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Title:
TREATMENT OF HYDROCARBON COMPONENT
Document Type and Number:
WIPO Patent Application WO/2016/170491
Kind Code:
A1
Abstract:
A biological process for treating a hydrocarbon component includes inoculating a hydrocarbon component with a bacterial component comprising at least one bacterial strain selected from the group consisting of the following bacterial strains deposited with the Microbial Culture Collection (MCC), Maharashtra, India on 21 October 2013 (KC620476), 4 August 2014 (KC620473), 20 October 2014 (KC620474, KC620478, KC620475) and 25 February 2015 (KC620477). The inoculated hydrocarbon component is incubated, thereby to allow the hydrocarbon component to biodegrade. The bacterial strain facilitates the biodegradation of the hydrocarbon component.

Inventors:
COWAN, Ashton, Keith (30C Saltvlei Road, 6170 Port Alfred, 6170, ZA)
EDEKI, Gerald, Oghenekume (2 Osawe Street, Ekpoma, NG)
IGBINIGIE, Eric, Egbe (24 Uwasota Road, Ugbowo, Benin City, NG)
ISAACS, Michelle, Louise (23 Ford Street, Kasoug, 6170 Port Alfred, 6170, ZA)
Application Number:
IB2016/052263
Publication Date:
October 27, 2016
Filing Date:
April 21, 2016
Export Citation:
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Assignee:
RHODES UNIVERSITY (Lucas Avenue, 6139 Grahamstown, 6139, ZA)
International Classes:
B09C1/10; C02F3/34
Foreign References:
US5427944A1995-06-27
Other References:
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STALLWOOD, B.; SHEARS, J.; WILLIAMS, P.A.; HUGHES, K.A.: "Low temperature bioremediation of oil-contaminated soil using biostimulation and bioaugmentation with a Pseudomonas sp. from maritime Antarctica", J. APPLMICROBIOL., vol. 99, 2005, pages 794 - 802
SU, D.; LI, P.; WANG, X.; STAGNITTI, F.; XIONG, X.: "Biodegradation of Benzo[a]pyrene in Soil by Immobilized Fungus", ENVIRONMENTAL ENGINEERING SCIENCE, vol. 25, 2008, pages 8, XP022858474, DOI: doi:10.1016/S1001-0742(06)60063-6
UENO, A.; HASANUZZAMAN, M.; YUMOYO, I.; OKUYAMA, H.: "Vertification of degradation of n-alkanes in diesel oil by Pseudomonas aeruginosa strain WatG in soil microcosms", CURR.MICROBIOL., vol. 52, 2006, pages 182 - 185, XP019365693
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WEBER JR, W.J.; CORSEUIL, H.X.: "Inoculation of contaminated subsurface soils with enriched indigenous microbes to enhance bioremediation rates", WATER RES., vol. 28, 1994, pages 1407 - 1414, XP000435990, DOI: doi:10.1016/0043-1354(94)90308-5
WOLICKA, D.; SUSZEK, A.; BORKOWSKI, A.; BIELECKA, A.: "Application of aerobic microorganisms in bioremediation in situ of soil contaminated by petroleum products", BIORESOURCE TECHNOLOGY, vol. 100, 2009, pages 3221 - 3227, XP026063273, DOI: doi:10.1016/j.biortech.2009.02.020
Attorney, Agent or Firm:
KOTZE, Gavin, Salomon (Adams & Adams, PO Box 101, 0001 Pretoria, 0001, ZA)
Download PDF:
Claims:
CLAIMS

1 . A biological process for treating a hydrocarbon component, which process includes

inoculating a hydrocarbon component with a bacterial component comprising at least one bacterial strain selected from the group consisting of the following bacterial strains deposited with the Microbial Culture Collection (MCC), Maharashtra, India on 21 October 2013 (KC620476), 4 August 2014 (KC620473), 20 October 2014 (KC620474, KC620478, KC620475) and 25 February 201 5 (KC620477) under the accession numbers as given in Table A:

TABLE A

and

incubating the inoculated hydrocarbon component, thereby to allow the hydrocarbon component to biodegrade, with the bacterial strain facilitating the biodegradation of the hydrocarbon component. 2. The process according to Claim 1 , wherein the bacterial component comprises a consortium of two or more of the bacterial strains listed in Table A.

