- Email: [email protected]
Hydrophobised sawdust as a carrier for immobilisation of the hydrocarbon-oxidizing bacterium Rhodococcus ruber
Available online at www.sciencedirect.com
Bioresource Technology 99 (2008) 2001–2008
Hydrophobised sawdust as a carrier for immobilisation of the hydrocarbon-oxidizing bacterium Rhodococcus ruber Elena A. Podorozhko a, Vladimir I. Lozinsky a,*, Irena B. Ivshina b, Maria S. Kuyukina b, Anastasiya B. Krivorutchko b, Jim C. Philp c, Colin J. Cunningham d a Institute of Organoelement Compounds, Russian Academy of Sciences, 28 Vavilov Street, 119991 Moscow, Russia Institute of Ecology and Genetics of Microorganisms, Ural Branch, Russian Academy of Sciences, 13 Golev Street, 614081 Perm, Russia c School of Life Sciences, Napier University, 10 Colinton Road, Edinburgh EH 10 5DT, UK Contaminated Land Assessment and Remediation Research Centre, Crew Building, The King’s Buildings, University of Edinburgh EH9 3JN, Scotland, UK b
d
Received 18 October 2006; received in revised form 12 March 2007; accepted 13 March 2007 Available online 3 May 2007
Abstract Pine sawdust treated by a series of hydrophobising agents (drying oil, organosilicon emulsion, n-hexadecane and paraffin) was examined as carrier for adsorption immobilisation of hydrocarbon-oxidizing bacterial cells Rhodococcus ruber. It was shown that hydrophobising agents based on drying oil turned out to be optimal (among the other modifiers examined) for the preparation of sawdust carriers suitable for the efficient immobilisation. The results obtained demonstrate promising possibilities in developing a wide range of available and cheap, biodegradable cellulose-containing carriers that possess varying surface hydrophobicity. 2007 Elsevier Ltd. All rights reserved. Keywords: Hydrophobised sawdust; Cell immobilisation; Rhodococcus ruber; Biodegradation of hydrocarbons
1. Introduction Whereas the efficacy of the practice of bioaugmentation during bioremediation of hydrocarbon contamination is questionable (e.g. Atlas, 1991; Pritchard, 1992; Vogel, 1996), in cold climates this approach may be of justification (Mohn et al., 2001). Arctic (Robinson and Wookey, 1997) and Antarctic (Aislabie et al., 1998, 2001) soils are considered to be deficient of microorganisms compared to other soils. Low numbers of hydrocarbon-oxidizing bacteria in pristine, cold climate soils (Aislabie et al., 1998, 2001) and short summer seasons may limit the spontaneous enrichment of oil-contaminated soils with autochthonous hydrocarbon-oxidizers when biostimulation (the application of fertilizers) alone is practiced. There are reports in the literature of the use of immobilised cells to enhance bioremediation (e.g. Cunningham *
Corresponding author. Tel.: +7 095 135 64 92; fax: +7 095 135 50 85. E-mail address: [email protected] (V.I. Lozinsky).
0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.03.024
et al., 2004; Jerabkova et al., 1997; Oh et al., 2000; Samsonova et al., 2003). Immobilisation may offer various advantages over the use of liquid cultures for bioaugmentation in contaminated soils. Immobilisation is known to reduce competition with indigenous microorganisms (Lin and Wang, 1991), offer protection from predation, extremes of pH and toxic compounds in the contaminated soil (Pritchard, 1992). There is also evidence of increased biological stability, including plasmid stability, in immobilised cells (Cassidy et al., 1996). The immobilisation matrix may also act as a bulking agent in contaminated soil (Cunningham et al., 2004). This latter feature may be of increased importance in cold climates, where oil-spills have increased viscosity and freezing in soils may occlude voids for oxygen, water and nutrient transfer. For bioaugmentation at full scale on contaminated sites, the immobilisation carrier has to be readily available locally, and be inexpensive material, be ecologically harmless, yet slowly biodegradable and be able to firmly bind microbial cells. The cellulose-containing materials,
2002
E.A. Podorozhko et al. / Bioresource Technology 99 (2008) 2001–2008
particularly, wastes of the wood processing industry (sawdust, shavings, wood chips) meet these requirements for bioremediation of Northern Russian oil-spills. Moreover, such materials possess a labyrinthine, macroporous structure to provide a very high surface area for cellular attachment. However, high hydrophilicity and low resistance to fungal decay could hamper the implementation of these carriers for the adsorption immobilisation of oil-degrading microorganisms. To improve the oil and oil-degrading microbe sorption abilities of such cellulose-containing materials, modification is required in order to increase their hydrophobicity, and to impart water-repellent properties, allowing better flotation and resistance to fungi. It is known (Nikitin, 1962) that to impart the hydrophobic properties to wood materials, their available surface hydroxyl groups should be blocked. Thermal treatment and coating with thermo-reactive resins are widely used for this purpose. These approaches, however, are not suitable for developing carriers to immobilise bacterial cells as such treatments may lead to reduced cell viability, in particular as a result of acid liberation in the former case and toxicity of chemicals used in the latter. In the present work, we attempted to form on the wood dispersed material, sawdust, a non-toxic hydrophobic coating, which is tightly adhered to such a carrier and could not be removed in aqueous medium. The effect of relative hydrophobicity of a cellulose-containing carrier on its ability to immobilise the hydrocarbon-oxidizing bacterium Rhodococcus ruber was studied. The rhodococci have a highly hydrophobic cell surface and R. ruber produces biosurfactants that impart greater hydrophobicity to the cell surface than the corresponding molecules in Rhodococcus erythropolis (Philp et al., 2002), which may extend the substrate range of R. ruber. This could help to determine physico-chemical criteria useful in selecting the appropriate hydrophilic–hydrophobic balance of carriers for the adsorption immobilisation of various bacterial cells. Moreover, there is evidence that rhodococcal strains may be of greater benefit in cold climate bioremediation: members of the alkane-degrading Nocardiaceae are widely distributed in Antarctic soils, whereas Pseudomonas strains are considered to be rare (Vishniac, 1993). It has been suggested that rhodococci are predominant alkanotrophs in pristine and contaminated polar soils (Aislabie et al., 2004). For the rhodococcal cells immobilised on the carriers thus prepared, we studied the hydrocarbon-oxidizing activity, which was evaluated by monitoring the biodegradation of n-hexadecane, considered to be a model medium chain-length n-alkane which is readily biodegradable. 2. Methods 2.1. Materials Pine sawdust with particle size of 1–3 mm were used as carriers for bacterial cells. Natural drying oils based either on sunflower seed oil (Obushki Co., Russia) or linseed oil
