B018_Nur Hidayatul Alami

3rd International Conferences and Workshops on Basic and Applied Sciences 2010 ISBN: 978-979-19096-1-7

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The Effectiveness of Pseudomonas putida T1(8) Biosurfactant in Bioremediation of Crude Oil

Contaminated Soil

Nur Hidayatul Alami *, Nimatuzahroh , Tini Surtiningsih

Department of Biology Airlangga University, Surabaya, Indonesia

e-mail: [email protected]

Abstract

Crude oil contaminated soil was a serious problem. Bioremediation method was a viable alternative method to remedy oil contaminated soil. A possible way to increase bioremediation rate was the application of biosurfactant. Pseudomonas putida T1(8) was the native isolate which can produce extracellular biosurfactant. The aim of this research was to know the effectiveness of biosurfactant P. putida T1(8) when it was added in bioremediation test. The effects of biosurfactant addition in the bioremediation can be known from the increasing of the microbial heterotrophic total count (cfu/g-soil) and the decreasing of the oil residue (g/g-soil). The type of the research was experimental with 3 controls, including non sterile soil without biosurfactant (KI), sterile soil without biosurfactant (KII), non sterile soil with biosurfactant at CMC level (KIII) and 4 treatment, including non sterile soil with adding of microbial consortium without biosurfactant (B0), non sterile soil with adding of microbial consortium and biosurfactant below the CMC (B1), non sterile soil with adding of microbial consortium and biosurfactant at CMC level (B2), and non sterile soil with adding of microbial consortium and biosurfactant above the CMC (B3). The incubation times were 0, 2, and 4 weeks. The resulted data of the logarithm microbial total count and the crude oil residue (g/g-soil) were analyzed descriptively and statistically by two-way analysis of variances (two-way ANOVA) and Duncan test (p=0.05). The results of this research indicated that biosurfactant gave an effect on bioremediation process. Biosurfactant at CMC value at final incubation time (fourth week) was known to be the most effective to increase microbial growth and to decrease the oil residue with the amount of 12.68%. Keywords: soil bioremediation, biosurfactant,

Pseudomonas putida T1(8)

1 Introduction

As the increasing of explorative activities and oil utilization, oil spills on the land has also been on the increase (Ekpo and Udofia, 2008). The greatest impact of oil spills is the disruption of land function as a natural media to support vegetations, animals, and human life. The disruption of land function will influence all of the ecosystem stability (Lynch and Poole, 1978; Reginawanti and Simarmata, 2004). Bioremediation was developed to solve that problem. Bioremediation has become a valuable alternative technology as it is a cost effective, cheap, and environmentally friendly treatment (Alamri, 2009; Mariano et al., 2007). These processes rely on the natural ability of microorganisms to carry out the mineralization of organic chemicals, leading ultimately to the formation of CO2, H2O and biomass. There are some methods in bioremediation, that are; biostimulation, bioaugmentation, and bioavailability. Biostimulation is strategy to accelerate the biological breakdown of hydrocarbons in soil include stimulation of the indigenous microorganisms by optimizing the nutrients. Bioaugmentation is a strategy through inoculation of an enriched mixed microbial consortium into soil, whereas bioavailability is a strategy to increase accessibility between substrate and microbes (Mariano et al., 2007). The combination of that all methods are necessary needed to advance bioremediation process.

The effort to increase availability of hydrocarbon substrate for microbes is extremely important, that because of its hydrophobic characteristic. So, it is difficult to absorb into the soil matrix. Providing a way to reduce the sorption of the hydrophobic organic contaminants to the soil matrix can increase the rate and extent of biodegradation. For this purpose, the addition of biosurfactant into the soil aims to enhance the emulsification of hydrocarbons

Nur Hidayatul Alami, The Effectiveness of Pseudomonas putida T1(8) Biosurfactant in Bioremediation of Crude Oil Contaminated Soil

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and therefore they have the potential to solubilize hydrocarbons and increase their bioavailability, thus it possible to increase accessibility between substrate and microbes. Finally, it can increase microbial growth and biodegradation rate (Tuleva et al., 2001; Jagadevan and Mukherji, 2004; Mariano et al., 2007).

