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Research Article
Effect of Mechanical Mixing and Microbial Population Dynamics in Slurry-Phase Bioremediation
Samuel C. Eziuzor1,2* and Gideon C. Okpokwasili2
1Department of Biological Science, Rhema University, Nigeria
2Department of Microbiology, University of Port Harcourt, Nigeria

KEYWORDS
Bioremediation; Niger delta; Bioreactor; Oil-contaminants; Removal efficiency; Microbial cells
Abstract
The need for high biodegradation rates requires application of microorganisms in controlled environmental and nutritional conditions. Soil slurry bioremediation is a powerful alternative to conventional methods in resolving environmental oil-contamination problems. This work studied the technical viability of treating soil slurry biologically by stimulation of native microorganisms. Oil contaminated mangrove soil from Alakiri, Nigeria was mixed with water at 1:3 ratio and treated in three different ways (A, B and C). Statistical increase in microbial population and hydrocarbon removal efficiency were observed. In terms of total petroleum hydrocarbon degradation, removal efficiency of 55.31% was achieved in Slurry B (amended and stirred slurry), closely followed by 53.21% of Slurry A (unamended and stirred slurry) and 31.58% for slurry C (amended and unstirred slurry). The importance of mechanical mixing on the microbial population dynamics and on the biodegradation of the oil was ascertained. This study has shown that slurry bioreactor is effective in the bioremediation of soils containing oil-contaminants.