(Olivesti Co., Russia), as well as a KE-30-04 50% organosilicon emulsion (Kserosil Co., Russia), n-hexadecane and paraffin (Reakhim, Russia) were used as hydrophobising agents. Chemically pure hexane was purchased from Laverna Co. (Russia), white spirit was from Reakhim (Russia). 2.2. Microorganisms Bacterial strain R. ruber IEGM 231 from the Regional Specialised Collection of Alkanotrophic Microorganisms (IEGM, 1994; Sugawara and Miyazaki, 1999) was used. The bacterial culture was grown in nutrient broth (Oxoid, Unipath Ltd., UK); the process was performed using orbital shaker (150 rpm) at 28 C for 3 days. 2.3. Procedures 2.3.1. Preparation of hydrophobised carriers Highly hydrophobised carriers for the immobilisation of bacterial cells were prepared by immersing the sawdust into drying oil, removing excess liquid, drying at room temperature for 4 days and further at 75 or 105 C, with an exposure time of 30, 60, 90 or 120 min. Slightly hydrophobised carriers were prepared by spraying of sawdust material with a white spirit solution of drying oil. The ratio of drying oil to sawdust was 5 or 10 g per 100 g of solid matter. Treated material was mixed thoroughly for 20 min to reach uniform distribution of the hydrophobising agent on the particles, which then were air dried for 24 h to remove the solvent, and heated at 75 or 105 C for 1–1.5 h. A KE-30-04 organosilicon emulsion was diluted with water to the final polymer concentration of 5% or 10%. This dilute emulsion was distributed evenly on the surface of the sawdust carrier, which was then air dried until the solvent was completely removed. Sawdust hydrophobisation by n-hexadecane or paraffin vapours was performed in a closed container. Sawdust sample was placed above the n-hexadecane or paraffin, heated up to 25 or 60 C, respectively, and incubated for 6–8 h. 2.3.2. Determination of relative hydrophobicity of sawdust-based carriers Determination of specific amount of water absorbed by a carrier upon swelling. The sawdust sample was immersed into water for 40 min at room temperature. Then the excess liquid was removed by pressing until there was no wet spot observed on dry paper filter. The wet pressed sample was weighed and dried at 105 C to constant weight. The specific amount of absorbed water was determined as the difference between the wet (pressed-off) and the dried samples weights divided by the dry sample weight. Determination of specific amount of water vapour adsorbed by a carrier at 100% humidity. The sawdust sample was placed above the water layer into a closed container at room temperature. The specific amount of water vapour
E.A. Podorozhko et al. / Bioresource Technology 99 (2008) 2001–2008
adsorbed by the carrier was measured gravimetrically and expressed as the difference between the wet and dry sample weights divided by dry sample weight. Determination of relative hydrophobicity of modified wood materials. Relative hydrophobicity of smooth surfaces of the initial and modified pine wood bars was evaluated by measuring contact wetting angles (Lipatov, 1980) of the two standard liquids. Measurements were performed using MMI-2 microscope (LOMO, Russia) at 10· magnification. The standard liquids used were water and formamide. 2.3.3. Immobilisation of bacterial cells A suspension of washed bacterial cells in sodium-phosphate buffer (pH 7.0) at a concentration corresponding to the optical density value (OD600) of 1.0 was used for immobilisation. The buffer contained (g/l): Na2HPO4, 3.53; KH2PO4, 3.39. Immobilisation of bacteria on the sawdust-based carriers was performed in 250 ml Erlenmeyer flasks containing 50 ml of cell suspension and 1 g of carrier. The process was carried out on the orbital shaker (130 rpm) at room temperature for 5 days. The adsorption immobilisation of the microorganisms was monitored nephelometrically at 600 nm. Adsorption capacities of carriers were calculated as follows (Medvedeva et al., 2001): C ¼ ðC init C res Þ V =m; where C is the adsorption capacity of the carrier, mg of dry cells/g carrier; Cinit is the concentration of cell suspension before immobilisation, mg dry cells/ml; Cres is the concentration of cell suspension after immobilisation, mg dry cells/ml; V is the volume of cell suspension; m is the carrier mass, g. Desorption of immobilised cells was performed using 1 M NaCl solution under conditions similar to those used in the immobilisation experiments. 2.3.4. Determination of hydrocarbon-oxidizing activity of immobilised cells Catalytic activity of immobilized bacterial cells was measured in the experiments on n-hexadecane biodegradation. n-Hexadecane was used as the model hydrocarbon compound. Biodegradation experiments using bacterial cells immobilised on sawdust-based carriers were performed on the orbital shaker (130 rpm) at 28 C for 10 days in 250 ml Erlenmeyer flasks containing 50 ml nutrient medium. This salt medium contained (g/l): KH2PO4, 1.0; K2HPO4, 1.0; NaCl, 1.0; KNO3, 1.0; MgSO4, 0.2; CaCl2, 0.02 and FeCl3. n-Hexadecane at a concentration of 3.0% (v/v) was used as the sole carbon source. Prior to inoculation, the medium was supplemented with the solution of trace elements (1.0 ml/l) and yeast extract (0.05 g/l). Blank (reference) experiments with non-inoculated carriers were carried out, as well. The efficacy of hydrocarbon oxidation was evaluated by measuring the rate of n-hexadecane biodegradation. For this end, residual hydrocarbon concentrations in cultural broth were determined gravimetrically after n-hexadecane extraction with hexane, which was done
2003
as follows. The liquid phase of cultural broth was transferred to a separation funnel supplemented with 30 ml of n-hexane, which was then shaken thoroughly for 1 min and then the organic phase was allowed to separate from the aqueous phase followed by the hexane phase removal. The extraction procedure was repeated three times. Water-free Na2SO4 was added to the resultant hexane extract for dehydration. Then the extract was filtered into a preliminary weighed round-bottom flask, and the solvent was removed by rotary evaporation at 50 C for 30 min. The amount of residual n-hexadecane was calculated after repeated weighing the flask. The obtained values were used to determine the mean rate of n-hexadecane biodegradation. The immobilisation of the cells and hexadecane biodegradation experiments were carried out in three repetitions, and the results obtained were averaged. 2.3.5. Electron microscopy Samples were mounted onto aluminium specimen stubs using double sided adhesive tape. They were then coated with gold using sputter Polaron E5100 series 2 (Quorum Technologies Ltd., UK) for a total of 6 min to prevent specimen charging under the electron beam. Specimens were subsequently assembled onto the specimen stage of the scanning electron microscope Cambridge S90 (Cambridge Instruments, UK), and the specimen chamber was evacuated. At vacuum threshold, the electron beam was initiated using a low accelerating voltage (10 kV) to avoid sample damage. The image was viewed, the image displayed on the SEM monitor (CRT), and stored using the image capture hardware/software (I-Scan) image archiving system. 3. Results and discussion 3.1. Preparation of carriers for cell immobilisation The ability of Rhodococcus cells to adhere to the nonpolar carriers can be explained by the hydrophobic properties of cell surfaces (Ivshina, 1994). Two methods were used to render hydrophobic properties for such cellulose-containing material, as sawdust, in order to achieve more efficient adsorption immobilisation of bacterial cells: (i) coating the material with a water-resistant hydrophobic polymer film followed by thermal treatment of the material; and (ii) adsorbing the vapours of low-molecular hydrocarbons. Initially, a continuous hydrophobic polymer film was formed on the surface of sawdust material by its treatment with an excess of drying oil (see Section 2.3), which is the liquid basis of oil-colours and possesses the ability to form films owing to the air-oxygen-initiated radical polymerization of the unsaturated fatty acids constituents in drying oil (Gandini and Belgacem, 1996). The results of the use of drying oil as a hydrophobising agent can be seen from the values of wetting contact angles (cos h) for the drying
2004
E.A. Podorozhko et al. / Bioresource Technology 99 (2008) 2001–2008
Table 1 The values of wetting contact angle (expressed in terms of cos h) for the pinewood bars treated with an excess of drying oil followed by air-drying at different temperatures Drying temperature, C
cos h for wetting liquids Water
Formamide
23 75 105
0.708 0.731 0.690
0.836 0.884 0.852
of an oil-treated smooth wood surface (Table 1). High values of cos h evidenced that the oil-treated wood surface acquired pronounced hydrophobic properties. Modified wood sample heated up at 75 C for 1 h demonstrated the highest hydrophobicity. For comparison, it was impossible to measure the contact wetting angles for the non-modified sample of wood bar, since both water and formamide drops spread instantly over the wood surface. For the optimisation of conditions of the sawdust hydrophobisation procedure the samples treated with dry-
Table 2 Effect of heating regime on water absorbing capacity of sawdust-based carriers treated with drying oil Sample no.