3. The process according to Claim 2, wherein the bacterial strains are enriched with the hydrocarbon component.

4. The process according to Claim 2 or Claim 3, wherein the bacterial strains are sourced from diesel contaminated soil.

5. The process according to any one of Claims 2 to 4 inclusive, wherein the consortium comprises one of the following combinations of bacterial strains/specie:

(i) KC620473 + KC620475

(ϋ) KC620473 + KC620474

(iii) KC620473 + KC620476

(iv) KC620473 + KC620477

(v) KC620473 + KC620474 + KC620475

(vi) KC620473 + KC620476 + KC620474

(vii) KC620473 + KC620475 + KC620476

(viii) KC620473 + KC620477 + KC620475

(ix) KC620475 + KC620476 + KC620477

6. The process according to any one of Claims 1 to 5 inclusive, wherein the hydrocarbon component is, or includes, a liquid hydrocarbon. 7. The process according to Claim 6, wherein the liquid hydrocarbon is an aromatic hydrocarbon.

8. The process according to any one of Claims 1 to 7 inclusive, wherein the hydrocarbon component is a waste hydrocarbon component.

9. The process according to Claim 8, wherein the waste hydrocarbon component is a dangerous pollutant selected from benzene, toluene, ethylbenzene and xylene; an organic halogenated compound; MTBE (methyl tert-butyl ether); a mineral oil; or an organic compound.

10. A biological process for treating a hydrocarbon component, which process includes

obtaining at least one bacterial strain from a liquid or solid medium containing a hydrocarbon component; working up the bacterial strain into a bacterial component comprising said at least one bacterial strain;

inoculating the same, or another, hydrocarbon component with the bacterial component; and

incubating the inoculated hydrocarbon component, thereby to allow the hydrocarbon component to biodegrade with the bacterial strain facilitating the biodegradation of the hydrocarbon component.

1 1 . The process according to Claim 10, wherein the obtaining of the bacterial strain from the liquid or solid medium containing the hydrocarbon component includes isolating the bacterial strain from the liquid or solid medium.

12. The process according to Claim 1 1 , wherein the bacterial strain is isolated from the solid medium, with the solid medium being in the form of soil contaminated with the hydrocarbon component.

13. The process according to any one of Claims 10 to 1 2 inclusive, wherein the bacterial strain is selected from the group consisting of the following bacterial strains deposited with the Microbial Culture Collection (MCC), Maharashtra, India on 21 October 2013 (KC620476), 4 August 2014 (KC620473), 20 October 2014 (KC620474, KC620478, KC620475) and 25 February 201 5 (KC620477) under the accession numbers as listed in Table A: TABLE A

MCC Accession Number Genbank Accession Number

MCC 0034 KC620473

MCC 0021 KC620474

MCC 0027 KC620475

MCC 0016 KC620476

MCC 0042 KC620477

MCC 0022 KC620478

14. The process according to Claim 13, wherein the bacterial component comprises a consortium of two or more of the bacterial strains listed in Table A. 15. The process according to any one of Claims 10 to 14 inclusive, wherein the hydrocarbon component is a liquid aromatic hydrocarbon.

16. The process according to any one of Claims 10 to 1 5 inclusive, wherein the inoculation of the bacterial component is in a liquid or solid medium containing the hydrocarbon component.

17. The process according to Claim 16, wherein the bacterial component is inoculated into the liquid medium, with the process then including, prior to the inoculation of the hydrocarbon component with the bacterial component, dispersing the bacterial component in a minimal mineral salt aqueous medium comprising one or more of K2HPO4, KH2P04, NHCI4, MgCI2 and CaCI2, to obtain an aqueous bacterial suspension-containing mineral salt medium which is then used to inoculate the hydrocarbon component.

18. The process according to Claim 1 7, wherein the mineral salt aqueous medium is enriched with a trace mineral solution comprising one or more of Na3C6H507, MnS04, CoS04, CoCI2, ZnS04, CuS04, AIK(S04)2, H3B04, Na2Mo04, NiCI2, Na2Se03, V3+CI, and Na2W04.

