- Email: [email protected]
Dibenzothiophene desulfurization by Gordonia alkanivorans strain 1B using recycled paper sludge hydrolyzate
Available online at www.sciencedirect.com
Chemosphere 70 (2008) 967–973 www.elsevier.com/locate/chemosphere
Dibenzothiophene desulfurization by Gordonia alkanivorans strain 1B using recycled paper sludge hydrolyzate Luı´s Alves a, Susana Marques a, Jose´ Matos a, Roge´rio Tenreiro b, Francisco M. Gı´rio b
a,*
a INETI, Departamento de Biotecnologia, Estrada do Pac¸o do Lumiar, 22, 1649-038 Lisboa, Portugal Universidade de Lisboa, Faculdade de Cieˆncias, Centro de Gene´tica e Biologia Molecular and Instituto de Cieˆncia Aplicada e Tecnologia, Portugal
Received 18 June 2007; received in revised form 8 August 2007; accepted 9 August 2007 Available online 25 September 2007
Abstract Enzymatic hydrolyzates of recycled paper sludge were tested as suitable feedstock for biological desulfurization by Gordonia alkanivorans strain 1B. Only the hydrolyzate obtained after enzymatic mixture dialysis (dialyzed hydrolyzate) allowed dibenzothiophene (DBT) desulfurization, in spite of faster bacterial growth did occur on non-dialyzed hydrolyzate. For dialyzed hydrolyzate, 250 lM DBT was consumed after 96 h displaying a maximum specific productivity of 2-hydroxybiphenyl of 1.1 lmol g 1(dry cell weight) h 1. A comparison of the kinetics of biodesulfurization was assessed according to the type of hydrolyzate supplementation. Complete consumption of DBT was observed upon the addition of only phosphates and ammonia although further addition of zinc did increase the 2-hydroxybiphenyl production by 14%. Strain 1B was able to desulfurize a model oil containing DBT, 4-methylDBT and 4,6-dimethylDBT, reducing by 63% the total sulfur content in 168 h. 2007 Elsevier Ltd. All rights reserved. Keywords: Biodesulfurization; Dibenzothiophene; Gordonia alkanivorans; Biocatalyst; Industrial wastes; Recycled paper sludge
1. Introduction The rising consumption of fossil fuels around the world, due to the growing industrial activity, provokes an increase on waste products causing atmospheric pollution. These wastes products are particulates and various gases such as sulfur dioxide, nitrogen oxides and volatile organic compounds that are produced due to impurities in the fuels (Gupta et al., 2005). Thus, environmental authorities along the world have recognized this problem and are imposing increasingly stringent restrictions on the maximum sulfur concentration allowed in the fossil fuels. The process currently utilized in refineries to remove sulfur from these fuels is called hydrodesulfurization. However, heterocyclic sulfur compounds such as substituted dibenzothiophenes (DBT) are very difficult to desulfurize by hydrodesulfurization.
*
Corresponding author. Tel.: +351 21 0924721; fax: +351 21 7163636. E-mail address: [email protected] (F.M. Gı´rio).
0045-6535/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2007.08.016
Biological desulfurization of fossil fuels may offer an alternative process to remove the recalcitrant sulfur. One of the limiting factors to apply this process on an industrial scale is the cost associated to the production of biocatalysts, mainly due to the costs associated to the culture media formulation. At present, there is no economically suitable method for large-scale preparation of biocatalysts (Ma et al., 2006). The utilization of alternative carbon sources derived from agro-industrial by-products or wastes may thereby represent an opportunity to cheaper culture media. These alternative substrates have widely been used as feedstock for several fermentation processes such as for the production of lactic acid (Bustos et al., 2005), polyhydroxybutyrate (Hu et al., 1999), ethanol (Karimi et al., 2006), pullulan (Israilides et al., 1998), xanthan gum (Yoo and Harcum, 1999), bacterial cellulose (Bae and Shoda, 2005), and xylanase (Nascimento et al., 2003). However, studies involving the DBT bacterial desulfurization have been carried out only using reagent-grade sugar-containing media. The utilization of alternative carbon sources by
968
L. Alves et al. / Chemosphere 70 (2008) 967–973
desulfurizing bacteria might raise a problem associated to the presence of readily bioavailable sulfur compounds. Desulfurization of DBT is completely inhibited in the presence of several sulfur compounds in the culture medium. The dsz promoter has been characterized from Rhodococcus erythropolis strain IGTS8 and it was found that the dsz genes expression is strongly repressed by sulfate or other sulfur compounds even in the presence of DBT (Li et al., 1996). Therefore, it is important to search for low-cost feedstocks containing low residual concentration or even a null content of sulfur. Pulp and paper industry generates large amounts of waste throughout the year (Thomas, 2000). Concentrated sludge generated by the wastewater treatment facilities of recycled paper plants is currently a major disposal problem concerning the paper industry and it has to be urgently solved (Oral et al., 2005). Recycled paper sludge (RPS) (after neutralization) is approximately made up of 35% cellulose, 10% xylan and 20% lignin (on a dry-weight basis), being the remaining mainly inorganic ash. Due to this high polysaccharide content RPS