1 2 3 4 5 6 7 8 9 10
Heating conditions Temperature, C 23 75
105
Time, min 0 30 60 90 120 220 30 60 90 120
Water absorbing capacitya, g of water/g of dry carrier
0.44 ± 0.02 0.44 ± 0.02 0.45 ± 0.02 0.54 ± 0.02 0.48 ± 0.02 0.39 ± 0.02 0.43 ± 0.02 0.51 ± 0.02 0.65 ± 0.02 0.62 ± 0.02
a Water absorption by the initial non-modified sawdust was equal to about 2.5 g/g.
ing oil were heated for different time periods. Table 2 shows the data on water absorbing capacities of highly hydrophobised sawdust samples treated thermally at 75 or 105 C for 30, 60, 90, 120 and 220 min. Upon increase in exposure time from 0 to 90 min the water absorbing capacity rose gradually, but then declined. For the sample which was not thermally treated, and the samples heated during short exposure time, the removal of non-reacted (free) hydrophobising agent into the water phase was observed, when the solvent was removed from the modified sawdust. Thermal treatment for 90 min and longer fixed the polymer film firmly on the sawdust surfaces, and no leaching of the hydrophobising agent was registered upon immersion of the samples in water. Thus, to promote the film-formation from the drying oil, further samples of hydrophobised sawdust were heated for at least 90 min. Table 3 summarises the data on absorption of bulk water and adsorption of water vapour by the highly hydrophobised sawdust (treated by an excess of drying oil). After such hydrophobisation, water absorbing properties of the sawdust carriers decreased by 5–6 times, with their ability to adsorb water vapour being decreased by about two times compared to those of non-treated material. Scanning electron microscopy revealed that hydrophobic film formed on the carrier surface covered almost completely the channels and pores in the sawdust (cf. Fig. 1a and b). This resulted in both decreasing hydration ability of the material and decrease in the surface area available for cell adsorption on the modified sawdust. Thus, the immobilisation experiments showed a significant decrease of the adsorption of bacterial cells on these highly hydrophobised carriers as compared to that of the initial non-modified sawdust (cf. Nos. 1 and 2 in Table 4). Therefore, in this study we decreased the concentration of the hydrophibising agent used to treat sawdust material further, and the treatment regimes have also been optimised. For this aim, either drying oil diluted with organic solvent (white spirit), or water-organosilicon emulsion diluted with additional water, or vapours of low molecular weight hydrocarbons were used.
Table 3 Water absorption and water vapour adsorption by the highly hydrophobised sawdust carriers Sample
Water absorbing capacity, g of water/g of dry carrier
Water vapour adsorbing capacity, g of water/g of dry carrier After 1 day
After 5 days
Non-modified (initial) sawdust
2.50 ± 0.02
0.16 ± 0.03
0.20 ± 0.02
Sawdust highly hydrophobised by the treatment with sunflower seed-based drying oil followed by heating: • at 75 C for 2 h • at 105 C for 2 h
0.39 ± 0.02 0.62 ± 0.02
0.09 ± 0.03 0.08 ± 0.03
0.11 ± 0.02 0.11 ± 0.02
Sawdust highly hydrophobised by linseed-based drying oil followed by heating: • at 23 C for 3 days • at 105 C for 2 h
0.30 ± 0.02 0.28 ± 0.02
0.08 ± 0.03 0.07 ± 0.03
0.10 ± 0.02 0.10 ± 0.02
E.A. Podorozhko et al. / Bioresource Technology 99 (2008) 2001–2008
Fig. 1. Micrographs (SEM) of initial non-modified sawdust (a) and highly hydrophobised sawdust treated with an excess of sunflower seed-based drying oil followed by heating at 105 C for 2 h (b).
3.2. Optimisation of hydrophobic/hydrophilic properties of sawdust-based carriers for the adsorption immobilisation of Rhodococcus bacteria It was found that the hydration capacity of sawdust treated with a small amount of hydrophobising agent was higher than the capacity of highly hydrophobic carriers, but 1.5–2 times lower than the capacity of non-treated sawdust (cf. Nos. 1, 3 and 4 in Table 4). The extent of water vapour adsorption by such slightly hydrophobised carriers was lower in comparison with the non-treated sawdust
2005
samples (cf. Nos. 1, 3 and 4 in Fig. 2). In addition, the non-modified and hydrophobised carriers differed markedly in the rate of water vapour adsorption (Fig. 2). For instance, if the non-modified sawdust (curve 1) for the first 1–2 days, i.e. a rather rapidly for such materials, adsorbed the vapour to the extent of 0.30–0.35 g per g of the carrier, highly hydrophobised sawdust (curve 2) for the same time adsorbed a threefold lower amount of water vapour, and slightly hydrophobised sawdust samples (curves 3–8) demonstrated intermediate adsorption rates. It is thought that these results could be associated with the discrete character of hydrophobic films formed on the slightly hydrophobised sawdust particles and also with the fact that the open pores remained in the treated material. Therefore, sawdust samples treated by limited amounts of the hydrophobising are characterized by a well-developed surface area, which, in principle, can be available for the adhesion of bacterial cells. Scanning electron microscopy data support this suggestion. Thus, Fig. 3 demonstrates the structure of organosilicon-treated sawdust as a typical representative of such slightly hydrophobised materials. Micrographs show the pores, which are similar to those in the non-treated sawdust (cf. Figs. 1a and 3a), while the hydrophobic coating (organosilicon-based polymer in this case) resembled a net-like film on the carrier surface, which is readily observed at higher magnification (Fig. 3b). Comparative studies of the immobilisation of Rhodococcus bacteria on various sawdust-based carriers demonstrated (Table 4) that cell adsorption capacity of slightly hydrophobised carriers was 1.2 and 3.0 times higher compared to that of the non-treated sawdust and of highly hydrophobised material, respectively. It is noteworthy that the cell surface of alkanotrophic rhodococci has a mosaic structure dominated by hydrophobic regions (Ivshina et al., 1997; Kuyukina et al., 2000). Hence, for the efficient adsorption of such cells on cellulose-containing carriers, a combination of hydrophilic and hydrophobic areas is required. To further optimise the techniques used for preparing cell immobilisation carriers, sawdust was subjected to additional alkaline treatment. The latter resulted in changes of hydration properties of the sawdust (No. 5 in Table 4). Decrease in the water binding capacity of sawdust after alkaline treatment was due to partial dissolution and extraction of lignin and hemicellulose that are readily hydrated substances. On the other hand, the observed increase in the adsorption of water vapour suggested loosening of the structure of sawdust material and increasing its specific surface area. Subsequent hydrophobisation of alkali-treated sawdust with small amounts of drying oil resulted in a marked decrease in both water absorption and water vapour adsorption, as well as an increase in adsorption capacity of the carrier for bacterial cells (No. 6 in Table 4). Apart from the polymer-forming hydrophobising agents applied from a liquid phase, we also used low molecular weight hydrocarbons such as n-hexadecane and paraffin
2006
E.A. Podorozhko et al. / Bioresource Technology 99 (2008) 2001–2008
Table 4 Physico-chemical properties of sawdust-based carriers and their use for the immobilisation of hydrocarbon-oxidizing bacteria No.
Modifying agent
Water vapour absorbing capacity, g of water/g of dry carrier
Water vapour absorbing capacity, g of water/g of dry carrier For 1 day
For 6 days
1 2 3 4 5 6
None Drying oil in an excess Drying oil in a shortage Organiosilicon emulsion NaOH solution NaOH solution + drying oil in a shortage n-Hexadecane vapours Paraffin vapours
2.55 ± 0.15 0.39 ± 0.02 1.24 ± 0.02 1.93 ± 0.02 1.66 ± 0.02 1.02 ± 0.02
0.28 ± 0.03 0.09 ± 0.01 0.22 ± 0.02 0.27 ± 0.02 0.27 ± 0.02 0.24 ± 0.02
1.68 ± 0.12 1.85 ± 0.02
0.29 ± 0.01 0.27 ± 0.02
7 8
Total bacterial adsorption, mg of dry cells/g of dry carrier
Average rate of n-hexadecane biodegradation, mg/l/h
0.34 ± 0.02 0.12 ± 0.02 0.31 ± 0.02 0.31 ± 0.02 0.38 ± 0.03 0.32 ± 0.02
39.0 ± 4.5 15.5 ± 1.5 46.5 ± 1.0 46.0 ± 3.0 18.0 ± 1.5 46.5 ± 1.0
71.0 ± 7.0 46.0 ± 6.0 104.0 ± 4.0 44.0 ± 3.5 26.0 ± 4.0 107.0 ± 6.0
0.31 ± 0.02 0.31 ± 0.02
41.0 ± 4.0 42.5 ± 4.0
42.5 ± 5.0 65.0 ± 2.5
0.45 0.4
g Water / g dry carrier
0.35 0.3 0.25 1
0.2
2 3
0.15
4 5
0.1
6 7
0.05
8
0 0
1
2
3
4
5
6
7
8
Time, days Fig. 2. Isotherms of water vapour adsorption at 100% humidity by the samples of sawdust-based carriers: 1 – initial non-modified sawdust; 2 – highly hydrophobised sawdust treated with an excess of sunflower seedbased drying oil followed by heating at 105 C for 2 h; 3 – slightly hydrophobised sawdust treated with the white spirit solution of drying oil followed by heating at 105 C for 1.5 h; 4 – slightly hydrophobised sawdust treated with the dilute water-organosilicon emulsion followed by air-drying; 5 – sawdust treated with the 1 N NaOH at 100 C for 2 h followed by rinsing with water and air-drying; 6 – slightly hydrophobised sawdust alkali-treated like the sample No.5 and then hydrophobised like the sample No. 3; 7 – slightly hydrophobised sawdust treated with the nhexadecane vapours; 8 – slightly hydrophobised sawdust treated with the paraffin vapours.