19 The process according to Claim 16, wherein the bacterial component is inoculated into the solid medium, with the solid medium being enriched with fertilizer to stimulate the activity of the bacterial strain(s). 20. The process according to Claim 19, wherein the bacterial component is a liquid suspension, with the concentration of the bacterial strain(s) in the suspension being in the range 3.24 - 3.62 χ 109 cfu/ml. 21 The process according to Claim 19, wherein the bacterial component is in granulated/pellet form, with the granules/pellets also comprising an inert carrier, with the concentration of the bacterial strains in the carrier being about 1 .5 mg/kg.

22. The process according to any one of Claims 10 to 21 inclusive, wherein the incubation of the inoculated hydrocarbon component is effected for at least 1 day.

Description:
TREATMENT OF A HYDROCARBON COMPONENT

THIS INVENTION relates to the treatment of a hydrocarbon component. More particularly, it relates to a biological process for treating a hydrocarbon component.

The management of hazardous wastes in soil, water and air has attracted a lot of concern and has resulted in an increased interest in determining the effectiveness of various treatment technologies. Among the hazardous wastes, petroleum hydrocarbons have proven to be the most common, persistent, and recalcitrant environmental contaminants with a tendency to bioaccumulate (Su et al., 2008). Petroleum contamination can either be due to accidental spillage or leakage of the raw materials or products (Lin et al., 2010). Petroleum hydrocarbon pollution accidents have become a common phenomenon and have caused ecological and social catastrophes (Das and Mukherjee, 2007). This is based on the fact that they contain many kinds of organic compounds such as n-alkanes (aliphatics), aromatics, resins and asphaltenes (Liu et al, 201 0). Low molecular weight hydrocarbons such as monoaromatic benzene, toluene, ethylbenzene and xylenes (BTEX) have become the focus of most environmental studies because of their toxic and carcinogenic potentials (Wolicka et al., 2009; Nikolova and Nenov, 2005). Remediation of BTEX-contaminated soil and groundwater is desirable in order to avoid public health hazards (Dou et al., 2008). Various remediation technologies have been employed over the years in treating contaminated environments. Bioremediation which involves the use of bacteria in cleaning up contaminated sites, has been preferred to other technologies such as physico-chemical approaches (volatilization, adsorption, chemical oxidation, photo-decomposition, steam injection and electrical heating pump and treat method, vapor extraction) (Fellie et al., 201 2). The reason for its preference is based thereon that it is an economic, energy efficient and environmentally sound approach (Duo, 2008; Vasudevan and Rajaram, 2001 ; Ferrari et al., 1996 and Nicholas, 1987). Microorganisms such as bacteria, fungi and yeast have been used in carrying out bioremediation of BTEX since 1 908 (Mazzeo et al., 2010). Although most microorganisms have detoxifying abilities such as mineralization, transformation and/or immobilization, bacteria are particularly known to play a crucial role in it (Diaz, 2004). Several bacteria which possess the ability to degrade petroleum hydrocarbons have been isolated and characterized. Some of these bacteria include Moraxella sp. (Hogx and Jaenicee, 1972), Nocarida sp., Alcaligenes denitrificans, Micrococcs sp., Arthrobacter sp. (Weber and Corseuil, 1994), Thermus sp. (Chen and Taylor, 1 997), Rhodococcus rhodochrous (Deeb and Alvarez-Cohen, 1999) and Pseudomonas spp. (Brusa et al., 2001 ). These bacteria were the most commonly reported bacteria for degrading BTEX compounds (Brusa et al., 2001 ). Bioremediation experiments on hydrocarbons using single strains of bacteria have been reported (Niu et al., 2009; Ueno et al., 2006; Stallwood et al., 2005; Jernberg and Jansson, 2002). Experiments involving bacteria consortia have also been reported (Silva et al., 2009; Jacques et al., 2008; Das and Mukherjee, 2007). Reports by many scientists have shown that mixed bacteria populations with overall broad enzymatic capacities are required to degrade complex mixtures of hydrocarbons such as crude oil in soil (Bartha and Bossert, 1 984), fresh water (Cooney, 1 984.), and marine environments (Atlas, 1 985; Floodgate, 1984).