Introduction
There is growing public concern as the high rate of oil-related activities in the Niger Delta region of Nigeria are being associated with frequent oil spills, as a result of sabotage, corrosion of pipes, storage tanks, eruption during oil production operation and oil tankers accidents. The region is notably characterized by mangrove swamps and narrow creeks which are flooded by tidal action [1]. Microorganisms are powerful alternatives to conventional methods in resolving environmental problems since they are able to use these oily substances as carbon and energy sources [2,3]. The need for high biodegradation rates requires application of specialized microorganisms in controlled environment and nutritional conditions [4,5]. The basic slurry approach involves the suspension of soil in an aqueous solution in a reaction vessel that allows for mechanical mixing [4,6,7]. Mixing facilitates aeration and enhances the rate of chemical exchange between soil particles and the solution [4,6,8].
Effective bioremediation of oil pollution relies heavily on a fundamental understanding of microbial population dynamics [6]. There is a need to integrate information about biodegradation processes and microbial populations [9]. The ability to characterize microbial communities, such as those involved in biodegradation, by analysis of biochemical markers improves the study of population dynamics involved in the biodegradation of these contaminants [10].
Environmental biostimulation such as through fertilizer addition and aeration is the commonly used approach for slurry bioremediation [2,4]. The main goal of the bioremediation design was the creation of the most favourable conditions for microbial growth and activities. The biodegradation potential of hydrocarbons can be assessed by using slurry reactors (10-30% soil : water w/v), which offer several advantages over soil microcosms [4,7]. Due to more efficient mixing, aeration and improved substrate bioavailability, the duration of a bioremediation process can be significantly reduced [11]. However, the degradation rates of hydrocarbons are site specific and are limited by the metabolic capabilities of the hydrocarbon-degrading microbial populations and also by a wide range of environmental factors [2]. The effectiveness of the bioremediation depends therefore on the success in identifying the rate-limiting factors and optimizing them.
This work encompasses the study of the effect of mechanical mixing on oil-contaminated bioremediation by microbial population dynamics, previously adapted in a mangrove soil medium containing oil as the only carbon and energy source.
Materials and Methods
Samples Collection
The oil-contaminated soil was obtained from a mangrove swamp close to SPDC Alakiri flow station, accessible via Port Harcourt Marine Base, Nigeria. The representative soil composite sample was excavated with a spade and collected in a plastic pail. The water was collected using 20L plastic water can for the preparation of soil slurries. The water can was first rinsed thoroughly with the samples before final collection.
Nutrient Supplement
Nitrogen-Phosphorus-Potassium (NPK/ 20:10:10) agricultural fertilizer (National Fertilizer Company of Nigeria, Onne, Rivers State, Nigeria) was used as the nutrient supplement at 0.01% concentration in different treatments.
Treatment Design
The design comprised of three treatment options: The 25% slurry mixes were introduced into a locally fabricated aerobic bioreactor system constructed using stainless steel material, with a working volume of 30L and powered by an electric motor of 2.0KW power and 50Hz frequency.
The treatments were:
  • Slurry A Un-amended and stirred slurry (25% soil slurry + nil NPK fertilizer + mixing in reaction vessel).
  • Slurry B Amended and stirred slurry (25% soil slurry + 0.01% NPK fertilizer + mixing in reaction vessel).
  • Slurry C Amended and unstirred slurry (25% soil slurry + 0.01% NPK fertilizer + without mixing in reaction vessel).
Enumeration, Isolation and identification of Bacteria
The total aerobic heterotrophic bacterial population present in the samples was determined using nutrient agar. Serial tenfold dilution using sterile normal saline water (0.85% NaCl) as diluent was prepared and 0.1ml aliquot of appropriate dilutions were spread plated in triplicate, incubated for of 24 – 48 hours at 37°C aerobically. After incubation the plates were observed for growth and colony count taken.
The enumeration of hydrocarbon utilizing bacteria was done by vapour-phase transfer method. This was done by using of the modified minerals salts medium containing the following in gl-1: MgSO4.7H2O, 0.42; KH2PO4, 0.83; NaCl, 10.0; KCl, 0.29; Na2HPO4, 1.25; NH4NO3, 0.42; agar, 15.0 and deionized water. Before incubation of the plates filter paper (Whatman No. 1) saturated with Bonny Light crude oil was aseptically placed inside the cover and inverted over the agar plate. The filter papers supplied the hydrocarbons by vapour-phase transfer to the inoculum. After incubation at room temperature for 5-7 days, colony counts were taken and recorded [1,3].
Phenanthrene overlay method (for enumeration of non-volatile hydrocarbon utilizers) was used. The count clearing method using a modified estuarine salt water agar of [12] comprises the following in gl-1 was used; sodium glycerophosphate, 0.05; proteose peptone, 0.1; yeast extract, 0.1; glycerol, 0.5ml; agar, 15.0 and aged tidal water. The plates were poured and dried overnight, then 0.2ml of a filtered (0.2m) solution containing 0.5g phenanthrene per 100ml of 95% ethanol was uniformly spread on the surface of the agar plates (while gently spinning around). The plates were further dried overnight at room temperature to allow the ethanol to evaporate before inoculation. Plates prepared in this manner had an opaque/opalescent film of phenanthrene on the agar surface (West et al., 1984). Appropriate dilutions of the samples were inoculated and incubated at room temperature for 4 to 7 days. Phenanthrene-utilizing (Phn+) bacteria appeared as colonies surrounded by zones of clearing in the opaque phenanthrene film.
Colonies of the hydrocarbon-utilizing bacteria were randomly picked, isolated and purified by streaking on nutrient agar. The isolated colonies were then stored on nutrient agar slants and later examined for their biochemical and phenotypic characteristics. These included Gram-stain reaction, colonial appearance, motility, catalase, indole, oxidase, citrate, methyl red and voges – proskaeur, sugar fermentation and starch hydrolysis. The tests were performed following the procedures of [13] and identification based on [14].
Temperature, pH and Conductivity
The temperature at each withdrawal was measured using a standard mercury thermometer. The pH of the withdrawn samples was determined by using a digital pH meter (Jenway 3075 UK), whereas conductivity values were determined by using Jenway 4010 UK meter, with temperature adjusted to 25±0.1°C.
Total Petroleum Hydrocarbon (TPH) and Gas Chromatographic Analysis
TPH analysis was carried out using American Society of Testing and Materials, ASTM D3921 method [15]. The extent of hydrocarbon degradation during the experimental set up was analyzed by gas chromatography, GC. The gas chromatography (GC – FID) used was a HP 6890 series (Hewlett Packard, USA) equipped with a flame ionization detector, FID. The initial and final temperatures within the oven were 40°C and 325°C respectively and pressure at 4.2Psi. The carrier gas was nitrogen while the combustible (support) gas was hydrogen. An aliquot of each of the samples was extracted by transferring into a separatory funnel and 10ml of pentane (extraction solvent) was added. The solvent extract was carefully cleaned up and concentrated to 1ml for analysis. One microlitre (1µl) of the concentrated sample was rapidly injected into the column. After separation between the gas and liquid phases, the sample was automatically detected as it emerges from the column (at a constant flow rate) with FID detector at 38.3 minutes run time.
Results
Table 1 displays the summary of the properties of oil-contaminated soil from Alakiri mangrove. It is evident from the table that there is a gross oil-contamination in the mangrove swamp. The three parameters of microorganisms tested indicated the population dynamism in the environment.
Table 1 Summary of the properties of the oil-contaminated soil.

Table 1

Parameters

Values

pH

6.90

Conductivity (mscm-1)

5690

Total petroleum hydrocarbon (mgl-1)

112.60

Total heterotrophic bacteria (Log10CFUg-1)

5.88

Hydrocarbon-utilizing bacteria (Log10CFUg-1)

5.70

Phenanthrene utilizing bacteria (Log10CFUg-1)

5.45

Table 1 Summary of the properties of the oil-contaminated soil.

×
Figure 1 Changes in the total aerobic heterotrophic bacterial population of the soil slurries during thebioremediation process.