that were adsorbed by the sawdust from the gaseous phase (see Section 2.3). Implementing this simple technique, we expected to obtain an appropriate carrier for bacterial cells, in which the hydrophobising agent would be adsorbed uniformly as a monolayer in the available regions of sawdust, thus maximising the preservation of the porosity and surface area of the material. Analysing the data in Table 4 (Nos. 7 and 8), the hydration properties of these latter carriers seemed to be similar to the carriers treated by the
Fig. 3. Micrographs (SEM) at low (a) and high (b) magnifications of slightly hydrophobised sawdust treated with the dilute water-organosilicon emulsion followed by air-drying.
small amounts of hydrophobising agents. However, the cell adsorption capacity in the case of carriers being hydrophobised by gaseous n-hexadecane or paraffin was slightly lower than for the carriers treated by small amounts of drying oil or organosilicon emulsion.
E.A. Podorozhko et al. / Bioresource Technology 99 (2008) 2001–2008
3.3. Hydrocarbon-oxidizing activity of immobilised rhodococci cells Comparative studies revealed certain positive correlation between the amounts of rhodococci cells immobilised on sawdust-based carriers and the catalytic activity of these cells. Thus, maximum amount of biomass (46.5 g of dry cells per g of a carrier) was adsorbed on the sawdust treated with small amounts of drying oil, and this resulted in the immobilised systems (Nos. 3 and 6 in Table 4) exhibiting the highest rate of n-hexadecane biodegradation – around 100 mg/l/h. However, in spite of relatively high amount of rhodococcal cells immobilised on the sawdust treated with organosilicon emulsion, either by the n-hexadecane or paraffin vapours (46.0, 41.0 or 42.5 mg/l/h, respectively Nos. 4, 7 and 8 in Table 4), the hydrocarbon-oxidizing activities of such immobilised systems were not high enough, probably as a result of the inhibitory effect on the biodegradation process caused by the former hydrophobising agent itself (or, more exactly, traces of residual minor additives of organosilicon emultion), or because of another nature of hydrophobic coating on the carriers in the latter cases (hydrophobisation of sawdust by the competitive hydrocarbons that could be consumed by the cells). The efficiency of hydrocarbon oxidation (and, hence, the removal of n-hexadecane from the liquid medium) depends on both the oxidation activities of immobilised bacterial cells and hydrocarbon-adsorbing capabilities of a carrier. Highly hydrophobised sawdust, in particular, was able to accumulate over 70% of n-hexadecane from water medium due to physical adsorption by the carrier, while the same values for other carriers varied from 10% to 30% depending on the hydrophobicity degree of respective material (the data are not shown). Therefore, it may be supposed that highly hydrophobised sawdust can be used as an adsorbent, which can be added to water and soil highly contaminated with oil, whose bioremediation is carried out with the cells immobilised on the slightly hydrophobised sawdust-based carriers. 4. Conclusions The results obtained in this study show promising possibilities in developing a wide range of available and cheap, biodegradable cellulose-containing carriers that possess varying surface hydrophobicity. The latter parameter, depending on the microorganisms to be immobilised, could be easily adjusted till required level. Hydrophobising agents based on drying oil proved to be optimal (among the other modifiers examined) for the preparation of sawdust carriers for the efficient immobilisation of hydrocarbon-oxidizing bacterial cells (Pororozhko et al., 2005). Acknowledgements INTAS Grant # 01-2151 is acknowledged for the financial support.
2007
References Aislabie, J.M., McLeod, M., Fraser, R., 1998. Potential for biodegradation of hydrocarbons in soil from Ross dependency, Antarctica. Appl. Microbiol. Biotechnol. 49, 210–214. Aislabie, J.M., Fraser, R., Duncan, S., Farrell, R.L., 2001. Effects of oil spills on microbial heterotrophs in Antarctic soils. Polar Biol. 24, 308– 313. Aislabie, J.M., Balks, M.R., Foght, J.M., Waterhouse, E.J., 2004. Hydrocarbon spills on Antarctic soils: effects and management. Environ. Sci. Technol. 38, 1265–1274. Atlas, R.M., 1991. Microbial hydrocarbon degradation – bioremediation of oil spills. J. Chem. Technol. Biotechnol. 52, 149–156. Cassidy, M.B., Lee, H., Trevors, J.T., 1996. Environmental applications of immobilised microbial cells: a review. J. Ind. Microbiol. 16, 79–101. Cunningham, C.J., Ivshina, I.B., Lozinsky, V.I., Kuyukina, M.S., Philp, J.C., 2004. Bioremediation of diesel contaminated soil by microorganisms immobilised in a polyvinyl alcohol cryogel. Int. Biodeterior. Biodegrad. 54, 167–174. Gandini, A., Belgacem, M.N., 1996. In: Salamone, J.C. (Ed.), Polymeric Materials Encyclopaedia, V.11. CRC Press, Inc., Boca Raton e.a, pp. 8518–8530. IEGM, WFCC No. 768; Catalogue of Strains, 1994.. Ivshina, I.B. (Ed.), 1994. Catalogue of Strains of the Regional Specialized Collection of Alkanotrophic Microorganisms. Nauka, Moscow, p. 63. Ivshina, I.B. 1997. Bacteria of the genus Rhodococcus: biodiversity, detection and immunodiagnostics. Professor’s Thesis, Institute of Ecology and Genetics of Microorganisms, Perm, 98p. (in Russian). Jerabkova, H., Kralova, B., Krejeu, V., Sanchez, J.L.I., Roig, M.G., 1997. Use of polyurethane foam for the biodegradation of n-alkanes by immobilised cells of Pseudomonas. Biotechnol. Tech. 11, 391–394. Kuyukina, M.S., Ivshina, I.B., Ritchkova, M.I., Chumakov, O.B., 2000. Effect of cell lipid composition on the formation of nonspecific antibiotic resistance in alkanotrophic rhodococci. Microbiology 69, 51–57. Lin, J.-E., Wang, H.Y., 1991. Use of co-immobilised biological systems to degrade toxic organic compounds. Biotechnol. Bioeng. 38, 273–279. Lipatov, Yu.S., 1980. Interfacial effects in polymers, Naukova Dumka, Kiev, p. 80 (in Russian). Medvedeva, N.G., Gridneva, Yu.A., Lysenko, A.A., Sukharevich, V.I., 2001. Immobilisation of acetic bacteria on carbon fibers and their use for thiodiglycol transformation. Russ. J. Biotechnol. (4), 266–270. Mohn, W.W., Radziminski, C.Z., Fortin, M.-C., Reimer, K.J., 2001. On site bioremediation of hydrocarbon-contaminated Arctic tundra soils in inoculated biopiles. Appl. Microbiol. Biotechnol. 57, 242–247. Nikitin, N.I., 1962. Chemistry of wood and cellulose, USSR Academy of Sciences, Moscow-Leningrad, p. 711 (in Russian). Oh, Y.-S., Maeng, J., Kim, S.-J., 2000. Use of microorganism-immobilised polyurethane foams to absorb and degrade oil on water surface. Appl. Microbiol. Biotechnol. 54, 418–423. Philp, J.C., Kuyukina, M.S., Ivshina, I.B., Dunbar, S.A., Christofi, N., Lang, S., Wray, V., 2002. Alkanotrophic Rhodococcus ruber as a biosurfactant producer. Appl. Microbiol. Biotechnol. 59, 318–324. Pororozhko, E.A., Kuyukina, M.S., Ivshina, I.B., Philp, J.C., Lozinsky, V.I., 2005. Composition for the preparation of carrier of immobilized microorganisms capable of destructing of hydrocarbons and the method for the carrier preparation. Russ. Patent Appl. No. 2005111376. Pritchard, P.H., 1992. Use of inoculation in bioremediation. Curr. Opin. Biotechnol. 3, 232–243. Robinson, C.H., Wookey, P.A., 1997. Microbial ecology, decomposition and nutrient cycling. In: Woodin, S.J., Marquiss, M. (Eds.), Ecology of Arctic Environments. Blackwell, Oxford, pp. 41–68. Samsonova, A.S., Aleschenkova, Z.M., Li, E., Lim, B.-R., Khvan, S.V., Syemotchkina, N.F., Pintchuk, L.S., Makarevitch, A.B., Kratsov,
2008
E.A. Podorozhko et al. / Bioresource Technology 99 (2008) 2001–2008
A.G., . Wastewater decontamination from diesel oil using immobilised degradative microorganisms. Biotechnol. Russ. 4, 80–84. Sugawara, H., Miyazaki, S. (Eds.), 1999. World Directory of Collections of Cultures of Microorganisms. World Data Centre for Microorganisms, WFCC-MIRCEN, p. 140.