However, a drawback remains that known bioremediation processes are time consuming, typically taking from 75 to 90 days to achieve a desired degree of remediation or degradation.

An object of the present invention is thus to provide a means whereby a hydrocarbon component, e.g. when present as a contaminant in soil, can effectively be biodegraded more quickly. This is provided by the process of the invention. Thus, according to a first aspect of the invention, there is provided a biological process for treating a hydrocarbon component, which process includes

inoculating a hydrocarbon component with a bacterial component comprising at least one bacterial strain selected from the group consisting of the following bacterial strains deposited with the Microbial Culture Collection (MCC), Maharashtra, India on 21 October 2013 (KC620476), 4 August 2014 (KC620473), 20 October 2014 (KC620474, KC620478, KC620475) and 25 February 201 5 (KC620477) under the accession numbers as given in Table A:

TABLE A

and

incubating the inoculated hydrocarbon component, thereby to allow the hydrocarbon component to biodegrade, with the bacterial strain facilitating the biodegradation of the hydrocarbon component.

The bacterial component may comprise a consortium of two or more of the bacterial strains listed in Table A above. In other words, the bacterial component or inoculum may comprise a consortium of different bacterial strains selected from Table A. The bacterial strains may be enriched in the hydrocarbon component or in a hydrocarbon of the hydrocarbon component when the hydrocarbon component comprises more than one hydrocarbon, For example, the bacterial strains may be enriched with diesel.

More particularly, the bacterial strains may be as specified in Table B, and may be sourced from diesel contaminated soil: TABLE B

Thus, the consortium may comprise one of the following combinations of bacterial strains/specie:

(i) KC620473 + KC620475

(ϋ) KC620473 + KC620474

(iii) KC620473 + KC620476

(iv) KC620473 + KC620477

(v) KC620473 + KC620474 + KC620475

(vi) KC620473 + KC620476 + KC620474

(vii) KC620473 + KC620475 + KC620476

(viii) KC620473 + KC620477 + KC620475

(ix) KC620475 + KC620476 + KC620477

The hydrocarbon component may be, or may include, a liquid hydrocarbon. More particularly, the liquid hydrocarbon may be an aromatic hydrocarbon. The aromatic hydrocarbon may be benzene, toluene, xylene, naphthalene, or the like, or two or more thereof. The hydrocarbon may be petroleum based or derived. It will be appreciated that the hydrocarbon component may comprise a mixture of the liquid hydrocarbons, rather than only a single hydrocarbon. The hydrocarbon component may be a waste hydrocarbon component. By degrading waste hydrocarbons using the process of the invention, they become more readily disposable. In particular, the waste hydrocarbon component may be a dangerous pollutant such as a BTEX (acronym of benzene, toluene, ethylbenzene and xylene), an organic halogenated compound, MTBE (methyl tert-butyl ether), a mineral oil, an organic compound, or a hydrocarbon in general.

More particularly, the process can thus involve obtaining the bacterial strain(s) from a liquid or solid medium containing a hydrocarbon component, and then using the bacterial strain(s) to effect the inoculation of the same, or another, hydrocarbon component and the incubation thereof, as hereinbefore described. Typically, the bacterial strain(s) are obtained from a solid medium in the form of soil, i.e. soil contaminated with a hydrocarbon component such as diesel.

The process thus then involves using a novel bacterial strain obtained from a hydrocarbon contaminated source or, preferably, a consortium of two or more such novel bacterial strains to treat that, or another, hydrocarbon contaminated site, thereby to biodegrade the hydrocarbon component with which that site is contaminated.

Thus, according to a second aspect of the invention, there is provided a biological process for treating a hydrocarbon component, which process includes

obtaining at least one bacterial strain from a liquid or solid medium containing a hydrocarbon component;

working up the bacterial strain into a bacterial component comprising said at least one bacterial strain;

inoculating the same, or another, hydrocarbon component with the bacterial component; and

incubating the inoculated hydrocarbon component, thereby to allow the hydrocarbon component to biodegrade with the bacterial strain facilitating the biodegradation of the hydrocarbon component. The obtaining of the bacterial strain from the liquid or solid medium containing the hydrocarbon component may include isolating the bacterial strain from the liquid or solid medium. In particular, the bacterial strain may be isolated from a solid medium in the form of soil contaminated with the hydrocarbon component.