The advantage of biosurfactant if compared to their chemically synthesized counterparts is environmentally friendly. Biosurfactant is low toxicity, can be degraded naturally by microbes, also can be produced from renewable sources (microbes) (Kosaric, 2001).

In another country, application of biosurfactant has already been developed to support bioremediation process. This strategy is of paramount concern (Banat et al., 2000). However in Indonesia, the application of biosurfactant in bioremediation is still rare. May be it is because of the cost of biosurfactant production (Hamme et al., 2003).

In the previous study, Nimatuzahroh et al. (2009) has been explored bacteria and yeast for bioremediation of hydrocarbon contamination purpose from contaminated area close to the storage and distribution center of oil products in Surabaya and Wonocolo refinery, East Java Indonesia. One of those microbes is Pseudomonas putida T1(8). Alami et al. (2010) has been reported that Pseudomonas putida T1(8) which was grown on a molasses substrate and was extracted by (NH4)2SO4 precipitation, gave the highest yield of biosurfactant about 5.0467 g/l, had the lowest CMC value about 665 ppm, and decrease surface tension more than 10 mN/m (21.6 mN/m). It also showed the best emulsifier activity in diesel oil and stable at the temperature range 30-70C (100-93.18%), pH 4-7 (100-82.41%), and NaCl concentration 0-3 M (100-98.60%).

In addition, according to Bordoloi and Konwar (2008), carbon source like molasses which was used to produce biosurfactant Pseudomonas putida T1(8) can be an alternative to compress biosurfactant production cost. So, it can be of great potential in the remediation of oil polluted soil.

The acceleration of bioremediation process adding by biosurfactant is influenced with the effectiveness of its biosurfactant. One of the factors that influence the effectiveness of biosurfactant is CMC value (Nimatuzahroh et al., 2004). Beside that, another factors, like: biosurfactant toxicity, the compatibility of biosurfactant against hydrocarbonoclastics microbes, hydrocarbon component, and environmental factors also influence the effectiveness of biosurfactant (Rouse et al., 1994).

Many study related with hydrocarbon biodegradation test often used various biosurfactant concentrations, which are: below the CMC, at CMC, and above the CMC. Each concentration give different response depends on the type of biosurfactant. In some cases reported that biosurfactant concentration above the CMC give inhibition effect, thus another studies used biosurfactant concentration below the CMC to reduce inhibition effect (Rouse et al., 1994). While, according to Kim et al. (2001), the most commonly used of surfactant concentration are at CMC. Hence, the aim of this study was to know the effectiveness of biosurfactant P. putida T1(8) using various concentration (below the CMC, at CMC, above the CMC) when it was added in bioremediation test.

2 Methodology

2.1 Microbial cultural The bacteria producing biosurfactant which was used in this study was Pseudomonas putida T1(8). While, microbial consortium which was used as hydrocarbonoklastics microbes, consist of: Aeromonas hydrophyla, Pseudomonas putida T1(8), Pseudomonas aeruginosa, Acinetobacter faecalis type II, Pseudomonas cepacea, Actinobacillus sp., Pseudomonas stutzeri, Pseudomonas pseudomallei, Pseudomonas fluorescens-25, Azotobacter sp., Beijerinkia sp., Candida famata, Rhodotorulla mucilaginosa, and Candida parapsilopsis. Those microbes were storage in Microbiology Laboratory, Biology Departemen Airlangga University Surabaya. The bacterial isolate was maintained on nutrient agar (NA), while yeast isolate on Sabouroud Dextrose Agar (SDA).