appears as a promising feedstock for formulation of inexpensive culture media (Van Wyk and Mohulatsi, 2003) providing their polymeric carbohydrates are broken down into fermentable monosaccharides. This hydrolysis can be carried out by chemical or enzymatic methods. The latter is advantageous since it is more specific, it allows milder operation conditions leading to reduced production of biologically inhibitory compounds (such as sugar and lignin degradation products) and the biocatalysts are potentially reusable (Wen et al., 2004). In this context, the aim of the present work was to evaluate the performance of the hydrolyzates obtained by enzymatic saccharification of RPS, as nutrient source for low-cost DBT desulfurization by Gordonia alkanivorans strain 1B. To our knowledge, this is the first report on the utilization of alternative raw materials as substitutes for refined substrates (namely glucose) in biodesulfurization studies. 2. Materials and methods 2.1. Substrate 2.1.1. Recycled paper sludge The present study used pressed RPS consisting of the solids resulting from wastewater treatment facility of the paper-recycling mill of Renova (Torres Novas, PT). 2.1.2. Enzymatic hydrolysis After neutralization with hydrochloric acid (to reduce the carbonate content) RPS was suspended in 50 mM sodium citrate buffer, pH 5.5, for an initial consistency of 7.5% (w/v), expressed in terms of total carbohydrate mass, and it was steam sterilized by autoclaving (at 121 C, 101.3 kPa, for 15 min). This sludge suspension was incubated with the filter-sterilized enzyme solution containing
a mixture of two commercial enzyme preparations (cellulolytic and xylanolytic, from Novozymes, Denmark): Celluclast 1.5 L, applied on a dosage of filter paper activity (FPase) of 10 U g 1 carbohydrate; and Novozym 188, 0.4 ml g 1 carbohydrate on sludge. The hydrolysis was carried out at 35 C in an orbital shaker (150 rpm) for 120 h, under aseptic conditions. A control enzyme mixture was subjected to the same hydrolysis conditions but in the absence of sludge. In an alternative approach in order to remove any sulfur compounds present in the commercial enzymatic mixture, this mixture was dialyzed overnight (cut-off = 12–14 kDa; Spectra/Por membranes, Spectrum Laboratories, Inc., Rancho Dominguez, CA, USA) against milli-Q water at 4 C. The hydrolyzates obtained were filter sterilized and analyzed for sugar composition or used for supplementation of culture media. 2.2. Bacterial strain and growth conditions 2.2.1. General conditions G. alkanivorans strain 1B, originally isolated from oil-contaminated soil (Alves et al., 2005), was used in all cultivation assays. Unless otherwise stated, strain 1B was cultured in sulfur-free mineral (SFM) medium supplemented with a trace elements solution as described by Alves et al. (2007). DBT, 4-methyl DBT (4-mDBT), and/ or 4,6-dimethyl DBT (4,6-dmDBT), dissolved in dimethylformamide, were added as a source of sulfur. All G. alkanivorans liquid cultivations were carried out in duplicate shake-flasks, at 30 C and initial pH = 7.5, with 150 rpm (orbital shaking). Harvested samples were analyzed for cell growth, organic compounds involved in desulfurization and sugar contents. 2.2.2. DBT desulfurization on RPS hydrolyzate The RPS hydrolyzates obtained either with dialyzed enzymes (dialyzed hydrolyzate) or non-dialyzed enzymes (non-dialyzed hydrolyzate), were used as carbon source in a concentration of 10 g l 1 glucose in a medium containing 0.25 mM DBT. Growth controls were carried out using reagent-grade glucose, xylose or cellobiose on a concentration of 10 g l 1. 2.2.3. Effect of supplementation of RPS hydrolyzate In order to investigate the possibility of using the RPS hydrolyzate obtained with dialyzed enzymes as the component of culture medium (on the concentration previously described), cultivations were carried out on the following formulations: (1) RPS hydrolyzate; (2) RPS hydrolyzate + phosphates (KH2PO4 and Na2HPO4 Æ 2H2O); (3) RPS hydrolyzate + ammonia (NH4Cl); (4) RPS hydrolyzate + phosphates + ammonia; (5) RPS hydrolyzate + phosphates + magnesium (MgCl2 Æ 6H2O); (6) RPS hydrolyzate + phosphates + ammonia + magnesium; (7) RPS hydrolyzate + phosphates + ammonia + magnesium + Zinc
L. Alves et al. / Chemosphere 70 (2008) 967–973
(ZnCl2); (8) RPS hydrolyzate + phosphates + ammonia + magnesium + trace elements solution. Phosphates, ammonia and magnesium were added in the concentration present in SFM medium and Zn2+ was used on a final concentration of 10 mg l 1. DBT was used as a sulfur source, in a concentration of 0.25 mM. 2.2.4. Desulfurization of model oil The model oil used consisted of n-heptane with DBT, 4mDBT and 4,6-dmDBT dissolved on a concentration of 2 mM each. In this assay a water/oil ratio of 10:1 was used. The culture medium used was the formulation 7 described in Section 2.2.3.
969
Table 1 Major carbohydrate composition of both hydrolyzates obtained from recycled paper sludge (RPS) Concentration (g l 1)
RPS hydrolyzate Control Dialyzed RPS hydrolyzate Control
Hydrolysis yield (%)
Glucose
Xylose
Cellobiose
Total sugars
56.2
12.9
6.8
75.9
93
5.1 36.3
0.9 12.7
0.4 4.9
6.4 53.9
– 72
0.0
0.0
0.0
0.0
–
Control assays (with dialyzed or non-dialyzed enzyme mixtures) were performed in the absence of RPS.