The working up of the bacterial strain to produce the bacterial component may comprise combining or formulating at least two bacterial strains obtained as hereinbefore described, into a consortium, with the bacterial component thus comprising a consortium of two or more of the bacterial strains.

The bacterial strains may be as listed or specified hereinbefore in Tables A and B. The bacterial component may comprise a consortium of two or more of the bacterial strains listed in Table A. In particular, the consortium may comprise one of combinations of bacterial strains/species as hereinbefore set out.

The hydrocarbon component may be as hereinbefore described. The inoculation of the bacterial component may be in a liquid or solid medium containing the hydrocarbon component.

In one embodiment of the invention, the bacterial component may thus be inoculated into a liquid medium.

The process may then include, prior to the inoculation of the hydrocarbon component with the bacterial component, dispersing the bacterial component in a minimal mineral salt aqueous medium comprising one or more of K 2 HP0 4 , KH 2 P0 4 , NHCI 4 , MgCI 2 and CaCI 2 , to obtain an aqueous bacterial suspension-containing mineral salt medium which is then used to inoculate the hydrocarbon component.

The mineral salt aqueous medium may be enriched with a trace mineral solution comprising one or more of Na3C 6 H 5 07, MnS0 4 , CoS0 4 , CoCI 2 , ZnS0 4 , CuS0 4 , AIK(S0 4 ) 2 , H 3 B0 4 , Na 2 Mo0 4 , NiCI 2 , Na 2 Se0 3 , V 3+ CI, and Na 2 W0 . The trace mineral solution may then comprise Na 3 C 6 H 5 07-2H 2 0 2.1 g, MnS0 4 -2H 2 0 0.5g, CoS0 4 or CoCI 2 -6H 2 0 0.1 g, ZnS0 4 -7H 2 0 0.1 g, CuS0 4 -5H 2 0 0.01 g, AIK(S0 4 ) 2 0.01 g, H 3 B0 4 0.01 g, Na 2 Mo0 4 -2H 2 0 0.1 g, NiCI 2 -6H 2 0 0.025g, Na 2 Se0 3 0.2g, V(III)CI 0.01 g, Na 2 W0 4 -2H 2 0 0.0033g)/100ml.

The process may include adjusting the pH of the enriched mineral salt aqueous medium to about 7 using a suitable acid or base.

In another embodiment of the invention, the bacterial component may be inoculated into a solid medium. The solid medium may then be enriched with fertilizer to stimulate the activity of the bacterial strain(s). The solid medium may be soil. In other words, the process is then applied to soil contaminated with the hydrocarbon component.

The bacterial component may then be applied as a liquid suspension. The concentration of the bacterial strain(s) in the suspension may in the range 3.24 - 3.62 x 10 9 cfu/rml but typically 3.48 x 10 9 cfu/ml. The suspension may be applied to the solid medium at a rate of between 300-400 L/Ha surface area of the solid medium.

The bacterial component may instead be applied in granulated/pellet form using an inert carrier which may include clay particles, compost, various zeolites and similar. The concentration of the bacterial strains in the carrier may be about 1 .5 mg/kg.

The process may include adding a neutralizing agent e.g. lime to the hydrocarbon-containing solid medium, for pH control.

The incubation of the inoculated hydrocarbon component may be effected for at least 1 day, preferably for at least 2 days, more preferably for 3 to 6 days, typically for 4 to 5 days. The invention will now be described in more detail with reference to the following non-limiting Example and the accompanying drawings.

In the drawings,

FIGURE 1 shows, for the Example, the experimental set up designed to check volatilization of hydrocarbons in liquid medium (A), and growth of bacteria in liquid medium on day 3 utilizing BTEX as a carbon source;

FIGURE 2 shows, for the Example, percentage degradation of BTEX in liquid medium using single bacterium isolate (B and C); the isolates of Figure 2 were the best performing organisms in their group; the biodegradation effectiveness of P.aeroginosa and B.subtilis in BTEX was compared to the various strains isolated from diesel contaminated soil; and

FIGURE 3 shows, for the Example, rate of degradation of BTEX using selected consortia in comparison with best performing single isolate and a positive control comprised of all 3 isolates obtained from the Department of Biochemistry, Microbiology and Biotechnology of Rhodes University in Grahamstown, SA.