2.2 Biosurfactant production For biosurfactant production, a modification of a mineral salts medium developed by Pruthi and Comeotra (1997) was used. It contained per liter distilled water, KH2PO4, 5.0g; K2HPO4, 2.0g; FeSO4.7H2O, 0.0006g; (NH4)2SO4, 3.0g; NaCl, 10g; MgSO4. 7H2O, 0.2g; CaCl2, 0.01g;MnSO4. H2O, 0.001g; H3BO3, 0.001g, ZnSO4. 7H2O, 0.001g; CuSO4. 5H2O, 0.001g; CoCl2.6H2O, 0.005g; and Na2M0O4. 2H2O, 0.001 g, pH was adjusted to 7.0. The flasks containing molasses 2% v/v as sole carbon source were inoculated with Pseudomonas putida T1(8) at 4 % (v/v) OD=0.5 at 610. Cultivations were performed in 10 flasks (1000 mL), each flask contains 200 mL mineral salts medium. The mixture was placed on a reciprocal shaker at 150rpm for 3 days at 30C. Afterwards, the culture was centrifuged and the supernatant was extracted by (NH4)2SO4 precipitation. The crude biosurfactant product will be added into


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bioremediation experiment with concentration below the CMC (332.5 ppm), at CMC (665 ppm), and above the CMC (997.5 ppm). 2.3 Preparation of soil A soil sample in this study was obtained from garden soil and sandy soil mixture. Before mixed those, to both garden soil and sandy soil was filtered by mess that has a measurement of 1 mm2.Then, each of those was weighted about 750 g. After that, the soils were mixed and homogenized. An initial determination of C:N concentrations was performed to estimate the experimental supplement of nutrients to ensure a C:N ratio of 100:10. Initial pH was adjusted at 7. For moisture content, additional water was added to bring the final moisture content of soil mixture to 77%.

2.4 Bioremediation experiments Bioremediation experiments were carried out in flasks (100 mL). This study was assessed using the full factorial experimental design, consists of 3 controls and 4 treatments, with the details like the following sentences:

- Control I (KI) contains of non sterile soil + crude oil

- Control II (KII) contains of sterile soil + crude oil

- Control III (KIII) contains of non sterile soil + crude oil + biosurfactant at CMC concentration

- Treatment I (B0) contains of non sterile soil + crude oil + microbial consortium

- Treatment II (B1) contains of non sterile soil + crude oil + microbial consortium + biosurfactant with concentration above the CMC

- Treatment III (B2) contains of non sterile soil + crude oil + microbial consortium + biosurfactant with concentration at CMC

- Treatment IV (B3) contains of non sterile soil + crude oil + microbial consortium + biosurfactant with concentration below the CMC

Each of control and treatment consist of 3 replicates for Total Plate Count (TPC) analysis and 3 replicates for Total Petroleum Hydrocarbon (TPH) analysis. To ensure suitable moisture, each microcosm was monitored periodically at the end of weeks (0 week, 1st week, 2nd week, 3rd week, and 4th week). Each of the flasks was filled by 10 g soil, whereas for the control with a sterile soil, the soil was dried by oven at 160C for 2 hours to sterilize it completely. The flasks were then added by crude oil 0.6 mL. Afterwards, 2 mL of consortium suspension OD=1 at 600 also was added into the flasks with consortium adding. Then for the groups with biosurfactant adding, biosurfactant was added into the flasks. The soil was then continuously hand mixed for 5 minutes with a stainless steel spatula.

The microcosms were kept at room temperature, approximately 30C for 0, 2, and 4 weeks. 2.5 Biodegradation measurement 2.5.1 Heterotrophic microbial calculation Total heterotrophic microbial were numbered by using the pour plate technique on plate count agar (NA for total bacteria and SDA for total yeast). Plate count of the soil microbial population was performed as follows: samples of 10 g of soil were added with 90 mL sterile saline solution and agitated mechanically, and then were precipitated for 5 minutes. After appropriate serial dilutions, 1 mL of the suspension was added into the petri dishes, and then NA and SDA were also added into the Petri dishes. The cultures were then incubated for 24 h (for bacteria) and 72 h (for yeast) at 35C. At the end of the incubation time, the counting of bacteria was performed for all treatments.