Vishniac, H.S., 1993. Microbiology of Antarctic soils. In: Friedmann, E.I. (Ed.), Antarctic Microbiology. Wiley, New York, pp. 297–341. Vogel, T.M., 1996. Bioaugmentation as a soil bioremediation approach. Curr. Opin. Biotechnol. 7, 311–316.
Bioresource Technology 99 (2008) 2001–2008
Hydrophobised sawdust as a carrier for immobilisation of the hydrocarbon-oxidizing bacterium Rhodococcus ruber Elena A. Podorozhko a, Vladimir I. Lozinsky a,*, Irena B. Ivshina b, Maria S. Kuyukina b, Anastasiya B. Krivorutchko b, Jim C. Philp c, Colin J. Cunningham d a Institute of Organoelement Compounds, Russian Academy of Sciences, 28 Vavilov Street, 119991 Moscow, Russia Institute of Ecology and Genetics of Microorganisms, Ural Branch, Russian Academy of Sciences, 13 Golev Street, 614081 Perm, Russia c School of Life Sciences, Napier University, 10 Colinton Road, Edinburgh EH 10 5DT, UK Contaminated Land Assessment and Remediation Research Centre, Crew Building, The King’s Buildings, University of Edinburgh EH9 3JN, Scotland, UK b
d
Received 18 October 2006; received in revised form 12 March 2007; accepted 13 March 2007 Available online 3 May 2007
Abstract Pine sawdust treated by a series of hydrophobising agents (drying oil, organosilicon emulsion, n-hexadecane and paraffin) was examined as carrier for adsorption immobilisation of hydrocarbon-oxidizing bacterial cells Rhodococcus ruber. It was shown that hydrophobising agents based on drying oil turned out to be optimal (among the other modifiers examined) for the preparation of sawdust carriers suitable for the efficient immobilisation. The results obtained demonstrate promising possibilities in developing a wide range of available and cheap, biodegradable cellulose-containing carriers that possess varying surface hydrophobicity. 2007 Elsevier Ltd. All rights reserved. Keywords: Hydrophobised sawdust; Cell immobilisation; Rhodococcus ruber; Biodegradation of hydrocarbons
1. Introduction Whereas the efficacy of the practice of bioaugmentation during bioremediation of hydrocarbon contamination is questionable (e.g. Atlas, 1991; Pritchard, 1992; Vogel, 1996), in cold climates this approach may be of justification (Mohn et al., 2001). Arctic (Robinson and Wookey, 1997) and Antarctic (Aislabie et al., 1998, 2001) soils are considered to be deficient of microorganisms compared to other soils. Low numbers of hydrocarbon-oxidizing bacteria in pristine, cold climate soils (Aislabie et al., 1998, 2001) and short summer seasons may limit the spontaneous enrichment of oil-contaminated soils with autochthonous hydrocarbon-oxidizers when biostimulation (the application of fertilizers) alone is practiced. There are reports in the literature of the use of immobilised cells to enhance bioremediation (e.g. Cunningham *
Corresponding author. Tel.: +7 095 135 64 92; fax: +7 095 135 50 85. E-mail address: [email protected] (V.I. Lozinsky).
0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.03.024
et al., 2004; Jerabkova et al., 1997; Oh et al., 2000; Samsonova et al., 2003). Immobilisation may offer various advantages over the use of liquid cultures for bioaugmentation in contaminated soils. Immobilisation is known to reduce competition with indigenous microorganisms (Lin and Wang, 1991), offer protection from predation, extremes of pH and toxic compounds in the contaminated soil (Pritchard, 1992). There is also evidence of increased biological stability, including plasmid stability, in immobilised cells (Cassidy et al., 1996). The immobilisation matrix may also act as a bulking agent in contaminated soil (Cunningham et al., 2004). This latter feature may be of increased importance in cold climates, where oil-spills have increased viscosity and freezing in soils may occlude voids for oxygen, water and nutrient transfer. For bioaugmentation at full scale on contaminated sites, the immobilisation carrier has to be readily available locally, and be inexpensive material, be ecologically harmless, yet slowly biodegradable and be able to firmly bind microbial cells. The cellulose-containing materials,
2002
E.A. Podorozhko et al. / Bioresource Technology 99 (2008) 2001–2008
particularly, wastes of the wood processing industry (sawdust, shavings, wood chips) meet these requirements for bioremediation of Northern Russian oil-spills. Moreover, such materials possess a labyrinthine, macroporous structure to provide a very high surface area for cellular attachment. However, high hydrophilicity and low resistance to fungal decay could hamper the implementation of these carriers for the adsorption immobilisation of oil-degrading microorganisms. To improve the oil and oil-degrading microbe sorption abilities of such cellulose-containing materials, modification is required in order to increase their hydrophobicity, and to impart water-repellent properties, allowing better flotation and resistance to fungi. It is known (Nikitin, 1962) that to impart the hydrophobic properties to wood materials, their available surface hydroxyl groups should be blocked. Thermal treatment and coating with thermo-reactive resins are widely used for this purpose. These approaches, however, are not suitable for developing carriers to immobilise bacterial cells as such treatments may lead to reduced cell viability, in particular as a result of acid liberation in the former case and toxicity of chemicals used in the latter. In the present work, we attempted to form on the wood dispersed material, sawdust, a non-toxic hydrophobic coating, which is tightly adhered to such a carrier and could not be removed in aqueous medium. The effect of relative hydrophobicity of a cellulose-containing carrier on its ability to immobilise the hydrocarbon-oxidizing bacterium Rhodococcus ruber was studied. The rhodococci have a highly hydrophobic cell surface and R. ruber produces biosurfactants that impart greater hydrophobicity to the cell surface than the corresponding molecules in Rhodococcus erythropolis (Philp et al., 2002), which may extend the substrate range of R. ruber. This could help to determine physico-chemical criteria useful in selecting the appropriate hydrophilic–hydrophobic balance of carriers for the adsorption immobilisation of various bacterial cells. Moreover, there is evidence that rhodococcal strains may be of greater benefit in cold climate bioremediation: members of the alkane-degrading Nocardiaceae are widely distributed in Antarctic soils, whereas Pseudomonas strains are considered to be rare (Vishniac, 1993). It has been suggested that rhodococci are predominant alkanotrophs in pristine and contaminated polar soils (Aislabie et al., 2004). For the rhodococcal cells immobilised on the carriers thus prepared, we studied the hydrocarbon-oxidizing activity, which was evaluated by monitoring the biodegradation of n-hexadecane, considered to be a model medium chain-length n-alkane which is readily biodegradable. 2. Methods 2.1. Materials Pine sawdust with particle size of 1–3 mm were used as carriers for bacterial cells. Natural drying oils based either on sunflower seed oil (Obushki Co., Russia) or linseed oil