2.3. Analytical methods Cell growth was directly monitored by measuring the optical density (OD) of the culture broth samples at 600 nm. Dry cell weight (DCW) was determined using 0.22 lm cellulose acetate membranes, after drying for 18 h at 100 C to constant weight. FPase, describing the cellulolytic activity, was assayed using Whatman number 1 filter paper as substrate. Enzyme activity was expressed in international units (U) as the amount of enzyme required to release 1 lmol min 1 of glucose reducing equivalent under the assay conditions. Reducing sugars were estimated by the dinitrosalicylic acid method (Miller, 1959). Sugars were measured by high-performance liquid chromatography using a Waters LC1 module 1 plus (Millford, LA) instrumentation equipped with a differential refractive index detector. An Aminex HPX-87H column (Bio-Rad Laboratories, Hercules, CA, USA) was used, operated at 50 C with 5 mM H2SO4 as a mobile phase at a flow rate of 0.4 ml min 1. The determination of the organic compounds involved in desulfurization was performed as described by Alves et al. (2007). In all GC measurements, anthracene was used as internal standard to minimize the variation. 3. Results and discussion 3.1. DBT desulfurization on RPS hydrolyzate Table 1 shows the sugar composition of both RPS hydrolyzates obtained. The sludge hydrolysis yield was calculated based on the total sugar concentration in the hydrolyzate (corrected for concentration in the control assay) relatively to the content of polysaccharides (potential glucose and xylose) of the feedstock. Since hydrolysis conditions were previously optimized a very high yield (93%) was obtained. A lower hydrolysis yield (72%) was obtained for dialyzed RPS hydrolyzate. This difference reflects the lower extension of hydrolysis of the cellulosic fraction resulting in a reduction on the efficiency of the cellulase formulation due to the dialysis process. The fact that both enzyme formulations (non-dialyzed and dialyzed)
were applied at the same dosage in terms of cellulase activity implies that the initial hydrolysis rate should be the same. However, it is not possible to assure that enzymatic activities have equivalent stabilities. Hence, this reduction on efficiency observed for dialyzed enzymes might be due to a reduction on enzymes stability, resulting in a loss of some small-molecule stabilizer components during dialysis. Despite the difference on sugar concentrations, both RPS hydrolyzates used in this work were composed of glucose, xylose and cellobiose (Table 1). Since strain 1B has no ability to use xylose and cellobiose (data not shown), the assays were performed using diluted RPS on an initial concentration of 10 g l 1 of glucose. Fig. 1. shows the time course of G. alkanivorans strain 1B cultivation using RPS hydrolyzates as carbon source. The bacterium attained maximum growth at 100–120 h of cultivation, and the turbidity at 600 nm at this time was around 10 for both cases (Fig. 1a and b). The maximum specific growth rates, lmax, for growth with non-dialyzed hydrolyzate (Fig. 1a) and dialyzed hydrolyzate (Fig. 1b) were 0.051 h 1 and 0.035 h 1, respectively. It is concluded that strain 1B grows better using hydrolyzates in comparison with the corresponding culture medium containing commercial grade glucose as the only carbon source (lmax = 0.019 h 1) (Alves et al., 2005). The RPS hydrolyzates not only contain the products of the RPS conversion but also all the components provided by the enzymatic formulation. Commercial enzyme preparations are obtained by growth of microbial organisms, followed by concentration of the culture cellfree broth, for obtaining an extract with a high enzymatic activity. Hence, not only protein concentration is achieved, but also most of all residual components present in culture media are also concentrated and these nutrients could be used by strain 1B, acting as growth promoters. This explains the faster growth achieved for the culture media containing RPS hydrolyzates. The slower growth of strain 1B with the dialyzed hydrolyzate is also justified by the removal of some of these nutrients present in the enzymatic formulation due to dialysis, attenuating this nutritional effect. Although this effect was already predicted, dialysis was performed aiming to remove possible sulfur compounds present on the enzymatic formulation that could
970
L. Alves et al. / Chemosphere 70 (2008) 967–973
ment with a previous study involving the same strain (Alves et al., 2005). The maximum specific productivity of 2-HBP was 1.1 lmol g 1 (DCW) h 1. 3.2. Effect of supplementation of RPS hydrolyzate In the previous assays, it was shown that dialyzed RPS hydrolyzate could be used as carbon source in a process of DBT desulfurization by G. alkanivorans strain 1B. Subsequently, and having in mind the already stated rich composition of the enzymatic formulation used, the possibility of using this hydrolyzate as a complete culture medium was investigated. In these assays, dialyzed RPS hydrolyzate was used in an initial glucose concentration of 10 g l 1, being supplemented using different nutrient formulations as stated in Section 2.2.3.
Fig. 1. Time course of G. alkanivorans strain 1B cultivation in SFM medium using as carbon source: Panel a, non-dialyzed hydrolyzate; Panel b, dialyzed hydrolyzate. d, strain 1B growth; j, 2-HBP concentration; m, glucose concentration.
be more easily assimilated than DBT by bacterial cells. The absence of DBT desulfurization in the non-dialyzed hydrolyzate (Fig. 1a) might be explained by the presence of other S-sources of faster assimilation than DBT. It has been reported (Takada et al., 2005) that the presence of other sulfur sources in the culture medium, even in small concentrations, inhibits the desulfurization of DBT. The dialysis of the enzyme mixture is therefore essential for the occurrence of DBT desulfurization, unless a recombinant strain (lacking dsz substrate repression) is used. As shown in Fig. 1b, the detection of 2-HBP only occurs after 50 h of cultivation with the dialyzed hydrolyzate, probably due to the presence in the culture medium of a residual concentration of sulfur contaminants that were not completely removed by dialysis. In this cultivation, 250 lM of DBT was consumed after 96 h of culture (data not shown), but less than 125 lM of 2-HBP was determined for this time, which means that only 50% of the 2HBP produced was detected, possibly due to the volatile characteristics of this compound. This result is in agree-
Fig. 2. Time course of growth (Panel a) and 2-HBP production (Panel b) for all the formulations tested in the supplementation assays using RPS hydrolyzate as culture medium: , formulation 1 (RPS hydrolyzate); m, formulation 2 (RPS hydrolyzate and phosphates); s, formulation 3 (RPS hydrolyzate and ammonia); j, formulation 4 (RPS hydrolyzate, phosphates and ammonia); h, formulation 5 (RPS hydrolyzate, phosphates and magnesium); +, formulation 6 (RPS hydrolyzate, phosphates, ammonia and magnesium); d, formulation 7 (RPS hydrolyzate, phosphates, ammonia, magnesium and zinc); ·, formulation 8 (RPS hydrolyzate, phosphates, ammonia, magnesium and trace elements solution).
