Comparative Heavy Metal Removal Eciencies of Biosurfactants Produced by Odoribacter Splanchnicus DSM 20712, Bacterium Clone JX981747 and Soil Washing Agents

The application of two biosurfactants in the removal of copper, zinc, and lead from waste metal dumpsite soil with their eciencies was compared to soil washing agents as KNO 3 , Ca(NO 3 ) 2 and NaOH. The test soil samples were also spiked separately with different concentrations (50 mg/L, 250 mg/L, 750 mg/L and 1500 mg/L ) of CuSO 4, ZnSO 4 and Pb(NO 3 ) 2 . The biosurfactants used were produced by Odoribacter splanchnicus DSM 20712 (WBS1) and an unidentied bacterium clone JX981747 (CMS). Five different treatment set up comprising of different ratios (20:1, 15:1, 10:1, 5:1, and 1:1) with the soil solution constant was respectively used. The heavy metal contents were measured using Atomic Absorption Spectrophotometer and the percentage heavy metal removal eciency was calculated. The highest concentrations of biosurfactant (20:1) at different spiked concentrations of metallic salts recorded the highest values of copper (95.47%, 95.73%, 91.69%, 78.82%); zinc (97.98%, 98.98%, 97.29%, 96.78%) and lead (97.68%, 93.09%, 88.12%, 84.98%) removal. The percentage metal removed in each treatment increased with increasing concentration of the biosurfactants and washing agents (1:1 to 20:1). The chemical structure of the two biosurfactants analyzed using Gas Chromatography Mass Spectroscopy (GC-MS) depict the major component of biosurfactants produced from Odoribacter splanchnicus DSM 20712 to be Di-n- amyl phthalate while 9, Octadecanoic acid, methyl ester was from unidentied bacterium clone JX981747. The one dimensional paper chromatography showed presence of galactose/glucose, mannose, ribose, rhamnose in the biosurfactants produced from Odoribacter splanchnicus DSM 20712 whereas the unidentied bacterium clone JX981747 produced biosurfactants that contained all sugars except mannose. The test biosurfactants studied showed high levels of copper and lead removal than zinc when compared with the test soil washing agents (KNO 3 ), Ca(NO3) 2 and NaOH used in this study. Biosurfactants have thus shown to have the ability to remove metals hence its use requires scaling up for environmental applications.