(Olivesti Co., Russia), as well as a KE-30-04 50% organosilicon emulsion (Kserosil Co., Russia), n-hexadecane and paraffin (Reakhim, Russia) were used as hydrophobising agents. Chemically pure hexane was purchased from Laverna Co. (Russia), white spirit was from Reakhim (Russia). 2.2. Microorganisms Bacterial strain R. ruber IEGM 231 from the Regional Specialised Collection of Alkanotrophic Microorganisms (IEGM, 1994; Sugawara and Miyazaki, 1999) was used. The bacterial culture was grown in nutrient broth (Oxoid, Unipath Ltd., UK); the process was performed using orbital shaker (150 rpm) at 28 C for 3 days. 2.3. Procedures 2.3.1. Preparation of hydrophobised carriers Highly hydrophobised carriers for the immobilisation of bacterial cells were prepared by immersing the sawdust into drying oil, removing excess liquid, drying at room temperature for 4 days and further at 75 or 105 C, with an exposure time of 30, 60, 90 or 120 min. Slightly hydrophobised carriers were prepared by spraying of sawdust material with a white spirit solution of drying oil. The ratio of drying oil to sawdust was 5 or 10 g per 100 g of solid matter. Treated material was mixed thoroughly for 20 min to reach uniform distribution of the hydrophobising agent on the particles, which then were air dried for 24 h to remove the solvent, and heated at 75 or 105 C for 1–1.5 h. A KE-30-04 organosilicon emulsion was diluted with water to the final polymer concentration of 5% or 10%. This dilute emulsion was distributed evenly on the surface of the sawdust carrier, which was then air dried until the solvent was completely removed. Sawdust hydrophobisation by n-hexadecane or paraffin vapours was performed in a closed container. Sawdust sample was placed above the n-hexadecane or paraffin, heated up to 25 or 60 C, respectively, and incubated for 6–8 h. 2.3.2. Determination of relative hydrophobicity of sawdust-based carriers Determination of specific amount of water absorbed by a carrier upon swelling. The sawdust sample was immersed into water for 40 min at room temperature. Then the excess liquid was removed by pressing until there was no wet spot observed on dry paper filter. The wet pressed sample was weighed and dried at 105 C to constant weight. The specific amount of absorbed water was determined as the difference between the wet (pressed-off) and the dried samples weights divided by the dry sample weight. Determination of specific amount of water vapour adsorbed by a carrier at 100% humidity. The sawdust sample was placed above the water layer into a closed container at room temperature. The specific amount of water vapour
E.A. Podorozhko et al. / Bioresource Technology 99 (2008) 2001–2008
adsorbed by the carrier was measured gravimetrically and expressed as the difference between the wet and dry sample weights divided by dry sample weight. Determination of relative hydrophobicity of modified wood materials. Relative hydrophobicity of smooth surfaces of the initial and modified pine wood bars was evaluated by measuring contact wetting angles (Lipatov, 1980) of the two standard liquids. Measurements were performed using MMI-2 microscope (LOMO, Russia) at 10· magnification. The standard liquids used were water and formamide. 2.3.3. Immobilisation of bacterial cells A suspension of washed bacterial cells in sodium-phosphate buffer (pH 7.0) at a concentration corresponding to the optical density value (OD600) of 1.0 was used for immobilisation. The buffer contained (g/l): Na2HPO4, 3.53; KH2PO4, 3.39. Immobilisation of bacteria on the sawdust-based carriers was performed in 250 ml Erlenmeyer flasks containing 50 ml of cell suspension and 1 g of carrier. The process was carried out on the orbital shaker (130 rpm) at room temperature for 5 days. The adsorption immobilisation of the microorganisms was monitored nephelometrically at 600 nm. Adsorption capacities of carriers were calculated as follows (Medvedeva et al., 2001): C ¼ ðC init C res Þ V =m; where C is the adsorption capacity of the carrier, mg of dry cells/g carrier; Cinit is the concentration of cell suspension before immobilisation, mg dry cells/ml; Cres is the concentration of cell suspension after immobilisation, mg dry cells/ml; V is the volume of cell suspension; m is the carrier mass, g. Desorption of immobilised cells was performed using 1 M NaCl solution under conditions similar to those used in the immobilisation experiments. 2.3.4. Determination of hydrocarbon-oxidizing activity of immobilised cells Catalytic activity of immobilized bacterial cells was measured in the experiments on n-hexadecane biodegradation. n-Hexadecane was used as the model hydrocarbon compound. Biodegradation experiments using bacterial cells immobilised on sawdust-based carriers were performed on the orbital shaker (130 rpm) at 28 C for 10 days in 250 ml Erlenmeyer flasks containing 50 ml nutrient medium. This salt medium contained (g/l): KH2PO4, 1.0; K2HPO4, 1.0; NaCl, 1.0; KNO3, 1.0; MgSO4, 0.2; CaCl2, 0.02 and FeCl3. n-Hexadecane at a concentration of 3.0% (v/v) was used as the sole carbon source. Prior to inoculation, the medium was supplemented with the solution of trace elements (1.0 ml/l) and yeast extract (0.05 g/l). Blank (reference) experiments with non-inoculated carriers were carried out, as well. The efficacy of hydrocarbon oxidation was evaluated by measuring the rate of n-hexadecane biodegradation. For this end, residual hydrocarbon concentrations in cultural broth were determined gravimetrically after n-hexadecane extraction with hexane, which was done
2003
as follows. The liquid phase of cultural broth was transferred to a separation funnel supplemented with 30 ml of n-hexane, which was then shaken thoroughly for 1 min and then the organic phase was allowed to separate from the aqueous phase followed by the hexane phase removal. The extraction procedure was repeated three times. Water-free Na2SO4 was added to the resultant hexane extract for dehydration. Then the extract was filtered into a preliminary weighed round-bottom flask, and the solvent was removed by rotary evaporation at 50 C for 30 min. The amount of residual n-hexadecane was calculated after repeated weighing the flask. The obtained values were used to determine the mean rate of n-hexadecane biodegradation. The immobilisation of the cells and hexadecane biodegradation experiments were carried out in three repetitions, and the results obtained were averaged. 2.3.5. Electron microscopy Samples were mounted onto aluminium specimen stubs using double sided adhesive tape. They were then coated with gold using sputter Polaron E5100 series 2 (Quorum Technologies Ltd., UK) for a total of 6 min to prevent specimen charging under the electron beam. Specimens were subsequently assembled onto the specimen stage of the scanning electron microscope Cambridge S90 (Cambridge Instruments, UK), and the specimen chamber was evacuated. At vacuum threshold, the electron beam was initiated using a low accelerating voltage (10 kV) to avoid sample damage. The image was viewed, the image displayed on the SEM monitor (CRT), and stored using the image capture hardware/software (I-Scan) image archiving system. 3. Results and discussion 3.1. Preparation of carriers for cell immobilisation The ability of Rhodococcus cells to adhere to the nonpolar carriers can be explained by the hydrophobic properties of cell surfaces (Ivshina, 1994). Two methods were used to render hydrophobic properties for such cellulose-containing material, as sawdust, in order to achieve more efficient adsorption immobilisation of bacterial cells: (i) coating the material with a water-resistant hydrophobic polymer film followed by thermal treatment of the material; and (ii) adsorbing the vapours of low-molecular hydrocarbons. Initially, a continuous hydrophobic polymer film was formed on the surface of sawdust material by its treatment with an excess of drying oil (see Section 2.3), which is the liquid basis of oil-colours and possesses the ability to form films owing to the air-oxygen-initiated radical polymerization of the unsaturated fatty acids constituents in drying oil (Gandini and Belgacem, 1996). The results of the use of drying oil as a hydrophobising agent can be seen from the values of wetting contact angles (cos h) for the drying
2004
E.A. Podorozhko et al. / Bioresource Technology 99 (2008) 2001–2008
Table 1 The values of wetting contact angle (expressed in terms of cos h) for the pinewood bars treated with an excess of drying oil followed by air-drying at different temperatures Drying temperature, C
cos h for wetting liquids Water
Formamide
23 75 105
0.708 0.731 0.690
0.836 0.884 0.852
of an oil-treated smooth wood surface (Table 1). High values of cos h evidenced that the oil-treated wood surface acquired pronounced hydrophobic properties. Modified wood sample heated up at 75 C for 1 h demonstrated the highest hydrophobicity. For comparison, it was impossible to measure the contact wetting angles for the non-modified sample of wood bar, since both water and formamide drops spread instantly over the wood surface. For the optimisation of conditions of the sawdust hydrophobisation procedure the samples treated with dry-
Table 2 Effect of heating regime on water absorbing capacity of sawdust-based carriers treated with drying oil Sample no.