Figure 1

Figure 1 Changes in the total aerobic heterotrophic bacterial population of the soil slurries during thebioremediation process.

×
Microbial Analysis
The population of total heterotrophic bacteria was 5.88 Log10 CFU (colony forming units) g-1 as initial property of oil-contaminated soil (Table 1). The populations of total heterotrophic bacteria of all slurries were between 5.97-5.99 Log10CFUml-1 at zero time before biotreatment (Figure 1). Then rose rapidly to between 6.24-6.43 Log10 CFUml-1 after 28-days of study.
The volatile hydrocarbon-utilizing population responded positively as their units increased from 5.76-5.98 Log10CFUml-1 at zero time to 6.18-6.29 Log10CFUml-1 after 28-day of study for all slurries (Figure 2). While the phenanthrene-utilizing bacteria showed a less than other populations Figure 3). The slurries show between 6.13-6.25 Log10CFUml-1 at the end of 28-days of treatment with Slurry B recording 6.14 Log10CFUml-1 (Table 2).
Figure 2 Changes in the volatile hydrocarbon-utilizing bacterial population of the soil slurries during the bioremediation process.

Figure 2

Figure 2 Changes in the volatile hydrocarbon-utilizing bacterial population of the soil slurries during the bioremediation process.

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Figure 3 Changes in the phenanthrene-utilizing bacterial population of the soil slurries during the bioremediation process.

Figure 3

Figure 3 Changes in the phenanthrene-utilizing bacterial population of the soil slurries during the bioremediation process.

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Figure 4 Changes in pH profiles of the soil slurries during the bioremediation process.

Figure 4

Figure 4 Changes in pH profiles of the soil slurries during the bioremediation process.

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Physicochemical Analysis
There are considerable fluctuations in the pH as the values obtained were acidic values (Figure 4). The pH readings after 28-days were 6.41 – 6.68 (Table 2), which are considerably steady with the zero time readings. The conductivity profile showed an observable decrease in values throughout the study (Figure5).
Table 2 Effect of mechanical mixing and nutrient addition on soil slurries during the 28-days study.

Table 2

Slurry

A

B

C

Chemical Properties

pH

6.41

6.42

6.68

Conductivity (mscm-1)

2840

2860

2940

Total petroleum hydrocarbon (mgl-1)

55.88

44.44

66.43

Microbiological Properties

Total heterotrophic bacteria (Log10CFUml-1)

6.43

6.35

6.24

Hydrocarbon-utilizing bacteria (Log10CFUml-1)

6.27

6.29

6.18

Phenanthrene utilizing bacteria (Log10CFUml-1)

6.25

6.14

6.13

Table 2 Effect of mechanical mixing and nutrient addition on soil slurries during the 28-days study.

×
The hydrocarbon removal efficiency of the various treatments was investigated (Figure 6). It showed that slurry B (amended and stirred slurry) had the highest removal percentage, closely followed by slurry A (unamended and stirred slurry) and lastly slurry C. The quantity of residual total hydrocarbon content remaining at the end differed according to the treatment option employed. These changes in chemical properties were correlated to the microbial activities of the slurries (Table 3).
Figure 5 Changes in conductivity profiles of the soil slurries during the bioremediation process.

Figure 5

Figure 5 Changes in conductivity profiles of the soil slurries during the bioremediation process.

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Figure 6 Relationship between microbial population and hydrocarbon removal efficiency of TPH during the bioremediation process.

Figure 6

Figure 6 Relationship between microbial population and hydrocarbon removal efficiency of TPH during the bioremediation process.

×
Table 3 Correlation coefficients among chemical and microbial parameters on oil-contaminated soil.a
aCorrelations were based on triplicate data of the soil slurry and at 0.05 probability levels. THB = total heterotrophic bacteria; HUB = hydrocarbon utilizing bacteria; PUB = phenanthrene-utilizing bacteria.

Table 3

 Parameters

THB

HUB

PUB

pH

Conductivity

THB

1

 

 

 

 

HUB

0.87821

1

 

 

 

PUB

0.985005

0.803464

1

 

 

pH

0.153893

0.244583

0.092348

1

 

Conductivity

-0.94598

-0.93231

-0.91936

-0.11227

1

Table 3 Correlation coefficients among chemical and microbial parameters on oil-contaminated soil.a
aCorrelations were based on triplicate data of the soil slurry and at 0.05 probability levels. THB = total heterotrophic bacteria; HUB = hydrocarbon utilizing bacteria; PUB = phenanthrene-utilizing bacteria.