Introduction
Soils may become contaminated by the accumulation of heavy metals and metalloids through emissions from the rapidly expanding industrial areas, mine tailings, disposal of high metal wastes, leaded gasoline and paints, land application of fertilizers, animal manures, sewage sludge, pesticides, wastewater irrigation, coal combustion residues, spillage of petrochemicals, hospital e uents, hospital wastes and atmospheric deposition (Roane et al., 1996;Khan et al., 2008;Zhang et al., 2010). Heavy metals constitute an ill-de ned group of inorganic chemical hazards, and those most commonly found at contaminated sites are lead (Pb), chromium (Cr), arsenic (As), zinc (Zn), cadmium (Cd), copper (Cu), mercury (Hg), and nickel (Ni) (Ogbulie, 2011). Soils are the major sink for heavy metals released into the environment by aforementioned anthropogenic activities (Ogbulie et al., 2010) and unlike organic contaminants which are oxidized to carbon (IV) oxide by microbial action, most metals do not undergo microbial or chemical degradation (Kirpichtchikova et al., 2006), and their total concentration in soils persists for a long time after their introduction (Adriano et al., 2003). There may be changes in their chemical forms (speciation) and bioavailability of which soil characterization would provide an insight into these heavy metal speciation and bioavailability (Zhao and Kaluarachchi, 2002).
The effectiveness of washing is closely related to the ability of the extracting solution to dissolve the metal contaminants in soils. However, the strong bonds between the soil and metals make the cleaning process di cult (Gombert, 1994). Owing to the different nature of heavy metals, extracting solutions that can optimally remove them must be carefully sought during soil washing. Several classes of chemicals used for soil washing include surfactants, cosolvents, cyclodextrins, chelating agents, and organic acids (USEPA, 1990;Wood et al., 1990;Chu and Chan, 2003;Gao et al., 2003;Maturi and Reddy, 2008;Zhang et al., 2009). All these soil washing extractants have been developed on a case-by-case basis depending on the contaminant type at a particular site. A few studies have indicated that the solubilization/exchange/extraction of heavy metals by washing solutions differs considerably for different soil types. Surfactants are used on a large scale basis worldwide in everyday household use to industrial cleaning and textile manufacturing. Surfactants are best known for their solubility and cleaning properties which secured them a place among detergents and other cleaning products (Ivanković and Hrenović, 2010). Synthetic detergents used to clean up spillages have often led to more destruction of the environment from an environmental view point (Ogbulie et al., 2008). It is important that the remediation process be as non destructive and environmentally benign as possible if the end product is intended to be a healthy productive ecosystem. Metal-contaminated sites vary according to location, the source of metal contamination, and the history and age of the metal contamination. We had studied the removal of metals from metal dumpsite soil using biosurfactants and other soil washing agents. The metal dumpsite soil is a sink for metals (copper, zinc, lead) and it has a long history of contamination with these metals (Okore, 2017a;b).
Chelators are chemical compounds that form complexes with metal ions or other substances and also called chelating agents or sequestering agents. The chelating agent has a ring like center, which forms a complex with the metal ion/substance by two or more bindings and the metal ion is bound and extracted.
There are a great number of metal chelators few of these materials are environmentally benign; the acids, alkalis, complexants, other solvents and surfactants examples KNO 3, Ca(NO 3 ) 2 and NaOH (Dermont et al., 2008). The synthetic chemicals such as nitrilotriacetic (NTA), ethylenediamine-tetraacetic acid (EDTA), and diethyl triamine pentaacetic acid (DTPA) are extremely effective at metal complexation. Their xenobiotic nature makes persist in the environment long after they are applied for a remedial measure, pose a threat to the aquatic environment and some are comparatively more toxic to human health. Their use in the eld for in-situ removal is questionable because of their demonstrated toxicity effects. The strong acids attack and degrade the soil crystalline structure at extended contact times (Dehghan-Noudeh et al., 2005).
The aqueous solutions of biosurfactants can be used to release compounds characterized by low solubility from soil and other media in process called washing. Soil washing performance is highly sensitive to site conditions. The process is most e cient when applied to soils and sediments containing large proportions of sand and gravel and is relatively ineffective when applied to soils having a high silt and clay content. Further, soils with a relatively high cation exchange capacity (the capacity to exchange cations for those in the polluting substances) tend to bind pollutants more tightly, which can limit the ability of the soil washing process to effectively separate the pollutants from the soil (Oberbremer et al., 1990;Fiebig et al., 1997;Yuste et al., 2000;Pacwa-Plociniczak et al., 2011;Kumari et al., 2012). Therefore, only extractants capable of dissolving large quantities of metals would be suitable for cleaning purposes (Tejowulan and Hendershot, 1998). The identi cation and quanti cation of coexisting solid metal species in the soil before and after treatment are essential to design and assess the e ciency of soil-washing technology (Kirpichtchikova et al., 2006).
The usefulness of biosurfactants for bioremediation of heavy metal contaminated soil is mainly based on their ability to form complexes with metals (Ogbulie, 2011). Metal ions are bound to oppositely charged ions or replace the same charged ions (electrostatic interactions or ion exchange) or complex with agents forming chelates on the polar head groups of micelle surface. This makes the metals more soluble in water. Biosurfactants used for soil treatment are also required to have minimal sorptive interactions applied to the soil system; in other words, most of the biosurfactant should remain in the aqueous phase. Biosurfactant sorption in general is likely the reason that high biosurfactant concentrations are required for effective metal removal (Asci et al., 2007;. Logically, the adsorptive behavior of a biosurfactant will depend on their molecular characteristics, such as charge and hydrophobicity, as well as on the soil characteristics. It is important that all substances released into the environment be degradable. Their potential for causing environmental damage should be assessed and the possibility of future harm due to build-up in the environment should be taken into consideration. This study was designed to characterize the biosurfactants produced from Odoribacter splanchnicus DSM20712 and unidenti ed bacterium clone JX981747 with a view to ascertain their metal removal e ciencies and to compare the metal removal e ciencies with some soil washing agents as KNO 3, Ca(NO 3 ) 2 and NaOH.