L. Alves et al. / Chemosphere 70 (2008) 967–973
Fig. 2 shows the time course of growth and production of 2-HBP by strain 1B obtained for each medium formulation. RPS hydrolyzate without additional nutrients (formulation 1), with phosphates (formulation 2) or with phosphates + magnesium (formulation 5) did not allow a significant growth neither 2-HBP production. Although formulation 3 (RPS hydrolyzate with ammonia) has allowed a high initial bacterial growth, this ended after the first 72 h of culture, probably due to the absence of some nutrients (Fig. 2a). The same behavior occurred with 2-HBP production after 96 h of cultivation. A complete consumption of the glucose and DBT present in culture medium (data not shown) was observed when formulations 4, 6, 7 and 8 were used. This indicates that these formulations can be used as complete culture medium for DBT desulfurization by G. alkanivorans strain 1B. Growth occurred throughout 120 h and a lmax value of 0.031 h 1 was achieved for formulation 7 and 0.027 h 1 for formulations 4, 6 and 8. The desulfurization patterns, in terms of 2-HBP productivity, were also very similar for formulations 4, 6, 7 and 8. However, formulation 7 seems to be more favorable to a faster desulfurization since the 2-HBP maximum productivity obtained was 14% higher (5.7 lM h 1) comparatively to the value of about 5 lM h 1 obtained for formulations 4, 6 and 8 (Fig. 2b). Formulation 7 consists of the composition of SFM medium supplemented with zinc. In a previous work it was found that this metal ion has an important role for the DBT desulfurization ability of G. alkanivorans strain 1B (data not shown).
971
Fig. 3. Time course of G. alkanivorans strain 1B cultivation with model oil: d, strain 1B growth; j, total sulfur concentration; m, glucose concentration.
3.3. Desulfurization of model oil Desulfurization of DBT, 4-mDBT and 4,6-dmDBT in a model oil system (containing 2 mM of each sulfur source dissolved in n-heptane) was carried out with G. alkanivorans strain 1B used as culture medium dialyzed RPS hydrolyzate supplemented with phosphates, ammonia, magnesium and zinc, corresponding to formulation 7. The volume ratio of oil-to-water was 0.1. Strain 1B cannot use n-heptane as carbon source (data not shown). Fig. 3. shows the bacterial growth and the sulfur and glucose consumption for 192 h of cultivation. The results show that strain 1B consumed almost all the glucose present in the culture medium after 96 h of culture, with a lmax of 0.062 h 1 and total sulfur was decreased 2.7-fold, to 2.23 mM after 168 h of cultivation. The specific desulfurization rates after 24, 48 and 72 h were 22.2, 11.1 and 4.8 lmol g 1 (DCW) h 1, respectively. Two alkylated DBTs, which are recalcitrant to hydrodesulfurization technology, were used in this work. In fact, DBTs substituted in positions 4 and 6 are the most recalcitrant to hydrodesulfurization (Okada et al., 2002). In Fig. 4, the time course of each individual sulfur source degradation is shown. Strain 1B simultaneously utilized all sulfur sources present in model oil desulfurizing, although DBT is preferentially utilized relatively to the alkylated DBTs. On the contrary, Prince and Grossman
Fig. 4. Time course of sulfur sources degradation during G. alkanivorans strain 1B cultivation with model oil: j, 4-mDBT concentration; m, 4,6dmDBT concentration; , DBT concentration.
(2003) stated that these alkylated compounds were removed in preference relatively to their unalkylated parent. The specific desulfurization rates in the model oil for DBT, 4-mDBT and 4,6-dmDBT were 0.78, 0.68 and 0.41 lmol g 1 (DCW) h 1, respectively. 4-mDBT has also been removed more extensively than 4,6-dmDBT, in the work performed by Prince and Grossman (2003). Okada et al. (2002) have also verified that desulfurization activities against alkyl DBTs decreased with increasing molecular weight of DBT derivatives. Fig. 5. shows the chromatograms obtained by GC analysis for an initial (Fig. 5a) and a final (Fig. 5b) sample of a model oil desulfurization with G. alkanivorans strain 1B. The peak with a retention time of 19.1 min corresponds to the internal standard used (anthracene). The peaks at 18.1, 20.7 and 23.2 min correspond to DBT, 4-mDBT and 4,6-dmDBT, respectively. The peak with retention time of 11.8 min corresponds to 2-HBP resulting from DBT desul-
972
L. Alves et al. / Chemosphere 70 (2008) 967–973
of 4-mDBT by strain 1B occurs by a different mechanism than the one for 4,6-dmDBT, since 2 compounds were produced, in a proportion of 1:5, detected at retention times of 13.9 and 14.5 min. This could suggest the cleavage of some carbon–sulfur bonds of 4-mDBT by strain 1B. 4. Conclusions Altogether, these results clearly show that RPS hydrolyzates can be employed as nutrients for DBT desulfurization by G. alkanivorans strain 1B (even with a low enrichment of the medium), providing process efficiencies similar to those achieved when using conventional medium containing commercial grade sugars. This procedure has a double profit because a harmful environmental waste is removed and RPS can be used as an alternative carbon source for a biotechnological process, as already reported for other industrial applications such as lactic acid (Lin et al., 2005) and ethanol production (Lark et al., 1997). Realistically, for a potential industrial application, an inexpensive culture medium would have to be employed in a large-scale process. Therefore, the cost of commercial enzyme formulations might economically hamper the present process, unless the enzymes added in the hydrolysis step are recovered and reused. This might be achieved by developing a process based on an enzymatic membrane reactor, in which a semi-permeable ultrafiltration membrane is used to retain the enzymes in the reactor while preserving their activity. Acknowledgement This work has been partially supported by Fundac¸a˜o para a Cieˆncia e Tecnologia (Contract POCTI/AMB/ 59108/04). References
Fig. 5. GC chromatograms obtained for the initial (Panel a) and final (Panel b) samples taken from G. alkanivorans strain 1B cultivation with model oil.
