and VT with PS at the precomposting stage has enhanced the ultimate

removal of TPH. However the enhancement was more with only CD addition than in

combination of CD with VT. Similarly the addition of CD as additional feed in the

vermireactors has also improved the percentage removal of TPH. For instance, the result of

30 day precompost when subjected to vermicomposting, 90 % and 88.55% removal of TPH

was recorded for the precompost with CD alone and with CD+VT respectively (Figure 9.3) in

E.eugeniae inoculated reactors. The same feed gives a result of 92% and 88.53% removal

when the vermireactors were supplemented with additional feed of CD. Similar reactors fed

with 50 day precompost have resulted in 93% removal (Figure 4.3.3). This is much higher

than the reported studies in the literature in which removal rates of 90% was achieved after

155 days by using microbes alone (Margesin and Schinner 1996). The impact of CD

addition at the vermicomposting stage for a better removal of TPH can also be attained

without CD addition provided the precomposting durations are prolonged to 60 days. For

example, 92% of TPH removal was noticed in a vermireactor fed with 30 day precomposted

soil (precomposting done with a combination of CD+I) with additional feed of cowdung, while

about 88.86 % removal of TPH could be noticed in the vermireactors fed with 60 day

precomposted soil (precomposting done with a combination of CD+I) as only feed without

any additional feed of cowdung. In summary, the better removal of TPH at a shorter duration

could be achieved with a supplement feed of cowdung at vermicomposting stage (Table

4.3.16).




When precomposted soil was subjected to vermicomposting with Eisenia fetida better

percent reduction was observed in reactors with additional inoculum and feed (Table 4.3.4).

However the effect of inoculum was best noted with 15d and 30day precomposted soil

samples. Only 19% and 15%reduction was noted in 15d precompost without feed in

Figure 4.3.3: Comparative evaluation of % removal of Soil TPH in E. eugeniae inoculated soil

A: Precomposted soil; B: Precomposted soil with cowdung C: Precomposted soil with cowdung and vermitea

PS: Contaminated soil I: Microbial inoculum; CD: Cowdung VT: Vermitea

Part IV


contaminated soil alone, while the same reactors with additional feed recorded 28% and

23% recovery was noted in with and without the enrichment culture. Cowdung amended

soil with and without inocula and feed performed better. Vermitea amendment along with

cowdung has also improved conversion efficiencies (Figure 4.3.4 ).

As precomposting duration increases far better removal of TPH was noted in all

experimental reactors. For example, 68-81% reduction in TPH concentration was found in

reactors with cowdung additives and with or without feed (Figure 4.3. 4). Total reduction of

71-76 % was found in contaminated soil amended with cowdung and vermitea.




Figure 4.3.4: Comparative evaluation of % removal of TPH in Eisenia fetida inoculated vermireactors

A: Precomposted soil; B: Precomposted soil with cowdung C: Precomposted soil with cowdung and vermitea PS: Contaminated soil I: Microbial inoculum; CD: Cowdung VT: Vermitea

Part IV


All the vermireactors performed better TPH removal (more than 90%) in both

cowdung, and cowdung along with vermitea added soil. Contaminated soil alone without any

additives also showed better removal of TPH (Table 4.3.16)

Table 4.3.16: Comparison of total duration needed for removal of TPH with various

combinations of substrates and with various duration of precomposting

Substrate

combination

Duration Eudrilus

eugeniae

Eisenia fetida Total

duration

(days) Precom-

posting

Vermicom-

posting

Additional feed

as cowdung

Additional feed as

cowdung

Yes No Yes No

PS+I 15 30 29.14 23.94 28.57 19.14 45

PS+I 30 30 57.71 55.14 55.43 52.91 60

PS+I 60 30 72.86 70.86 80 78 90

PS-I 15 30 29.43 26.86 23.77 15.43 45

PS-I 30 30 57.14 54 44.86 38.86 60

PS-I 60 30 76.57 73.14 81.71 80 90

PS+CD+I 15 30 79.14 76 73.14 72.86 45

PS+CD+I 30 30 92 90 81.71 80 60

PS+CD+I 60 30 93.43 89.43 94.29 94 80

PS+CD-I 15 30 57.14 4.86 71.43 66.86 45

PS+CD-I 30 30 80.57 79.14 78.29 68.57 60

PS+CD-I 60 30 92.86 90 94 94.53 90

PS+CD+VT+I 15 30 64.29 65.14 67.43 66 45

PS+CD+VT+I 30 30 88.86 88.57 76 71.14 60

PS+CD+VT+I 60 30 93.14 92.86 94.57 94 90

PS+CD+VT-I 15 30 59.71 57.43 63.71 63.14 45

PS+CD+VT-I 30 30 88 87.14 76.29 73.14 60

PS+CD+VT-I 60 30 93.14 91.71 92.86 92 90




The impact of additional microbial inoculum was best reported in the 15 day and 30

day precompost. In the 60day precompost all most all reactor recorded better removal of soil

TPH.