2.5.2 Total Petroleum Hydrocarbon (TPH) analysis The soil samples were dried with oven for 3 days at 75 0C. After that, the dried soil was grinded. About 1 gram of grinded soil was then added into the tube. Afterwards, about 5 mL of toluene was added into the tube. And then was agitated using vortex for 10 seconds and leaving to stand for 10 minutes. The soluble crude oil within toluene was then filtered using filter papers number 1. The suspension was diluted by taking 0.5 mL, and then was added with 4.5 mL toluene. After that, Optical Density (OD) value of dilution suspension was measured using spectrophotometer at 420. The result of measurement were then calculate into standard curve, so that will be obtained crude oil content (mL/g tanah), then was converted into g/g soil (Okolo et al., 2005).

3 Result

The type of heterotrophic microbial which was analyzed in this study, consist of: bacteria and yeast. The results of Total Plate Count of heterotrophic microbial (log cfu/g) at each treatment condition during incubation time 0, 2, and 4 weeks were showed in Figure 1. The figure showed the increasing of total heterotrophic microbial during incubation time in almost all of the treatment condition (K1, K2, K3, BO, B2, CMC).


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0

2

4

6

8

10

12

14

Tota

l het

erot

roph

ic

mic

robi

al (l

og c

fu/g

)

KI KII KIII B0 B1 B2 B3 CMC

Treatment condition

0 week2 weeks 4 weeks

Figure 1: Total heterotrophic microbial (log cfu/g) of each treatment condition during

incubation times of 0, 2, and 4 weeks.

During 2 weeks of incubation time, Pseudomonas aeruginosa was dominant at treatment condition of KIII, B1, B2, and B3, although KIII was a treatment group without consortium adding. This showed that Pseudomonas aeruginosa was an indigenous bacteria of the soil. From the yeast group also showed the growth of Candida famata and Candida parapsilopsis on treatment condition of B0 and B2. In the other side, on treatment condition of B1 and B3 only showed the growth of Candida parapsilopsis. In almost all treatment, Candida famata was less dominates (Data was not shown). At fourth week of incubation time, it was shown that Pseudomonas aeruginosa was dominant at treatment condition of K1, KIII, B0, B3, CMC. Meanwhile, there is a change of microbial dominance at treatment condition of B0, B1 and B2, from Pseudomonas stutzeri at treatment condition of B0 in the second week to Pseudomonas aeruginosa in the fourth week, and from Pseudomonas aeruginosa at treatment condition of B1 and B2 in the second week to Pseudomonas pseudomallei and Pseudomonas putida T1(8) in the fourth week. At the fourth week, it was also showed the existence of bacteria that never exist before, like; Azotobacter sp. and Beijerenkia sp. While, Pseudomonas aeruginosa was begin exist at treatment condition of KI in the fourth week. On the other hand, the highly growth of Pseudomonas putida T1(8) was shown at treatment condition of B2 in the fourth week (Data was not shown).

The result of statistical analyses showed that there is an influence of biosurfactant concentration against total heterotrophic microbes, with F value about 6.051 and probability


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84.00%

86.00%

88.00%

90.00%

92.00%

94.00%

96.00%

98.00%

100.00%

0 week 2 weeks 4 weeks

Incubation time (week)

Perc

enta

ge o

f cru

de o

il (%

)

B0B1B2B3

Figure 2: Percentage of crude oil at each treatment condition during incubation times of

0, 2, and 4 weeks.

Although from descriptive analysis was known that there was a difference in crude oil decreasing within B1, B2, and B3. But, from the statistical analysis, there was no significant difference.

The statistical analysis of incubation time against crude oil residue obtained F value about 0.048 with the probability >0.05 (0.953), therefore H0 was accepted, or there is no influence of incubation time against crude oil residue. So, it did not need to be continued to the Duncan test. Although there was no difference between incubation time and crude oil residue, but descriptively, there was a decreasing of crude oil concentration until second week, after that there was no decreasing of crude oil percentage until fourth week.

The statistical analyses also showed the influence of interaction of biosurfactant concentration and incubation time against crude oil residue, with F value about 0.329 and probability >0.05 (0.065), therefore H0 was accepted, or there was no influence of interaction of biosurfactant concentration and incubation time against crude oil residue. Whatever the result of statistical analysis, if that was analyzed descriptively, the best combination was on combination of B2M2 and B2M4, those was a treatment condition with biosurfactant at CMC concentration and incubation time two and four weeks.