furization. The desulfurization of 4,6-dmDBT produces only one compound, detected at retention time of 16.7 min. The desulfurization of 4,6-dmDBT by Nocardia globerula R-9 also produces only one product that was identified by HPLC/GC–MS as monohydroxy dimethyl biphenyl (Luo et al., 2003). A different result was described for 4,6-dmDBT desulfurization by Sphingomonas paucimobilis strain TZS-7, which produces 3 compounds, suggesting that this compound is degraded through a ring-destructive pathway (Lu et al., 1999). In G. alkanivorans strain 1B, the degradation of 4,6-dmDBT was assumed to occur through the 4S pathway, which involves the carbon–sulfur bond cleavage reaction and generates hydroxybiphenyl compounds derived from the substrate. The desulfurization
Alves, L., Melo, M., Mendonc¸a, D., Simo˜es, F., Matos, J., Tenreiro, R., Gı´rio, F.M., 2007. Sequencing, cloning and expression of the dsz genes required for dibenzothiophene sulfone desulfurization from Gordonia alkanivorans strain 1B. Enzyme Microb. Technol. 40, 1598–1603. Alves, L., Salgueiro, R., Rodrigues, C., Mesquita, E., Matos, J., Gı´rio, F.M., 2005. Desulfurization of dibenzothiophene, benzothiophene and other thiophene analogues by a newly isolated bacterium, Gordonia alkanivorans strain 1B. Appl. Biochem. Biotechnol. 120, 199–208. Bae, S.O., Shoda, M., 2005. Production of bacterial cellulose by Acetobacter xylinum BPR2001 using molasses medium in a jar fermentor. Appl. Microbiol. Biotechnol. 67, 45–51. Bustos, G., Moldes, A.B., Cruz, J.M., Domı´nguez, J.M., 2005. Production of lactic acid from vine-trimming wastes and viticulture lees using a simultaneous saccharification fermentation method. J. Sci. Food Agri. 85, 466–472. Gupta, N., Roychoudhury, P.K., Deb, J.K., 2005. Biotechnology of desulfurization of diesel: prospects and challenges. Appl. Microbiol. Biotechnol. 66, 356–366. Hu, P.H., Chua, H., Huang, A.L., Ho, K.P., 1999. Conversion of industrial food wastes by Alcaligenes latus into polyhydroxyalkanoates. Appl. Biochem. Biotechnol. 77, 445–454. Israilides, C.J., Smith, A., Harthill, J.E., Barnett, C., Bambalov, G., Scanlon, B., 1998. Pullulan content of the ethanol precipitate from
L. Alves et al. / Chemosphere 70 (2008) 967–973 fermented agro-industrial wastes. Appl. Microbiol. Biotechnol. 49, 613–617. Karimi, K., Emtiazi, G., Taherzadeh, M.J., 2006. Ethanol production from dilute-acid pretreated rice straw by simultaneous saccharification and fermentation with Mucor indicus, Rhizopus oryzae, and Saccharomyces cerevisiae. Enzyme Microb. Technol. 40, 138–144. Lark, N., Xia, Y.K., Qin, C.G., Gong, C.S., Tsao, G.T., 1997. Production of ethanol from recycled paper sludge using cellulase and yeast, Kluveromyces marxianus. Biomass Bioenerg. 12, 135–143. Li, M.Z., Squires, C.H., Monticello, D.J., Childs, J.D., 1996. Genetic analysis of the dsz promoter and associated regulatory regions of Rhodococcus erythropolis IGTS8. J. Bacteriol. 178, 6409–6418. Lin, J.Q., Lee, S.M., Koo, Y.M., 2005. Modeling and simulation of simultaneous saccharification and fermentation of paper mill sludge to lactic acid. J. Microbiol. Biotechnol. 15, 40–47. Lu, J., Nakajima-Kambe, T., Shigeno, T., Ohbo, A., Nomura, N., Nakahara, T., 1999. Biodegradation of dibenzothiophene and 4,6dimethyldibenzothiophene by Sphingomonas paucimobilis strain TZS7. J. Biosci. Bioeng. 88, 293–299. Luo, M.F., Gou, Z.X., Xing, J.M., Liu, H.H., Chen, J.Y., 2003. Microbial desulfurization of model and straight-run diesel oils. J. Chem. Technol. Biot. 78, 873–876. Ma, C.Q., Feng, J.H., Zeng, Y.Y., Cai, X.F., Sun, B.P., Zhang, Z.B., Blankespoor, H.D., Xu, P., 2006. Methods for the preparation of a biodesulfurization biocatalyst using Rhodococcus sp.. Chemosphere 65, 165–169. Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31, 426–428.