1 2 3 4 5 6 7 8 9 10
Heating conditions Temperature, C 23 75
105
Time, min 0 30 60 90 120 220 30 60 90 120
Water absorbing capacitya, g of water/g of dry carrier
0.44 ± 0.02 0.44 ± 0.02 0.45 ± 0.02 0.54 ± 0.02 0.48 ± 0.02 0.39 ± 0.02 0.43 ± 0.02 0.51 ± 0.02 0.65 ± 0.02 0.62 ± 0.02
a Water absorption by the initial non-modified sawdust was equal to about 2.5 g/g.
ing oil were heated for different time periods. Table 2 shows the data on water absorbing capacities of highly hydrophobised sawdust samples treated thermally at 75 or 105 C for 30, 60, 90, 120 and 220 min. Upon increase in exposure time from 0 to 90 min the water absorbing capacity rose gradually, but then declined. For the sample which was not thermally treated, and the samples heated during short exposure time, the removal of non-reacted (free) hydrophobising agent into the water phase was observed, when the solvent was removed from the modified sawdust. Thermal treatment for 90 min and longer fixed the polymer film firmly on the sawdust surfaces, and no leaching of the hydrophobising agent was registered upon immersion of the samples in water. Thus, to promote the film-formation from the drying oil, further samples of hydrophobised sawdust were heated for at least 90 min. Table 3 summarises the data on absorption of bulk water and adsorption of water vapour by the highly hydrophobised sawdust (treated by an excess of drying oil). After such hydrophobisation, water absorbing properties of the sawdust carriers decreased by 5–6 times, with their ability to adsorb water vapour being decreased by about two times compared to those of non-treated material. Scanning electron microscopy revealed that hydrophobic film formed on the carrier surface covered almost completely the channels and pores in the sawdust (cf. Fig. 1a and b). This resulted in both decreasing hydration ability of the material and decrease in the surface area available for cell adsorption on the modified sawdust. Thus, the immobilisation experiments showed a significant decrease of the adsorption of bacterial cells on these highly hydrophobised carriers as compared to that of the initial non-modified sawdust (cf. Nos. 1 and 2 in Table 4). Therefore, in this study we decreased the concentration of the hydrophibising agent used to treat sawdust material further, and the treatment regimes have also been optimised. For this aim, either drying oil diluted with organic solvent (white spirit), or water-organosilicon emulsion diluted with additional water, or vapours of low molecular weight hydrocarbons were used.
Table 3 Water absorption and water vapour adsorption by the highly hydrophobised sawdust carriers Sample
Water absorbing capacity, g of water/g of dry carrier
Water vapour adsorbing capacity, g of water/g of dry carrier After 1 day
After 5 days
Non-modified (initial) sawdust
2.50 ± 0.02
0.16 ± 0.03
0.20 ± 0.02
Sawdust highly hydrophobised by the treatment with sunflower seed-based drying oil followed by heating: • at 75 C for 2 h • at 105 C for 2 h
0.39 ± 0.02 0.62 ± 0.02
0.09 ± 0.03 0.08 ± 0.03
0.11 ± 0.02 0.11 ± 0.02
Sawdust highly hydrophobised by linseed-based drying oil followed by heating: • at 23 C for 3 days • at 105 C for 2 h
0.30 ± 0.02 0.28 ± 0.02
0.08 ± 0.03 0.07 ± 0.03
0.10 ± 0.02 0.10 ± 0.02
E.A. Podorozhko et al. / Bioresource Technology 99 (2008) 2001–2008
Fig. 1. Micrographs (SEM) of initial non-modified sawdust (a) and highly hydrophobised sawdust treated with an excess of sunflower seed-based drying oil followed by heating at 105 C for 2 h (b).
3.2. Optimisation of hydrophobic/hydrophilic properties of sawdust-based carriers for the adsorption immobilisation of Rhodococcus bacteria It was found that the hydration capacity of sawdust treated with a small amount of hydrophobising agent was higher than the capacity of highly hydrophobic carriers, but 1.5–2 times lower than the capacity of non-treated sawdust (cf. Nos. 1, 3 and 4 in Table 4). The extent of water vapour adsorption by such slightly hydrophobised carriers was lower in comparison with the non-treated sawdust
2005
samples (cf. Nos. 1, 3 and 4 in Fig. 2). In addition, the non-modified and hydrophobised carriers differed markedly in the rate of water vapour adsorption (Fig. 2). For instance, if the non-modified sawdust (curve 1) for the first 1–2 days, i.e. a rather rapidly for such materials, adsorbed the vapour to the extent of 0.30–0.35 g per g of the carrier, highly hydrophobised sawdust (curve 2) for the same time adsorbed a threefold lower amount of water vapour, and slightly hydrophobised sawdust samples (curves 3–8) demonstrated intermediate adsorption rates. It is thought that these results could be associated with the discrete character of hydrophobic films formed on the slightly hydrophobised sawdust particles and also with the fact that the open pores remained in the treated material. Therefore, sawdust samples treated by limited amounts of the hydrophobising are characterized by a well-developed surface area, which, in principle, can be available for the adhesion of bacterial cells. Scanning electron microscopy data support this suggestion. Thus, Fig. 3 demonstrates the structure of organosilicon-treated sawdust as a typical representative of such slightly hydrophobised materials. Micrographs show the pores, which are similar to those in the non-treated sawdust (cf. Figs. 1a and 3a), while the hydrophobic coating (organosilicon-based polymer in this case) resembled a net-like film on the carrier surface, which is readily observed at higher magnification (Fig. 3b). Comparative studies of the immobilisation of Rhodococcus bacteria on various sawdust-based carriers demonstrated (Table 4) that cell adsorption capacity of slightly hydrophobised carriers was 1.2 and 3.0 times higher compared to that of the non-treated sawdust and of highly hydrophobised material, respectively. It is noteworthy that the cell surface of alkanotrophic rhodococci has a mosaic structure dominated by hydrophobic regions (Ivshina et al., 1997; Kuyukina et al., 2000). Hence, for the efficient adsorption of such cells on cellulose-containing carriers, a combination of hydrophilic and hydrophobic areas is required. To further optimise the techniques used for preparing cell immobilisation carriers, sawdust was subjected to additional alkaline treatment. The latter resulted in changes of hydration properties of the sawdust (No. 5 in Table 4). Decrease in the water binding capacity of sawdust after alkaline treatment was due to partial dissolution and extraction of lignin and hemicellulose that are readily hydrated substances. On the other hand, the observed increase in the adsorption of water vapour suggested loosening of the structure of sawdust material and increasing its specific surface area. Subsequent hydrophobisation of alkali-treated sawdust with small amounts of drying oil resulted in a marked decrease in both water absorption and water vapour adsorption, as well as an increase in adsorption capacity of the carrier for bacterial cells (No. 6 in Table 4). Apart from the polymer-forming hydrophobising agents applied from a liquid phase, we also used low molecular weight hydrocarbons such as n-hexadecane and paraffin
2006
E.A. Podorozhko et al. / Bioresource Technology 99 (2008) 2001–2008
Table 4 Physico-chemical properties of sawdust-based carriers and their use for the immobilisation of hydrocarbon-oxidizing bacteria No.