Methodology
Isolation, screening and identi cation biosurfactant producing-bacteria The bacteria species were isolated from waste battery dumpsite soil and cassava mill soil collected at Nekede Imo State, Nigeria. The bacteria species were identi ed by 16S rRNA gene in Iquaba, Pittsburg, South Africa. Both bacteria were screened for biosurfactant production using emulsi cation index test, oil displacement test and β hemolysis test using methods described by Okore et al., (2017 a,b,c) Production and extraction of crude biosurfactant The colonies of Odoribacter splanchnicus DSM 20712 isolated from WBS and an unidenti ed bacterium clone JX981747 form CMS were inoculated in 500 ml nutrient broth medium in 1000 ml Erlenmeyer ask and incubated at 30 o C for 7 days on a mechanical shaker. After 1 week of incubation, it was ltered by centrifugation for 45 min at 4,000 X g to obtain cell free supernatant and sediments of cell debris. The extraction was performed by acid precipitation with 1 M H 2 SO 4 of equal volume to attain a pH of 2.0 (54.3 ml conc. H 2 SO 4 poured into 1 L distilled water = 1 M H 2 SO 4 ). This was put in the fridge (4 o C) overnight for complete precipitation of the biosurfactant. After 24 h it was centrifuged at 4,000 X g for 45 min to sediment the biosurfactant. The supernatant was ltered off and the sediment stirred continuously for 20 min after which GC-MS chemical analysis and one dimensional paper chromatography was carried out.

Chemical characterization of produced biosurfactant i. Gas chromatography-Mass Spectrometer
The partially puri ed biosurfactant obtained as sediment was mixed with chloroform and the chemical component analyzed using Gas chromatography-Mass Spectrometer (GC-MS).
ii. Sugar detection One dimensional paper chromatography (AOAC, 2005) was employed. The crude biosurfactant was reconstituted in 1 ml of sterile distilled water and hydrolysed by boiling in 5 ml of bench sulphuric acid solution (2 M). The hydrolysate was spotted on Whatman chromatographic paper and ran in two separate solvents Butanol-Acetic acid-Water, 4:1:5 (BAW) and Butanol-Ethanol-Water, 4:1:2.2 (BEW). The separate chromatographs were allowed to run for 18-24 h and the paper was removed and dipped in aniline hydrogen phthalate solution in 1:1 ether: butanol solution. It was then dried for 5 min at 105˚C and viewed under UV light and the various spots were marked. The relative fronts (rfs) were calculated and matched against standard relative fronts for sugars (RFS). The matching sugars were deemed detected.

iii. Protein detection
The tests for protein by Ninhydrin and Biuret were based on the standard method of Association of O cial Analytical Chemists (AOAC 2005). In Ninhydrin test, 2 ml of sample mixed with 2 ml of Ninhydrin solution was heated, the development of liliac or faint blue colour is positive for presence of protein. In Biuret test, 2 ml of sample was mixed with 2 ml Biuret reagent, the development of purple or violet colour shows presence of protein.