973
Nascimento, R.P., Coelho, R.R.R., Marques, S., Alves, L., Girio, F.M., bom, E.P.S., Amaral-Collaco, M.T., 2003. Production and partial characterisation of xylanase from Streptomyces sp. strain AMT-3 isolated from Brazilian cerrado soil. Enzyme Microb. Technol. 31, 549–555. Okada, H., Nomura, N., Nakahara, T., Maruhashi, K., 2002. Analyses of substrate specificity of the desulfurizing bacterium Mycobacterium sp. G3. J. Biosci. Bioeng. 93, 228–233. Oral, J., Sikula, J., Puchyr, R., Hajny, Z., Stehlik, P., Bebar, L., 2005. Processing of waste from pulp and paper plant. J. Clean. Prod. 13, 509–515. Prince, R.C., Grossman, M.J., 2003. Substrate preferences in biodesulfurization of diesel range fuels by Rhodococcus sp. strain ECRD-1. Appl. Environ. Microb. 69, 5833–5838. Takada, M., Nomura, N., Okada, H., Nakajima-Kambe, T., Nakahara, T., Uchiyama, H., 2005. De-repression and comparison of oil–water separation activity of the dibenzothiophene desulfurizing bacterium, Mycobacterium sp G3. Biotechnol. Lett. 27, 871–874. Thomas, S., 2000. Production of lactic acid from pulp mill solid waste and xylose using Lactobacillus delbrueckii (NRRL B445). Appl. Biochem. Biotechnol., 455–468. Van Wyk, J.P.H., Mohulatsi, M., 2003. Biodegradation of wastepaper by cellulase from Trichoderma viride. Bioresource Technol. 86, 21–23. Wen, Z.Y., Liao, W., Chen, S.L., 2004. Hydrolysis of animal manure lignocellulosics for reducing sugar production. Bioresource Technol. 91, 31–39. Yoo, S.D., Harcum, S.W., 1999. Xanthan gum production from waste sugar beet pulp. Bioresource Technol. 70, 105–109.
Chemosphere 70 (2008) 967–973 www.elsevier.com/locate/chemosphere
Dibenzothiophene desulfurization by Gordonia alkanivorans strain 1B using recycled paper sludge hydrolyzate Luı´s Alves a, Susana Marques a, Jose´ Matos a, Roge´rio Tenreiro b, Francisco M. Gı´rio b
a,*
a INETI, Departamento de Biotecnologia, Estrada do Pac¸o do Lumiar, 22, 1649-038 Lisboa, Portugal Universidade de Lisboa, Faculdade de Cieˆncias, Centro de Gene´tica e Biologia Molecular and Instituto de Cieˆncia Aplicada e Tecnologia, Portugal
Received 18 June 2007; received in revised form 8 August 2007; accepted 9 August 2007 Available online 25 September 2007
Abstract Enzymatic hydrolyzates of recycled paper sludge were tested as suitable feedstock for biological desulfurization by Gordonia alkanivorans strain 1B. Only the hydrolyzate obtained after enzymatic mixture dialysis (dialyzed hydrolyzate) allowed dibenzothiophene (DBT) desulfurization, in spite of faster bacterial growth did occur on non-dialyzed hydrolyzate. For dialyzed hydrolyzate, 250 lM DBT was consumed after 96 h displaying a maximum specific productivity of 2-hydroxybiphenyl of 1.1 lmol g 1(dry cell weight) h 1. A comparison of the kinetics of biodesulfurization was assessed according to the type of hydrolyzate supplementation. Complete consumption of DBT was observed upon the addition of only phosphates and ammonia although further addition of zinc did increase the 2-hydroxybiphenyl production by 14%. Strain 1B was able to desulfurize a model oil containing DBT, 4-methylDBT and 4,6-dimethylDBT, reducing by 63% the total sulfur content in 168 h. 2007 Elsevier Ltd. All rights reserved. Keywords: Biodesulfurization; Dibenzothiophene; Gordonia alkanivorans; Biocatalyst; Industrial wastes; Recycled paper sludge
1. Introduction The rising consumption of fossil fuels around the world, due to the growing industrial activity, provokes an increase on waste products causing atmospheric pollution. These wastes products are particulates and various gases such as sulfur dioxide, nitrogen oxides and volatile organic compounds that are produced due to impurities in the fuels (Gupta et al., 2005). Thus, environmental authorities along the world have recognized this problem and are imposing increasingly stringent restrictions on the maximum sulfur concentration allowed in the fossil fuels. The process currently utilized in refineries to remove sulfur from these fuels is called hydrodesulfurization. However, heterocyclic sulfur compounds such as substituted dibenzothiophenes (DBT) are very difficult to desulfurize by hydrodesulfurization.
*
Corresponding author. Tel.: +351 21 0924721; fax: +351 21 7163636. E-mail address: [email protected] (F.M. Gı´rio).