The monitoring of moisture during incubation time was indicated that the moisture decreased from 77% to 59.41% at fourth week. It was mean that moisture content still too high until fourth week. While, from the monitoring of pH during incubation time, showed that pH was still in optimum range to support bioremediation process (pH 6-7). The results from to both descriptive analysis and statistical analysis showed that various concentrations of biosurfactant gave an influence to the growth of heterotrophic microbes. The most effective concentration was on CMC concentration (B2) with fourth week incubation time. According

to Rouse et al. (1994), biosurfactant at CMC concentration have an ability to form micelle. Hence, biosurfactant can emulsify the oil (disperse a complex hydrocarbon molecule into simple forms). Finally hydrocarbon component can soluble and microbes can access to the substrate easily. Therefore it can increase microbial growth. The increasing of microbial growth in the treatment condition with biosurfactant adding at CMC concentration indicated the compatibility and low toxicity of biosurfactant to the microbial consortium (Figure 1). As crude oil can be emulsified and mineralized by microbial activity, crude oil content can be decreased.

The results of descriptive and statistical analysis showed the influence of biosurfactant adding in various concentrations against total heterotrophic microbial and crude oil residue. Biosurfactant at CMC value was known to be the most effective to increase microbial growth and to decrease the oil residue.

The microbes were increasing significantly at incubation time between 0 week and two weeks, but the oil concentration was decreasing. Between two weeks up to four weeks of incubation time, the growth of microbes were decreasing, except to treatment condition of B2 (biosurfactant concentration at CMC value) (Figure 1). The growth in B2 may because of the availability of C substrate that was obtained by microbial activity during 0 week up to second week. Beside that, the microbes in B2 may be able to use biosurfactant as its growth substrate. Overall, the decreasing of microbes made static decreasing of oil concentration up to fourth week (Figure 2). Beside by microbial activity to degrade the substrate and the availability of the substrate for microbes, the rate and the completeness of bioremediation was also influenced by environmental factors likes; moisture, soil pH, oxygen content, nutrient content, temperature, even by soil type (Vidali, 2001 and Kosaric, 1996). That is way; the analysis of environmental factors during bioremediation process was extremely needed.

According to Vidali (2001), the range of optimal moisture of bioremediation is in 30-90%. This is possible in some cases, but in another cases, the high moisture of the soil decreased bioremediation rates. Baptista et al.(2005) and Cubitto et al. (2004) reported that the high moisture (above 50%) gave a negative effect to bioremediation rate, these correlated with the less of aeration in the soil. The lowest value of moisture in this study was 59.41 % at fourth week. That moisture was still too high. At the condition with too high moisture, the oil was inclined at the top of soil surface where the water content was also too high at the top of the soil. So, it takes a more time for both oil and water to absorb


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into the soil. Thus, the effectiveness of biosurfactant to increase the bioremediation process also needs a more time. In addition, according to Environmental Technologies Research Group and Spanish Association of Fishering Cities (2002), there is a complication of bioremediation in a wetland or the soil with the high moisture; Oil can penetrate into the anoxic layers of the sediment. Below only a few centimeters of depth, the environment becomes anaerobic, and crude oil biodegradation is likely to be much slower even in the presence of an adequate supply of nitrogen and phosphorus. In this specific case, the addition of oxygen may be required as part of the bioremediation strategy.

4 Conclusions

In conclusion, Pseudomonas putida T1(8) biosurfactant gave an effect on bioremediation process. Biosurfactant at CMC value was known to be the most effective to increase microbial growth and to decrease the oil residue about 12.68%, if compared with the control at final incubation time (fourth week).

References

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[18] Okolo, J. C., E. N. Amadi, and C. T. I. Odu, Effects of Soil Treatments Containing Poultry Manure on Crude Oil Degradation in a Sandy Loam Soil. Applied Ecology and Environmental Research. 3 (1): 47-53, 2005.

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B018_Nur Hidayatul Alami