A study was also conducted to assess the loss of TPH due to mere evaporation. The

results showed only very less amount of TPH was lost by evaporation. After 60 day

exposure only 2.03% TPH was lost (Table 4.3.11). This result also agrees with those

reported by Namkoong et al., (2000) where only 3% loss was occurred after 100 days of

exposure.

Chemical analysis showed the reduction of total petroleum hydrocarbon concentration in

the earthworm – processed reactors, whereas in control reactors, without worms no

significant reduction was observed. The positive influence of worms on the reduction of oil

pollution in the earthworm processed soil can be explained by a better aeration of the soil

due to the burrowing activities and the enhancement of microbial activity. Earthworms have

a complex interrelation ship with microorganisms and promote microbial biomass and

activity in soil in decaying organic matter by fragmenting it with microorganisms, and they

disperse microorganisms throughout the soil (Edward and Bohlen, 1996). Ubiquist

microorganisms are able to oxidize alkanes in three steps into carboxylic acids. Therefore

earthworms can be seen as promoters in terms of bioremediation of contaminated soil

(Schafer, 2001). As earthworms are known to increase the metabolic activity of soil

microorganisms, this stimulation may lead to an enhanced biodegradation of mineral oil

compound so earthworms are considered as additional organisms to support

bioremediation.

Bioremediation of contaminated soil relies on the stimulation of insitu microorganisms

present in the contaminated soil. The bioavailability and bioaccessibility of contaminants for

soil microorganisms, is often limited because the contaminants can be physically protected

within the soil matrix. Earthworms burrow through the soil, mixing it constantly in their gut

(Eijsackers et al., 2001). They facilitate and increase the contact between contaminants and

soil microorganisms (Hickman and Reid, 2008). So addition of the earthworms to the

contaminated soil enhance contact between contaminants and soil microorganisms thereby

increase the faster removal of contaminant hydrocarbons. The effects of earthworms on the

Part IV


removal of PAHs, which serve as a model for soil contaminated with petroleum or as a

remediation test, have been reported by several authors (Ma et al.,1995; Eijsackers et

al.,2001; Contreras-Ramos et al.,2008; Geissen et al.,2008; Hickman et al.,2008; Tejada

and Masciandaro, 2011).

A summary of benefits of the use of earthworm in petroleum contaminated soil are given

below: 1) May enhance the long term remediation of petroleum contaminated soil by

stimulating the microbial biomass and activity which would reduce cost as sites could be

used commercially sooner 2) improve soil quality( soil fertility, increased drainage and

aeration by earthworm dwelling activities) and 3) increase the bioavailability of oil

components for microbial degradation. Bioavailability of oil components in contaminated soil

is an important regulating factor for microbial degradation rates (De Jonge et al.,1997).

Petroleum hydrocarbons which are not bioavailable for microorganisms are either solids or

diffused into inaccessible soil pore. The biodegradation decreases when they strongly

bound to particulate as they are less available to microbial population which can utilize them

( Ivshina et al.,1998). Earthworms may increase the bioavailability of petroleum hydrocarbon

bound to soil matrix and those in inaccessible pores for microbial degradation, by

mechanically working up the soil matrix.

Dorn and Salinitro (2000) observed that a significant fraction of volatile hydrocarbon was

lost during mixing and weathering of the substrate. De Jonge et al.,(1997) reported that

oxygen was a limiting factor and therefore assumed that aeration and fertilization can be

very efficient during bioremediation. The positive effect of increased hydrocarbon removal in

earthworm treated soil is attributed to an increase in aeration and bioavailability of oil by

redistributing oil in soil which increased the area of exposure of oil to microorganisms.

Chaineu et al.,(2003) in their field studies after 480 days of experiments 70-80% reduction in

HC concentration was observed in soil with nutrients whereas natural attenuation without

added nutrients have only 56% reduction. Aeration is essential for composting of oil

contaminated soil because hydrocarbon contained in diesel are degraded by oxygenase

catalyzed reactions in the presence of molecular oxygen. Hwang et al., (2006) reported that

intermittent aeration resulted in better degradation of diesel contaminated soil. Although soil

microorganisms are responsible for the biochemical degradation of organic matter,

earthworms are important drivers of the process, conditioning the substrate and altering

biological activity (Schaefer et al.,2005). Earthworms maintain the aerobic conditions in soil

through continuous mixing thus favoring degradation of contaminants. They increase




organic substrate availability for autochthonous soil microorganisms thereby increasing

microbiological and biochemical soil activity (Wolters, 2000).