0045-6535/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2007.08.016
Biological desulfurization of fossil fuels may offer an alternative process to remove the recalcitrant sulfur. One of the limiting factors to apply this process on an industrial scale is the cost associated to the production of biocatalysts, mainly due to the costs associated to the culture media formulation. At present, there is no economically suitable method for large-scale preparation of biocatalysts (Ma et al., 2006). The utilization of alternative carbon sources derived from agro-industrial by-products or wastes may thereby represent an opportunity to cheaper culture media. These alternative substrates have widely been used as feedstock for several fermentation processes such as for the production of lactic acid (Bustos et al., 2005), polyhydroxybutyrate (Hu et al., 1999), ethanol (Karimi et al., 2006), pullulan (Israilides et al., 1998), xanthan gum (Yoo and Harcum, 1999), bacterial cellulose (Bae and Shoda, 2005), and xylanase (Nascimento et al., 2003). However, studies involving the DBT bacterial desulfurization have been carried out only using reagent-grade sugar-containing media. The utilization of alternative carbon sources by
968
L. Alves et al. / Chemosphere 70 (2008) 967–973
desulfurizing bacteria might raise a problem associated to the presence of readily bioavailable sulfur compounds. Desulfurization of DBT is completely inhibited in the presence of several sulfur compounds in the culture medium. The dsz promoter has been characterized from Rhodococcus erythropolis strain IGTS8 and it was found that the dsz genes expression is strongly repressed by sulfate or other sulfur compounds even in the presence of DBT (Li et al., 1996). Therefore, it is important to search for low-cost feedstocks containing low residual concentration or even a null content of sulfur. Pulp and paper industry generates large amounts of waste throughout the year (Thomas, 2000). Concentrated sludge generated by the wastewater treatment facilities of recycled paper plants is currently a major disposal problem concerning the paper industry and it has to be urgently solved (Oral et al., 2005). Recycled paper sludge (RPS) (after neutralization) is approximately made up of 35% cellulose, 10% xylan and 20% lignin (on a dry-weight basis), being the remaining mainly inorganic ash. Due to this high polysaccharide content RPS appears as a promising feedstock for formulation of inexpensive culture media (Van Wyk and Mohulatsi, 2003) providing their polymeric carbohydrates are broken down into fermentable monosaccharides. This hydrolysis can be carried out by chemical or enzymatic methods. The latter is advantageous since it is more specific, it allows milder operation conditions leading to reduced production of biologically inhibitory compounds (such as sugar and lignin degradation products) and the biocatalysts are potentially reusable (Wen et al., 2004). In this context, the aim of the present work was to evaluate the performance of the hydrolyzates obtained by enzymatic saccharification of RPS, as nutrient source for low-cost DBT desulfurization by Gordonia alkanivorans strain 1B. To our knowledge, this is the first report on the utilization of alternative raw materials as substitutes for refined substrates (namely glucose) in biodesulfurization studies. 2. Materials and methods 2.1. Substrate 2.1.1. Recycled paper sludge The present study used pressed RPS consisting of the solids resulting from wastewater treatment facility of the paper-recycling mill of Renova (Torres Novas, PT). 2.1.2. Enzymatic hydrolysis After neutralization with hydrochloric acid (to reduce the carbonate content) RPS was suspended in 50 mM sodium citrate buffer, pH 5.5, for an initial consistency of 7.5% (w/v), expressed in terms of total carbohydrate mass, and it was steam sterilized by autoclaving (at 121 C, 101.3 kPa, for 15 min). This sludge suspension was incubated with the filter-sterilized enzyme solution containing
a mixture of two commercial enzyme preparations (cellulolytic and xylanolytic, from Novozymes, Denmark): Celluclast 1.5 L, applied on a dosage of filter paper activity (FPase) of 10 U g 1 carbohydrate; and Novozym 188, 0.4 ml g 1 carbohydrate on sludge. The hydrolysis was carried out at 35 C in an orbital shaker (150 rpm) for 120 h, under aseptic conditions. A control enzyme mixture was subjected to the same hydrolysis conditions but in the absence of sludge. In an alternative approach in order to remove any sulfur compounds present in the commercial enzymatic mixture, this mixture was dialyzed overnight (cut-off = 12–14 kDa; Spectra/Por membranes, Spectrum Laboratories, Inc., Rancho Dominguez, CA, USA) against milli-Q water at 4 C. The hydrolyzates obtained were filter sterilized and analyzed for sugar composition or used for supplementation of culture media. 2.2. Bacterial strain and growth conditions 2.2.1. General conditions G. alkanivorans strain 1B, originally isolated from oil-contaminated soil (Alves et al., 2005), was used in all cultivation assays. Unless otherwise stated, strain 1B was cultured in sulfur-free mineral (SFM) medium supplemented with a trace elements solution as described by Alves et al. (2007). DBT, 4-methyl DBT (4-mDBT), and/ or 4,6-dimethyl DBT (4,6-dmDBT), dissolved in dimethylformamide, were added as a source of sulfur. All G. alkanivorans liquid cultivations were carried out in duplicate shake-flasks, at 30 C and initial pH = 7.5, with 150 rpm (orbital shaking). Harvested samples were analyzed for cell growth, organic compounds involved in desulfurization and sugar contents. 2.2.2. DBT desulfurization on RPS hydrolyzate The RPS hydrolyzates obtained either with dialyzed enzymes (dialyzed hydrolyzate) or non-dialyzed enzymes (non-dialyzed hydrolyzate), were used as carbon source in a concentration of 10 g l 1 glucose in a medium containing 0.25 mM DBT. Growth controls were carried out using reagent-grade glucose, xylose or cellobiose on a concentration of 10 g l 1. 2.2.3. Effect of supplementation of RPS hydrolyzate In order to investigate the possibility of using the RPS hydrolyzate obtained with dialyzed enzymes as the component of culture medium (on the concentration previously described), cultivations were carried out on the following formulations: (1) RPS hydrolyzate; (2) RPS hydrolyzate + phosphates (KH2PO4 and Na2HPO4 Æ 2H2O); (3) RPS hydrolyzate + ammonia (NH4Cl); (4) RPS hydrolyzate + phosphates + ammonia; (5) RPS hydrolyzate + phosphates + magnesium (MgCl2 Æ 6H2O); (6) RPS hydrolyzate + phosphates + ammonia + magnesium; (7) RPS hydrolyzate + phosphates + ammonia + magnesium + Zinc
L. Alves et al. / Chemosphere 70 (2008) 967–973
(ZnCl2); (8) RPS hydrolyzate + phosphates + ammonia + magnesium + trace elements solution. Phosphates, ammonia and magnesium were added in the concentration present in SFM medium and Zn2+ was used on a final concentration of 10 mg l 1. DBT was used as a sulfur source, in a concentration of 0.25 mM. 2.2.4. Desulfurization of model oil The model oil used consisted of n-heptane with DBT, 4mDBT and 4,6-dmDBT dissolved on a concentration of 2 mM each. In this assay a water/oil ratio of 10:1 was used. The culture medium used was the formulation 7 described in Section 2.2.3.