EXAMPLE

Methodology

Selection of microorganisms through enrichment Bacterial consortia were selected from strains of bacteria which were isolated from diesel contaminated soil sourced from Grahamstown in the Eastern Cape Province of South Africa. 1 g of diesel contaminated soil was inoculated in nutrient broth and incubation was done at 30 °C for 48hrs on a rotary shaker. Serial dilution of broth culture in 10 folds was carried out after 48hrs. Each dilution factor was plated on agar plates and incubated for 24hrs at 30 °C. Isolation of pure colonies was carried out after visible growth of the organisms was observed. Six bacterial strains were isolated and pure cultures of each, enriched with diesel, were stored in 80% glycerol medium at -20 S C. Bacteria {P.aeroginosa, P.putida and B.subtilis) which have been widely reported by researchers to degrade petroleum hydrocarbons were also used in this experiment and they were sourced from Department of Biochemistry, Microbiology and Biotechnology of Rhodes University in Grahamstown, SA. Preparation of BTEX substrate

BTEX mixture was prepared by carefully but thoroughly mixing equal volumes of benzene, toluene, ethylbenzene and xylene (BTEX) in the ratio of 1 :1 :1 :1 to form a composite substrate. The substrate comprising benzene (purity of 99% - CAS N Q 71 -43-2), toluene (purity of 99% - CAS N Q - 1 08-88-3), ethylbenzene (purity of 99.80% - CAS N Q 100-41 -4) and xylene (purity of 99%, mixture of isomers CAS N Q - 1330-20-7) were used in carrying out biodegradation assay.

Cultivation and incubation procedures

Mineral salt medium (MSM, containing K 2 HP0 4 1 .71 g/L, KH 2 P0 4 1 .32 g/L, NHCI 4 1 .26 g/L, MgCI 2 6H 2 0 0.01 1 g/L, CaCI 2 0.02 g /L) which was enriched with 4ml trace mineral solution (TMS containing per litre of water, Na 3 C 6 H 5 0 7 -2H 2 0 2.1 g, MnS0 4 -2H 2 0 0.5g, CoS0 4 or CoCI 2 -6H 2 0 0.1 g, ZnS0 4 -7H 2 0 0.1 g, CuS0 4 -5H 2 0 0.01 g, AIK(S0 4 ) 2 0.01 g, H 3 B0 4 0.01 g, Na 2 Mo0 4 -2H 2 0 0.1 g, NiCI 2 -6H 2 0 0.025g, Na 2 Se0 3 0.2g, V(III)CI 0.01 g, Na 2 W0 4 -2H 2 0 0.0033g)/100ml was prepared at pH 7.0 and aliquots dispensed into flasks and autoclaved at 121 °C for 15 min. After cooling to ambient, 1 ml of BTEX was added in each flask. Six different bacteria strains were inoculated in different flasks. P.aeroginosa, P.putida and B.subtilis were used as positive controls while un-inoculated flasks were used as negative controls. Flasks were sealed completely and incubated at 30 °C while biodegradation was continually monitored for 1 week. A comparative biodegradation experiment was also set up to compare the rate of degradation of different bacteria consortia to the best performing bacteria isolates from the first set up. Each consortium was formed from centrifuged and thoroughly washed bacterial pellets by carefully re-suspending each strain in an equal proportion in the ratio of either 1 :1 or 1 :1 :1 (v/v/v). Analytical methods

Gas chromatography mass spectrometry (GC-MS)

GC-MS analysis was carried out to determine the rate of degradation of BTEX in culture media. C18 columns were used to extract organics from culture media by homogenising the cultures in the flasks before passing 10ml of BTEX culture through the columns. Trapped BTEX in the columns was eluted in GC-MS vials using 1 ml acetone. Analysis was carried out using GC-MS; the oven temperature program was an initial temperature of 60 °C increased at a rate of 7°C/min to 220 °C followed by 15 °C/min to 280 °C and held at 280 °C for 5min. pH analysis

A WTW 330 pH meter which was calibrated with two standard buffer solutions of pH 7 and 10, was used to analyse the pH of each of the samples by measuring 1 0ml into a 1 50ml flask; in each case, the pH analysis was done in triplicate.