Earthworms enhance hydrocarbon degradation by stimulating microbial activity and

growth via excretion of readily degradable carbon. They also have an indirect effect on the

structure and activities of bacteria and fungal communities through grazing, gut passage

and aggregate formation (Aira et al., 2002). Matscheko et al., (2002) also reported

improvement of the aerobic conditions of the soil by earthworms through continuous mixing

thereby increasing the degradation of contaminants. Additionally, when the earthworms

ingest soil, rearrange soil particles and thereby increasing the availability of the

contaminants (Schaefer et al., 2005). The addition of earthworms increased the removal of

anthracene compared to the soil without earthworms. Contreras-Ramos et al.,(2006) found

a 42% reduction in anthracene contamination of soil with the autochthonous microorganisms

and 91% in soil with earthworms. Eijsackers et al.,(2001) also found that removal of

phenanthrene in soil was aided by the use of earthworms. Coutino-gonzalez et al., (2010)

used Eisenia fetida to remediate anthracene-contaminated soil. Anthracene-contaminated

soil was amended with or without Eisenia fetida and degradation products monitored. A 93%

removal of anthracene was found for treatments with earthworms and 41%for those without

earthworms.

The addition of earthworms to soil can stimulate degradation of hydrocarbons in different

ways. The secretion of mucus by the earthworm might stimulate the growth of hydrocarbon-

degrading microorganisms. Furthermore, beneficial microbes, such as nitrogen-fixing

bacteria, might have accumulated in the worm casts. At the same time, earthworm

burrowing improves aeration, mixes the soil and increases the soil surface area for microbial

interactions, can inhibit soil-borne pathogens, and neutralize pH (Edwards and Bohlen 1996;

Schaefer and Filser 2007). Morgan and Burrows (1982) reported that the internal fluid of E.

fetida cocoons contains bacteria of the genera Nocardia, Pseudomonas and Alcaligenes. In

addition, bacteria of genera Rhodococcus and Azotobacter have been found in burrows of L.

terrestris (Tiunov and Dobrovolskaya, 2002). The bacterium, Rhodococcus has been

observed to use anthracene, phenanthrene, pyrene and fluoranthene as its sole source of

carbon and energy (Dean-Ross et al.,2001). Singleton et al.,(2003) studied the bacteria

associated with the intestine and casts of the earthworms and found bacteria, such as

Part IV


Pseudomonas, Paenibacillus, Azoarcus, Burkholderia, Spiroplasm and Acidobacterium.

Some of these bacteria, such as Pseudomonas, Alcaligenes and Acidobacterium are known

to degrade hydrocarbons (Cerniglia 1993; Juhasz and Naidu 2000; Johnsen et al.,2005).

Furthermore, fungi, such as Penicillium, Mucor and Aspergillus have been isolated from

earthworm intestines (Pizl and Nováková, 2003) as well as fungi of the genus Penicillum

that are able degrade PAHs (Johnsen et al., 2005). Earthworms might also directly

contribute to the removal of hydrocarbons from soil by selecting and/or stimulating soil

microorganisms. It is known that earthworms can directly regulate microbial populations by

consuming large amount of soil (Drake and Horn 2007). This leads to elimination of some

microorganisms and proliferation of others in the digestive tract, drilosphere and faeces of

earthworms (Bonkowski et al.,2000; Tiunov and Scheu, 2000; Tiunov and Dobrovolskaya,

2002). Byzov et al.,(2007) found that the mid-gut fluid of earthworms possesses a selective

suppressive activity while stimulating certain soil microorganisms. For instance, spores of

some fungi that survived in the mid-gut environment (Alternaria alternata) started to

germinate and grew actively in fresh excrement. The fate of microorganisms passing the

digestive tract of earthworms is an important factor in the formation of the soil microbial

community

Earthworms might also directly contribute to the removal of hydrocarbons from soil

by selecting and/or stimulating soil microorganisms. It is known that earthworms can directly

regulate microbial populations by consuming large amount of soil (Drake and Horn 2007).