×
The gas chromatogram represented the peak area values of group hydrocarbons obtained from the various slurry analyzed (Figure 7).
Figure 7 Changes in gas chromatogram profiles of the soil slurries during the bioremediation process. (THB=total heterotrophic bacterial population; HUB=hydrocarbon-utilizing bacterial population; TPH=total petroleum hydrocarbon)

Figure 7

Figure 7 Changes in gas chromatogram profiles of the soil slurries during the bioremediation process. (THB=total heterotrophic bacterial population; HUB=hydrocarbon-utilizing bacterial population; TPH=total petroleum hydrocarbon)

×
Discussion
The activities of the resident microorganisms at the Alakiri mangrove swamp near Port Harcourt, Nigeria is greatly affirmed by their microbiological properties (Table 1). The population dynamics in the mangrove ecology is appreciated in the variety and changes noticed during the study [3,16]. The near neutral pH value of 6.90 is a considerable factor encouraging the flourish in the microbial activities in oil degradation. The Niger Delta region is noted for unstable physical parameters including pH, conductivity and temperature due largely to oil contamination effect via gas flaring [17]. The temperature values obtained were in the mesophilic range of 28 – 32°C, which agrees with [18].
The response of hydrocarbon-utilizing bacterial population was positive with high population growth occurring progressively as the time elapsed [16,19]. The percentage of hydrocarbon-utilizing bacteria to the total heterotrophic bacterial counts reflected the extent of native microbial and hydrocarbon degradation activities in the oil-contaminated site. [11] reported a strong correlation between microbial population and hydrocarbon degradation. The total microbial population in the form of total colony forming units was usually increased by higher orders of magnitude [2,4,20]. The identification of native bacteria showed a predominance of these organisms: Acinetobacter, Alcaligenes, Athrobacter, Bacillus, Citrobacter, Corynebacterium, Flavobacterium, Micrococcus, Pseudomonas and Vibrio species. Three bacterial genera; Alcaligenes, Pseudomonas and Vibrio species were also identified as phenanthrene utilizing bacteria (Phn+). This agrees with [21] and [20].
Biostimulation such as through fertilizer and aeration is the commonly used approach in soil slurry bioremediation [22,23], since the main goal of any biotreatment design is the creation of the most favourable conditions for microbial growth and activities [5]. [16] inferred that bacterial population changes occurred as a result of biostimulation, and it correlated appropriately with figures 1 – 3. The biostimulation of indigenous bacterial population using an NPK fertilizer was very invaluable as it provided for nitrogen and phosphorous required for metabolism [24]. Due to more efficient mixing, aeration and improved substrate bioavailability, the duration of the biotreatment was significantly reduced [8,25]. Oxygen is made more available to the organisms for growth thereby helping to eliminate anaerobic conditions, which usually occurs in static culture [20].
The high initial values of total petroleum hydrocarbon are due to large amounts of crude oil in the mangrove soil, which reflects the level of oil pollution in the Niger Delta [3,26,27]. The removal efficiencies of total petroleum hydrocarbon were in the order of 55.31%, 53.21% and 31.58% for slurry B, slurry A and slurry C, respectively. This can be attributed to vigorous mixing that increased the contact between microbial cells and the oil pollutants except for slurry C. [28] found out that bioaugmentation instead had a removal efficiency of about 65 – 93%, which is even a higher percentage, though it was for polyaromatic hydrocarbons in a bioslurry phase reactor. In addition, oxygen is made more available and the supplemental nutrient led to increase in biomass and biological activity. Slurry C (amended unstirred slurry) recorded lowest due to lack of mixing, even though there was a supplemental nutrient. The nutrient was not made readily available to the microbial cells. There was a negative correlation between oil and grease and nutrient levels in all the slurries [29].
The comparative high peak area of the slurries at zero time prior to treatment indicated high proportions of saturated hydrocarbon. A careful observation of the peaks showed evidently that slurries obtained after 28 day, had a comparable peak reduction. The results showed that high removal efficiency could be obtained as found by other workers [20,21,29]. Because of analytical limitations, polycyclic aromatic hydrocarbons (PAHs) were not determined. The works of [28] established a good removal efficiency of PAHs through the use of bioaugmentation.
This study has shown that slurry bioreactor can be effective in the bioremediation of soils containing oil-contaminants as mixing breaks up soil aggregates and enhanced the rate of exchange of the contaminant between soil suspension and microbial cells. The biostimualtion effect of NPK fertilizer addition was very instrumental to the bioslurry treatment results obtained.
Acknowledgement
The authors are grateful to Technical Partners International, Port Harcourt for the total petroleum hydrocarbon and gas chromatographic-flame ionization detection analysis in their laboratory.

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Cite this article: Eziuzor SC, Okpokwasili GC (2013) Effect of Mechanical Mixing and Microbial Population Dynamics in Slurry-Phase Bioremediation. JSM Microbiology 1(1): 1003.
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