Heavy metal removal from spiked contaminated soil
The experimental set up was done using the soil samples collected from waste metal dumpsite soil spiked with solutions of different concentrations of the metallic salts ( Pb(NO 3 ) 2 , Cu(SO) 4 , and Zn(SO) 4 using the modi ed methods of Mulligan et al., (1999) and Maier et al., (2001). The metallic solutions was prepared by dissolving 50 mg, 250 mg, 750 mg and 1500 mg each of Cu(SO 4 ) 2 , Pb(NO 3 ) 2 and Zn(SO 4 ) 2 separately in 1 L distilled water. The Atomic Absorption Spectrophotometer (AAS) reading of the various metallic concentrations was analyzed. Then 1 g of the soil sample from waste metal dump site was added to equal volumes of each concentrations of the metallic solution (5 ml). Thereafter it was centrifuged and supernatant subjected to AAS for the metal content determination. The results served as the initial metal content without biosurfactant treatment. The biosurfactant producing isolates Odoribacter splanchnicus DSM 20712 and the unidenti ed bacterium clone JX981747 were inoculated into nutrient broth and left for 7 days on the mechanical shaker. Thereafter the broth was centrifuged to separate the bacterial cells from the biosurfactant containing supernatant. The heavy metal removal of the spiked soils with biosurfactant was carried out using soil metallic solution to biosurfactant ratios (1:1, 1:5; 1:10; 1:15 and 1:20). The soil metallic solution with biosurfactant was shaken and left for 3 days. Then the soil was removed by centrifugation (4000 X g, 25 min) and dried. The supernatant was removed and placed into another centrifuge tubes acidi ed with 5 drops of HNO 3 to precipitate the biosurfactant then refrigerated for 24 h. After refrigeration the precipitated biosurfactant was removed from solution by centrifugation at (4000 X g, 25 min). Then the supernatant analyzed for the metal content using AAS. The amount of metals extracted from the soil by each biosurfactant was then calculated as a percentage of the original metal concentration without biosurfactant using the formula below.
Soil washing of heavy metal contaminated soils (un-spiked) This was carried out using the method of Mulligan et al., (1999) and Ru no et al., (2011) with modi cations. The test soil samples (10 g) dried at room temperature was digested over low ame for 15 min. This was done by initial heating for 3 min with10 ml of H 2 O 2 , then another 5 ml of H 2 O 2 for another 3 min, followed by 5 ml of H 2 O 2 and heating for 3 min. Thereafter 70 ml of concentrated H 2 SO 4 was added and heated for 6 min before the sample will be removed from the heat for cooling. Distilled water was added after cooling to make up the volume to 400 ml. The soil sample supernatant was collected after votexing to attain an equilibrium and centrifuged (5000 X g 10 min). The supernatant was then ltered through a 0.2 µm cellulose acetate lter prior to AAS analysis for metal concentration and this served as the amount of metal originally in the soil sample supernatant. The biosurfactant producing isolate was inoculated into 1000 ml nutrient broth and left for 7 days on mechanical shaker. Thereafter the broth was centrifuged at 4,000 X g 45 min to sediment the bacterial cell and the supernatant used for the soil washings studies. The soil washing with biosurfactant study was carried out using soil sample supernatant to biosurfactant ratios ranging from 1:1 to 20:1 in acid washed plastic centrifuge tubes of 50 ml without varying the pH. The comparison experiment was set up using aqueous solution of similar ionic strength like KNO 3, Ca(NO 3 ) 2 and NaOH. The experimental set up was left for 1 week before AAS analysis to ascertain the amount of heavy metal left in the supernatant after centrifugation. The percentage of metal removal was calculated using similar equation as above:

Screening and Identi cation
The E 24 of biosurfactants from Odoribacter splanchnicus DSM 20712 recorded is 78.13% on kerosene while the E 24 of the biosurfactants from an unidenti ed bacterium clone JX981747is 68.52% on power vegetable oil and 57.41% on crude oil. Both isolates exhibited positive β hemolysis and displaced hydrocarbons used in the oil displacement test (Table 1).

Production and extraction of crude biosurfactant
The quantity of crude biosurfactant produced from the 500 ml broth culture of biosurfactants from Odoribacter splanchnicus DSM 20712 and unidenti ed bacterium clone JX981747 was respectively 1.53 g and 1.33 g after acid precipitation (Table 2). Chemical characterization of produced biosurfactants 1) Gas chromatography analysis A chromatogram depicting the real time of peaks generated as the separate components pass through the detector was recorded. The GC-MS fatty acids analysis of Odoribacter splanchnicus DSM 20712 showed peaks each corresponding to long chain poly aliphatic and unsaturated compounds consistent with fatty acid methyl esters linked with benzene ring (Table 3). On the other hand, the GC-MS fatty acids analysis of the unidenti ed bacterium clone JX981747showed peaks corresponding to long chain poly aliphatic and unsaturated compounds consistent with fatty acid methyl esters linked mainly with Decanoic acid (Table 4).
The chromatograms of the biosurfactants produced by both test isolates are as shown in Figs. 1 and 2 respectively depicting a major peak of the likely compound at varying retention times (Odoribacter splanchnicus DSM 20712: Retention time of 12.470; an unidenti ed bacterium clone JX981747: Retention time of 13.705) whereas the other low peaks are the contaminants.
The Gas Chromatography library search result of biosurfactants produced by the test isolates showed the retention times of compounds detected, the components that exhibited peaks, their individual mass spectrum and chemical structures as shown on Tables 3, and Table 4 for each of the respective biosurfactants.