This leads to elimination of some microorganisms and proliferation of others in the digestive

tract, drilosphere and faeces of earthworms (Bonkowski et al., 2000; Tiunov and Scheu

2000; Tiunov and Dobrovolskaya 2002). Byzov et al. (2007) found that the mid-gut fluid of

earthworms possesses a selective suppressive activity while stimulating certain soil

microorganisms.The fate of microorganisms passing the digestive tract of earthworms is an

important factor in the formation of the soil microbial community and the degradation of

organic material and contaminants. Achazi et al. (1998) found enzymatic activity of

cytrochrome P450 in terrestrial earthworms (E. fetida) exposed to PAHs. Zhang et al. (2006)

observed a dose-depended increase in the total cytochrome P450 content in E. fetida in

response to rising concentrations of pyrene and BaP from 10−6 to 10−2mg ml−1.




Level of TPH in vermicast

The concentration of TPH present in the worm cast was assessed. Most of the

vermicasts obtained using precomposted soil as feed had TPH in them. The worm cast

obtained using the precompost having PS+I possessed high concentration of TPH. Fewer

amounts were present in the combination of PS with CD and feed. Therefore precautions or

preventive measures should be taken during the application of such worm casts as fertilizer

in the field in order to avoid the contamination of soil with TPH (Table 4.3.10).

Worm biomass and worm number

Change in biomass of earthworms in vermireactors fed with precomposted soils is

illustrated in figures 4.3.5-4.3.10. Worm biomass was found to be increased in those

reactors having an additional inoculum. An increase in worm biomass was observed in all

the three combinations with feed in the 30 day precomposted samples, whereas in 60 day

samples an increase in biomass was observed only for CD and VT supplemented

precompost. Maximum number of young ones production was observed with CD

supplemented precomposts of both 30 and 60 day precomposting durations when used as

feed in vermireactors (Tables 4.3.13 and 4.3.14). Maximum mortality of worms was

observed with reactors run with PS - precomposted without any supplement of CD or VT

(Tables 4.3.12 -4.3.14).

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Figure 4.3.5: Change in biomass of Eudrilus eugeniae in 15 day precomposted soil in the different combination of PS, PS with CD; and PS with CD and VT with or without microbial inoculum

Figure 4.3.6: Change in biomass of Eisenia fetida in 15 day precomposted soil in the different combination of PS, PS with CD; and PS with CD and VT with or without microbial inoculum




Figure 4.4.7: Change in biomass of Eudrilus eugeniae in 30 day precomposted soil in the

different combination of PS, PS with CD; and PS with CD and VT with or without microbial

inoculum

Figure 4.3.8: Change in biomass of Eisenia fetida in 30 day precomposted soil in the

different combination of PS, PS with CD; and PS with CD and VT with or without microbial

inoculum

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Figure 4.3.9: Change in biomass of Eudrilus eugeniae in 60 day precomposted soil in the different combination of PS, PS with CD; and PS with CD and VT with or without microbial inoculum

Figure 4.3.10: Change in biomass of Eisenia fetida in 60 day precomposted soil in the different combination of PS, PS with CD; and PS with CD and VT with or without microbial inoculum




Figure 4.3.11: Young one production in Eudrilus eugeniae reactors after 30 days of

vermicomposting period in three different precompost

Figure 4.3.12: Young one production in Eisenia fetida inoculated reactors after 30days of

vermicomposting period in three different precompost

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Several studies have found that organic material added as feed has a positive effect on

the survival of earthworms in PAHs contaminated soil while reducing weight loss (Table 1).

Tejada and Masciandaro (2011) found that the weight of E. fetida decreased 20% less

compared to the control when organic municipal solid waste was added to soil contaminated

with 50 mg kg−1 BaP and 22% poultry manure was applied. Contreras-Ramos et al.,(2009)

reported that when E. fetida had food (wastewater sludge) and was exposed to a mixture of

100 mg phenanthrene kg−1, 500 mg anthracene kg−1and 50 mg BaP kg−1, the survival rate

of the earthworms was 93% and their weight increased with 28% after 70 days. However,

when deprived of food (i.e. organic matter) earthworms lost 79% of their initial weight and

their survival rate was only 60%. Concordantly, Matscheko et al.,(2002) reported a 5%

decrease in weight of earthworms 19 days after being added to a soil contaminated with 16

different PAHs. Eijsackers et al.,(2001) reported that when worms (E. fetida) were added to

a soil contaminated with 10 mg phenanthrene kg−1 but also having 10% or 40% organic

material, their survival was 98% and 100%, respectively, with an average of 25% weight

loss. These findings indicate that organic material plays an important role in earthworm

survival by providing them with nutrients, which stimulates the microbial activity in their gut.