Results

Bacterial identification and molecular characterization Pure bacteria cultures were plated on agar medium and extraction of the DNAs was carried out which followed the method of Head et al., 1998. Molecular characterization of samples followed the method of Sambrook et al., 1989. PGR analysis was carried out followed by cloning and sequencing (Muyzer et al., 1993 and Santegoeds et al, 1998). PCR products were sent to Inqaba Biotec for Sanger sequencing which were converted into text format using Chromas and then put into the NCBI BLAST database (Bond et al., 2002). The sequences were submitted to GenBank using BankIT and assigned accession numbers which were to be released in 2014. Table 1 presents the identity of bacteria used as positive controls experiment and their source.

Table 1. Bacteria used as positive controls

Organisms Source

Pseudomonas Department of Biochemistry, Microbiology and aeroginosa Biotechnology,

Pseudomonas putida Rhodes University, Grahamstown, South Africa

Bacillus subtilis

Table 2 presents the accession number of each strain, the identity of the organism, and its source Table 2. Isolated organisms and their accession numbers.

Genbank accession Closest Related Source

numbers Species to isolates

KC620473 Bacillus massiliensis Diesel contaminated soil

KC620474 Seratia nematodiphila Diesel contaminated soil

KC620475 Proteus penneri Diesel contaminated soil

KC620476 Exiguobacterium Diesel contaminated soil aurantiacum

KC620477 Mycobacterium aurum Diesel contaminated soil

KC620478 Proteus mirabilis Diesel contaminated soil

Table 3 shows the different bacterial consortia formulated and the codes assigned to each consortium.

Table 3. Bacterial consortia composition and the codes assigned to each consortium Genbank accession number Codes for each Alternative bacteria codes consortium

(i) KC620473+ KC620475 EBRU Culture 1 ECCN 1b

(i) KC620473+ KC620474 EBRU Culture 2 ECCN 2b

(Hi) KC620473+ KC620476 EBRU Culture 3 ECCN 3b

(iv) KC620473+ KC620477 EBRU Culture 4 ECCN 4b

(v) KC620473+ KC620474+ KC620475 EBRU Culture 5 ECCN 5b

(vi) KC620473+ KC620476+ KC620474 EBRU Culture 6 ECCN 6b

(vii) KC620473+ KC620475+ KC620476 EBRU Culture 7 ECCN 7b

(viii) KC620473+ KC620477+ KC620475 EBRU Culture 8 ECCN 8b

(ix) KC620475+ KC620476+ KC620477 EBRU Culture 9 ECCN 9b

In the alternative codes, ECCN is derived from EBRU Culture Collection Number, and 'b' indicates that the culture is in bacterial form. During incubation of BTEX flasks, volatilization was prevented by sealing the flasks completely. Physical observation of each flask was also carried out during incubation process. Growth of bacteria was seen in different flasks when provided with a substrate of BTEX. By way of example, Figure 1 A demonstrates the experimental set up designed to check volatilization. Figures 1 B and 1 C show the growth of EBRU Culture 1 in the presence of BTEX and MSM.

Figure 2 shows the percentage degradation of BTEX using single bacteria strains over a period of 6 days. Pseudomonas aeroginosa and strain KC620478 were able to degrade benzene completely while other strains couldn't. However, with toluene, ethylbenzene and xylene, no strain was able to achieve 100% degradation except for strains KC620478 and KC620477. Figure 3 shows the rate of degradation of BTEX in MSM using different formulated bacteria consortia. The rate of degradation of the best performing single isolates from the first experiment was compared to that of different bacteria consortia. The results showed that the bacteria consortium which comprised of P.aeroginosa, P.putida and B.subtilis competed favourably with the formulated consortia. However, the rate of degradation of the consortium at the beginning of the experiment was very slow when compared to formulated consortia.