Sugar test
The presence of these sugars (Tables 5 and 6) Glucose/Galactose, Mannose, Ribose, Rhamnose, were found in biosurfactant from Odoribacter splanchnicus DSM 20712, Glucose/Galactose, Ribose and Rhamnose found in biosurfactant from the unidenti ed bacterium clone JX981747. This result was con rmed from the two mobile solvents used in the one dimensional paper chromatography method.

Protein test
Tests for protein by Ninhydrin and Biuret both show faint colours hence indicating traces of protein. The same result was obtained for both samples.

Metal analysis result on spiked soil
The percentage metal removed increased with higher concentrations of the biosurfactants used. Copper recovery rates were as high as 95.47%, 95.73%, 91.69% and 78.82% in solutions respectively spiked with 50 mg/L, 250 mg/L, 750 mg/L and 1500 mg/L of copper and treated with 20:1 concentration of biosurfactant produced by Odoribacter splanchnicus DSM 20712 (Fig. 3a).  (Fig. 4c ). Comparatively, there was obvious steady increase in copper removal rate in all the concentration ratios in relation to using biosurfactants from O. splanchnicus DSM 20712 than with BC JX981747 with the peak recorded in the highest treatment ratio (20:1) whereas Zinc and Lead removal was high in the later [Figures 3 (a-c) and 4(a-c)].    The percentage metal removal e ciency of the biosurfactants and other soil washing agents used are shown on Figs. 5a-c. The trend observed showed that the percentage metal removal e ciency recorded increased with higher concentrations of the biosurfactants and the test soil washing agents used.
The zinc removal percentage observed in this study is as shown in gure 5c. Comparatively, of the 20:1, 15:1, 10:1 and 5:1 concentrations NaOH removed highest concentration of zinc followed by KNO 3 , Ca(NO 3 ) 2 , CMS1 and WBS1 in that order except at 1:1 ratio where Ca(NO 3 ) 2 had the highest value.
Comparatively, the test biosurfactants used in this study removed copper and lead more than the soil washing agents whereas the later removed zinc more than the former.