Additionally, the application of organic material increases soil microbial activity in general,

thereby accelerating the removal of PAHs (Hamdi et al.,2007; Sinha et al.,2008; Juwarkar et

al.,2010).

Geissen et al. (2008) reported that the survival of E. fetida was not affected by 1%

petroleum, but mortality increased at 2%. Gomez-Eyles et al. (2011) found a survival rate of

97% for E. fetida when added to soil contaminated with a mixture of PAHs (from 2 to 6

rings); the earthworms also lost 31% of their initial weight after 56 days. Eisenia fetida had a

survival rate of 86% with a 72% weight loss in an anthracene-contaminated soil

(concentrations of 200, 500 and 1000 mg kg−1were used) and a 70% survival rate with a

70% weight loss in the same soil spiked with benzo(a)pyrene (BaP) (average concentrations

of 50, 100 and 150 mg kg−1) without feed after 77 days (Contreras-Ramos et al.,2006).

Phenanthrene was the most toxic of the three PAHs studied as no earthworm survived when

soil was contaminated with 150 mg phenanthrene kg−1 soil, but 83% did when contaminated

with 100 mg phenanthrene kg−1 soil. Schaub and Achazi (1996) found that 100 mg BaP kg−1

soil did not affect growth and survival of E. fetida.




Nutrient status of worm cast

The amount of total nitrogen, available phosphorous and potassium in the casts

obtained with precomposted soils were higher than that of the casts obtained from the

control reactors fed with cowdung as feed. The results of chemical parameters showed a

decrease in the concentration of total carbon, phosphorous and available forms of carbon

and nitrogen. This suggests a progressive degradation of organic compounds, including

pollutants, especially when microorganisms were added or authoctonous microorganisms

were stimulated with the addition of organic substrates such as cowdung. Earthworm

increases the availability of nitrate which is an electron acceptor thus helping in the

degradation of organic matter. Degradation of organic matter and immobilization of mineral

nutrients- by the reduction in assimilable phosphorus and ammonia created the conditions to

increase total microbial biomass. Also the NPK level in the casts obtained in the present

study is comparable with reported value in the literature (Cardoso et al., 2002). C/N ratio

was found to be less in the combinations of PS, with CD and feed when compared to other

combinations other than control reactors. Since the cast contain considerable amount of

nutrients in the available form they can be used as fertilizer, with a precaution of the TPH

content of the cast.

4.3.6. Summary and Conclusions

As a result of anthropogenic activities, huge quantities of hydrocarbons are released

into the environment. Petroleum hydrocarbons including Poly Aromatic Hydrocarbons

(PAHs) have been categorized as priority pollutants by United States Environmental

Protection Agency (USEPA). The use of bioremediation in the treatment of hazardous waste

such as petroleum hydrocarbon is a recent concept, and it is a rapidly growing trend in

environmental management. The significance of the present study lies on the use of

earthworms and associated microorganisms together in remediating oil contaminated soil.

Two epigeic earthworms- Eudrilus eugeniae and Eisenia fetida-were used in this study for

the bioremediation of oil contaminated soil. The contaminated soil was subjected to

precomposting for different time intervals (15 days, 30 days and 60 days) with the addition

Part IV


of different amendments such as cowdung (CD), vermitea (VT) and inoculum (I). Then, the

precompost obtained were used as bedding material for earthworms in vermireactors. The

vermireactors were operated with and without additional feed of cowdung. After 30 days of

vermicomposting, the percentage removal of TPH in the soil samples were analysed by

Soxhlet extraction followed by absorbance using UV spectrophotometer. The loss of TPH

due to mere evaporation from the reactors operated without any amendment and without

precomposting and without earthworms was also assessed. Worm biomass, mortality,

survival and young ones produced during the vermicomposting of precomposted soils were

recorded.

The findings of this study leads to the following significant conclusions:

1. The combination of microbes and earthworms resulted in a better- rather faster removal

of TPH than the experiments reported in the literature in which remediation was done

with microbes alone.

2. Cowdung (CD) supplements either at precomposting or at vermicomposting stage

increases the TPH removal rate, i.e. Addition of CD facilitates faster removal of TPH.

3. The CD supplement also enhances the number of juveniles production as well reduces

mortality in earthworms.

4. The C/N ratio of the vermicast produced is well within usable range, so that when the

casts are applied to the soil the plants can assimilate nutrients from the casts easily,

however the level of TPH contained in the vermicasts need to be assessed before

applying them in the field.

5. The impact of inoculum on the contaminant removal was better in 15d and 30d

precomposted soil. In the 60d precomposted samples no significant impact on TPH

removal was noticed between reactors operated with and without inocula.

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