Table 4 shows the effect of each isolate in liquid media in relation to pH changes. 10ml of culture medium was measured in 250ml Erienmeyer flask and the pH was determined over time. Table 4. pH changes over 5 days for the first set of treatments involving single bacterial isolates

Treatment Day O Day 1 Day 2 Day 3 Day 4 Day 5

Uninoculated 7.0 7.04±0.08 7.03±0.07 7.0±0.13 7.07±0.03 7.0210.05 control

Pseudomonas 7.0 7.43±0.05 7.49±0.07 7.57±0.10 7.62±0.12 7.61 +0.04 aeroginosa

Bacillus 7.0 7.37±0.03 7.42±0.06 7.45±0.05 7.50±0.04 7.50+0.06 subtilis

KC620473 7.0 7.31 ±0.05 7.39±0.05 7.47±0.08 7.49±0.10 7.50+0.12

KC620475 7.0 7.06±0.07 7.10±0.04 7.16±0.06 7.35±0.02 7.38+0.02

KC620477 7.0 7.21 ±0.04 7.22±0.05 7.34±0.02 7.38±0.06 7.38+0.03

KC620478 7.0 7.10±0.04 7.10±0.1 1 7.10±0.10 7.13±0.07 7.17+0.06

KC620476 7.0 7.13±0.06 7.14±0.08 7.16±0.02 7.25±0.04 7.29+0.12

KC620477 7.0 7.08±0.10 7.10±0.07 7.13±0.04 7.1610.1 1 7.17+0.08 In one embodiment of a practical application of the invention, one of the bacteria consortia (i) to (ix) listed hereinbefore may be prepared in glucose containing medium such as Luria broth. Bacteria biomass in the form of pellets is separated from the medium and transformed into granules; preferably into powdered form. The granulated or powdered form of bacteria may be mixed with nutrients which may be in the form of fertilizer containing nitrogen and phosphorus. Hydrocarbon contaminated soil is disked and teased apart for aerobic conditions to be met. Neutralizing agents such as lime may be applied to the top layer of the hydrocarbon contaminated soil. Application of the said neutralizing agent may be between 20 tons/Ha to 30 tons/Ha, preferably 25 tons/Ha. Bacteria consortium in the form of granules may be carried out by broadcasting in the range of 200 kg/Ha-300 kg/Ha, preferably 250 kg/Ha. Constant irrigation and churning of the hydrocarbon contaminated soil may be required and carried out fortnightly for speedy oxidation process.

In another embodiment of a practical application of the invention, one of the bacteria consortia (i) to (ix) may be cultured in a hydrocarbon containing mineral salt medium in a reactor. The mineral salt medium may be made solely of nitrogen, phosphorus and potassium. Incubation of the said consortium may be for a period of 20 to 30 days, preferably 25 days. Inoculation of the hydrocarbon containing medium into contaminated soil at the rate of 300 L/Ha to 500 L/Ha, preferably 400 L/Ha may be carried out after soil has been disked and neutralised by liming at a rate of 20 tons/Ha to 30 tons/Ha, preferably 25 tons/Ha.

The Applicant has surprisingly found that by means of the process of the invention, complete biological remediation (to within specified limits), e.g. of contaminated soil, can be effected within 35 days, as compared to prior art which typically teach remediation processes of about 90 days. For example, Silva et al (2009) teaches an 84 day bioremediation period, Das and Mykherjee (2007) teaches a 120 day remediation period, and Jacques et al (2000) teaches a 75 day remediation period. Thus, in accordance with the present invention, hydrocarbon degrading bacterial strains can be sought, isolated, screened, tested in liquid culture, consortia thereof formulated and retested, the consortia used to treat hydrocarbon polluted substances, e.g. contaminated soil, and complete remediation achieved, all within 35 days.

The invention further involves using novel organisms obtained from contaminated sites, i.e. organisms that are bioprospected rather than already available or known; screening the organisms; and rapidly formulating them into effective consortia.

The present invention is thus directed towards the treatment of petroleum hydrocarbon contaminated sites using bacteria consortia. More especially, it concerns isolating strains of bacteria from a diesel contaminated soil. In particular, it concerns formulating bacteria consortia from the single isolates and testing their abilities in utilising petroleum hydrocarbons as the only carbon source in comparison with single bacteria isolates. References

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