Discussions
Crude Biosurfactant production and characteristics Biosurfactants are indeed good bio-products with great diversity and broad spectrum of functions and environmental applications. There various sources could be from contaminated water, soil and most importantly from microorganisms (Reis et al., 2013). In this study, crude biosurfactants were obtained from two major isolates amongst others namely Odoribacter splanchnicus DSM 20712 and an unidenti ed bacterium clone JX981747. The possible production and recovery of crude biological surfactants from microorganisms as observed in this study corroborates with the ndings of Anandaraj and Thivakaran, (2010) who obtained 0.122 g of biosurfactant from Pseudomonas sp. grown in 50 ml of R2B broth with 1 ml of petrol as carbon source. It also supports the ndings of Maneerat et al., (2006) who also proved the possibility of extracting crude biosurfactant from isolates during his experiment with marine bacterium Myroides sp and extracted 2.64 g of a crude oil emulsi er from 10 L of cultured strain SMI. This current study is in agreement that different yields of crude biosurfactant can be precipitated and extracted from the culture broth.
The biosurfactants produced from Odoribacter splanchnicus DSM 20712 and the unidenti ed bacterium clone JX981747 in this study showed the presence of rhamnose sugar, and fatty acid component. The major component of O. splanchnicus, di-n-amyl phthalate as regards its structure will remove more metals by chelating and less of hydrocarbon because of the short aliphatic chain whereas the major component of bacterium clone JX981747, which is 9-Octadecenoic acid-a methyl ester, will remove less metals and function better as hydrocarbon removal because of the chain length.
Heavy metal removal from spiked soil using crude biosurfactant Heavy metal removal from terrestrial environment is one of the key areas in environmental management and clean up operations where the signi cance of biosurfactants has not been explored in depth (Luna et al., 2016). The result of this study evidently depict that the test heavy metals (copper, lead and zinc) removal rate was effective and high with increase in concentration of the biological surfactants tested.
This corroborates with the report made by Maier at al., (2001) who studied biosurfactant removal of metals from spiked sewage sludge which was anaerobically digested. They equally had high rate of copper recovery in solution spiked with 2000 mg/kg treated with 50 mM rhamnolipid. The ndings of this study equally established this fact, and support the ndings made by Mulligan et al., (2001;2004). In their study, they also observed high copper removal (70%) proving the feasibility metal removal with amnionic biosurfactants in batch washing experiments.
Furthermore, the high rate of metal removal as observed in this study have shown that surfactants could remove metals by ion exchange precipitation, dissolution and counterion association with the crude sugar surfactant, depicting superior performance similar to previous reports (Mulligan et al., 1999(Mulligan et al., , 2001, factor in uencing heavy metal removal e ciency in this study and elsewhere (Mulligan et al., 2001 andQi et al., 2018).
E ciencies of biosurfactant and soil washing agents in heavy metal removal Mulligan et al, (1999) showed the feasibility of removing cadmium, lead, and zinc with 2% anionic biosurfactants surfactin from soil that is low in exchangeable metal fractions. The use of NaOH alone removed 20% copper, 10% zinc while surfactin biosurfactant removed 70% copper and 25% zinc.
Chakrabarti, (2015) noted lead removal using biosurfactant produced by a Bacillus sp. SJ301 which removed nearly 2-3% lead from a highly toxic 100 ppm lead solution. This current study equally established the feasibility of Cu, Pb, Zn removal with biosurfactant depicting increase in the concentration of biosurfactant and its additional effect on the percentage metal removal e ciency. This also in agreement with the ndings of Hong et al., (2002) and Mulligan et al., (2007) who observed that the removal percentage of Cu, Zn and Ni in the soil increased linearly with increasing concentration of rhamnolipid used. The recorded values are indications that biosurfactant can be used to remove metals from contaminated samples. Maier et al., (2001) in their study also observed the effects of biosurfactants rhamnolipid and surfactin in metal removal in a historically contaminated soil by 10 mM puri ed rhamnolipid adjusted to pH 7.1 compared with other soil washing agents, KNO3 solution adjusted to the same ionic strength as the rhamnolipid solution and 50 mM Ca(NO3)2. The rhamnolipid removed a total of 14 to 15% of the lead from each historically contaminated soil in 10 extractions. Their study in consonance with the ndings of this study showed that rhamnolipid containing biological surfactant greatly enhanced metal removal than with chelators (KNO 3 or Ca(NO 3 ) 2 ). This study, irrespective of the source and history of contamination of soil established the feasibility of biosurfactant removal of copper, zinc and lead contaminants.

Conclusion
The two biosurfactants produced from Odoribacter splanchnicus DSM 20712 and unidenti ed bacterium clone JX981747 recorded the highest emulsi cation index and equally showed higher levels of percentage metal removal. Biosurfactant from O. splanchnicus e ciently removed copper more than that from BC JX981747 whereas that from unidenti ed bacterium clone JX981747 removed both zinc and lead e ciently more than does the former in the spiked soil. Nevertheless, both signi cantly removed the test metals with negligible difference achieved possibly by forming complex at the polar end of the micelles with the metals and as a result these metals are desorbed out of the contaminating matrix. Furthermore, the test biosurfactants studied showed high levels of copper and lead removal than zinc when compared with the test soil washing agents (KNO 3 ), Ca(NO3) 2 and NaOH used in this study.
Biosurfactants have therefore proved to be e cient in metal removal and since they are biodegradable, the culture medium for the optimum production of these biosurfactants should be formulated to enhance high production yield. In addition, its exploitation should be encouraged for large scale industrial and environmental applications.