Modifying agent
Water vapour absorbing capacity, g of water/g of dry carrier
Water vapour absorbing capacity, g of water/g of dry carrier For 1 day
For 6 days
1 2 3 4 5 6
None Drying oil in an excess Drying oil in a shortage Organiosilicon emulsion NaOH solution NaOH solution + drying oil in a shortage n-Hexadecane vapours Paraffin vapours
2.55 ± 0.15 0.39 ± 0.02 1.24 ± 0.02 1.93 ± 0.02 1.66 ± 0.02 1.02 ± 0.02
0.28 ± 0.03 0.09 ± 0.01 0.22 ± 0.02 0.27 ± 0.02 0.27 ± 0.02 0.24 ± 0.02
1.68 ± 0.12 1.85 ± 0.02
0.29 ± 0.01 0.27 ± 0.02
7 8
Total bacterial adsorption, mg of dry cells/g of dry carrier
Average rate of n-hexadecane biodegradation, mg/l/h
0.34 ± 0.02 0.12 ± 0.02 0.31 ± 0.02 0.31 ± 0.02 0.38 ± 0.03 0.32 ± 0.02
39.0 ± 4.5 15.5 ± 1.5 46.5 ± 1.0 46.0 ± 3.0 18.0 ± 1.5 46.5 ± 1.0
71.0 ± 7.0 46.0 ± 6.0 104.0 ± 4.0 44.0 ± 3.5 26.0 ± 4.0 107.0 ± 6.0
0.31 ± 0.02 0.31 ± 0.02
41.0 ± 4.0 42.5 ± 4.0
42.5 ± 5.0 65.0 ± 2.5
0.45 0.4
g Water / g dry carrier
0.35 0.3 0.25 1
0.2
2 3
0.15
4 5
0.1
6 7
0.05
8
0 0
1
2
3
4
5
6
7
8
Time, days Fig. 2. Isotherms of water vapour adsorption at 100% humidity by the samples of sawdust-based carriers: 1 – initial non-modified sawdust; 2 – highly hydrophobised sawdust treated with an excess of sunflower seedbased drying oil followed by heating at 105 C for 2 h; 3 – slightly hydrophobised sawdust treated with the white spirit solution of drying oil followed by heating at 105 C for 1.5 h; 4 – slightly hydrophobised sawdust treated with the dilute water-organosilicon emulsion followed by air-drying; 5 – sawdust treated with the 1 N NaOH at 100 C for 2 h followed by rinsing with water and air-drying; 6 – slightly hydrophobised sawdust alkali-treated like the sample No.5 and then hydrophobised like the sample No. 3; 7 – slightly hydrophobised sawdust treated with the nhexadecane vapours; 8 – slightly hydrophobised sawdust treated with the paraffin vapours.
that were adsorbed by the sawdust from the gaseous phase (see Section 2.3). Implementing this simple technique, we expected to obtain an appropriate carrier for bacterial cells, in which the hydrophobising agent would be adsorbed uniformly as a monolayer in the available regions of sawdust, thus maximising the preservation of the porosity and surface area of the material. Analysing the data in Table 4 (Nos. 7 and 8), the hydration properties of these latter carriers seemed to be similar to the carriers treated by the
Fig. 3. Micrographs (SEM) at low (a) and high (b) magnifications of slightly hydrophobised sawdust treated with the dilute water-organosilicon emulsion followed by air-drying.
small amounts of hydrophobising agents. However, the cell adsorption capacity in the case of carriers being hydrophobised by gaseous n-hexadecane or paraffin was slightly lower than for the carriers treated by small amounts of drying oil or organosilicon emulsion.
E.A. Podorozhko et al. / Bioresource Technology 99 (2008) 2001–2008
3.3. Hydrocarbon-oxidizing activity of immobilised rhodococci cells Comparative studies revealed certain positive correlation between the amounts of rhodococci cells immobilised on sawdust-based carriers and the catalytic activity of these cells. Thus, maximum amount of biomass (46.5 g of dry cells per g of a carrier) was adsorbed on the sawdust treated with small amounts of drying oil, and this resulted in the immobilised systems (Nos. 3 and 6 in Table 4) exhibiting the highest rate of n-hexadecane biodegradation – around 100 mg/l/h. However, in spite of relatively high amount of rhodococcal cells immobilised on the sawdust treated with organosilicon emulsion, either by the n-hexadecane or paraffin vapours (46.0, 41.0 or 42.5 mg/l/h, respectively Nos. 4, 7 and 8 in Table 4), the hydrocarbon-oxidizing activities of such immobilised systems were not high enough, probably as a result of the inhibitory effect on the biodegradation process caused by the former hydrophobising agent itself (or, more exactly, traces of residual minor additives of organosilicon emultion), or because of another nature of hydrophobic coating on the carriers in the latter cases (hydrophobisation of sawdust by the competitive hydrocarbons that could be consumed by the cells). The efficiency of hydrocarbon oxidation (and, hence, the removal of n-hexadecane from the liquid medium) depends on both the oxidation activities of immobilised bacterial cells and hydrocarbon-adsorbing capabilities of a carrier. Highly hydrophobised sawdust, in particular, was able to accumulate over 70% of n-hexadecane from water medium due to physical adsorption by the carrier, while the same values for other carriers varied from 10% to 30% depending on the hydrophobicity degree of respective material (the data are not shown). Therefore, it may be supposed that highly hydrophobised sawdust can be used as an adsorbent, which can be added to water and soil highly contaminated with oil, whose bioremediation is carried out with the cells immobilised on the slightly hydrophobised sawdust-based carriers. 4. Conclusions The results obtained in this study show promising possibilities in developing a wide range of available and cheap, biodegradable cellulose-containing carriers that possess varying surface hydrophobicity. The latter parameter, depending on the microorganisms to be immobilised, could be easily adjusted till required level. Hydrophobising agents based on drying oil proved to be optimal (among the other modifiers examined) for the preparation of sawdust carriers for the efficient immobilisation of hydrocarbon-oxidizing bacterial cells (Pororozhko et al., 2005). Acknowledgements INTAS Grant # 01-2151 is acknowledged for the financial support.
2007
References Aislabie, J.M., McLeod, M., Fraser, R., 1998. Potential for biodegradation of hydrocarbons in soil from Ross dependency, Antarctica. Appl. Microbiol. Biotechnol. 49, 210–214. Aislabie, J.M., Fraser, R., Duncan, S., Farrell, R.L., 2001. Effects of oil spills on microbial heterotrophs in Antarctic soils. Polar Biol. 24, 308– 313. Aislabie, J.M., Balks, M.R., Foght, J.M., Waterhouse, E.J., 2004. Hydrocarbon spills on Antarctic soils: effects and management. Environ. Sci. Technol. 38, 1265–1274. Atlas, R.M., 1991. Microbial hydrocarbon degradation – bioremediation of oil spills. J. Chem. Technol. Biotechnol. 52, 149–156. Cassidy, M.B., Lee, H., Trevors, J.T., 1996. Environmental applications of immobilised microbial cells: a review. J. Ind. Microbiol. 16, 79–101. Cunningham, C.J., Ivshina, I.B., Lozinsky, V.I., Kuyukina, M.S., Philp, J.C., 2004. Bioremediation of diesel contaminated soil by microorganisms immobilised in a polyvinyl alcohol cryogel. Int. Biodeterior. Biodegrad. 54, 167–174. Gandini, A., Belgacem, M.N., 1996. In: Salamone, J.C. (Ed.), Polymeric Materials Encyclopaedia, V.11. CRC Press, Inc., Boca Raton e.a, pp. 8518–8530. IEGM, WFCC No. 768; Catalogue of Strains, 1994.
2008
E.A. Podorozhko et al. / Bioresource Technology 99 (2008) 2001–2008
A.G., . Wastewater decontamination from diesel oil using immobilised degradative microorganisms. Biotechnol. Russ. 4, 80–84. Sugawara, H., Miyazaki, S. (Eds.), 1999. World Directory of Collections of Cultures of Microorganisms. World Data Centre for Microorganisms, WFCC-MIRCEN, p. 140.
Vishniac, H.S., 1993. Microbiology of Antarctic soils. In: Friedmann, E.I. (Ed.), Antarctic Microbiology. Wiley, New York, pp. 297–341. Vogel, T.M., 1996. Bioaugmentation as a soil bioremediation approach. Curr. Opin. Biotechnol. 7, 311–316.