969
Table 1 Major carbohydrate composition of both hydrolyzates obtained from recycled paper sludge (RPS) Concentration (g l 1)
RPS hydrolyzate Control Dialyzed RPS hydrolyzate Control
Hydrolysis yield (%)
Glucose
Xylose
Cellobiose
Total sugars
56.2
12.9
6.8
75.9
93
5.1 36.3
0.9 12.7
0.4 4.9
6.4 53.9
– 72
0.0
0.0
0.0
0.0
–
Control assays (with dialyzed or non-dialyzed enzyme mixtures) were performed in the absence of RPS.
2.3. Analytical methods Cell growth was directly monitored by measuring the optical density (OD) of the culture broth samples at 600 nm. Dry cell weight (DCW) was determined using 0.22 lm cellulose acetate membranes, after drying for 18 h at 100 C to constant weight. FPase, describing the cellulolytic activity, was assayed using Whatman number 1 filter paper as substrate. Enzyme activity was expressed in international units (U) as the amount of enzyme required to release 1 lmol min 1 of glucose reducing equivalent under the assay conditions. Reducing sugars were estimated by the dinitrosalicylic acid method (Miller, 1959). Sugars were measured by high-performance liquid chromatography using a Waters LC1 module 1 plus (Millford, LA) instrumentation equipped with a differential refractive index detector. An Aminex HPX-87H column (Bio-Rad Laboratories, Hercules, CA, USA) was used, operated at 50 C with 5 mM H2SO4 as a mobile phase at a flow rate of 0.4 ml min 1. The determination of the organic compounds involved in desulfurization was performed as described by Alves et al. (2007). In all GC measurements, anthracene was used as internal standard to minimize the variation. 3. Results and discussion 3.1. DBT desulfurization on RPS hydrolyzate Table 1 shows the sugar composition of both RPS hydrolyzates obtained. The sludge hydrolysis yield was calculated based on the total sugar concentration in the hydrolyzate (corrected for concentration in the control assay) relatively to the content of polysaccharides (potential glucose and xylose) of the feedstock. Since hydrolysis conditions were previously optimized a very high yield (93%) was obtained. A lower hydrolysis yield (72%) was obtained for dialyzed RPS hydrolyzate. This difference reflects the lower extension of hydrolysis of the cellulosic fraction resulting in a reduction on the efficiency of the cellulase formulation due to the dialysis process. The fact that both enzyme formulations (non-dialyzed and dialyzed)
were applied at the same dosage in terms of cellulase activity implies that the initial hydrolysis rate should be the same. However, it is not possible to assure that enzymatic activities have equivalent stabilities. Hence, this reduction on efficiency observed for dialyzed enzymes might be due to a reduction on enzymes stability, resulting in a loss of some small-molecule stabilizer components during dialysis. Despite the difference on sugar concentrations, both RPS hydrolyzates used in this work were composed of glucose, xylose and cellobiose (Table 1). Since strain 1B has no ability to use xylose and cellobiose (data not shown), the assays were performed using diluted RPS on an initial concentration of 10 g l 1 of glucose. Fig. 1. shows the time course of G. alkanivorans strain 1B cultivation using RPS hydrolyzates as carbon source. The bacterium attained maximum growth at 100–120 h of cultivation, and the turbidity at 600 nm at this time was around 10 for both cases (Fig. 1a and b). The maximum specific growth rates, lmax, for growth with non-dialyzed hydrolyzate (Fig. 1a) and dialyzed hydrolyzate (Fig. 1b) were 0.051 h 1 and 0.035 h 1, respectively. It is concluded that strain 1B grows better using hydrolyzates in comparison with the corresponding culture medium containing commercial grade glucose as the only carbon source (lmax = 0.019 h 1) (Alves et al., 2005). The RPS hydrolyzates not only contain the products of the RPS conversion but also all the components provided by the enzymatic formulation. Commercial enzyme preparations are obtained by growth of microbial organisms, followed by concentration of the culture cellfree broth, for obtaining an extract with a high enzymatic activity. Hence, not only protein concentration is achieved, but also most of all residual components present in culture media are also concentrated and these nutrients could be used by strain 1B, acting as growth promoters. This explains the faster growth achieved for the culture media containing RPS hydrolyzates. The slower growth of strain 1B with the dialyzed hydrolyzate is also justified by the removal of some of these nutrients present in the enzymatic formulation due to dialysis, attenuating this nutritional effect. Although this effect was already predicted, dialysis was performed aiming to remove possible sulfur compounds present on the enzymatic formulation that could
970
L. Alves et al. / Chemosphere 70 (2008) 967–973
ment with a previous study involving the same strain (Alves et al., 2005). The maximum specific productivity of 2-HBP was 1.1 lmol g 1 (DCW) h 1. 3.2. Effect of supplementation of RPS hydrolyzate In the previous assays, it was shown that dialyzed RPS hydrolyzate could be used as carbon source in a process of DBT desulfurization by G. alkanivorans strain 1B. Subsequently, and having in mind the already stated rich composition of the enzymatic formulation used, the possibility of using this hydrolyzate as a complete culture medium was investigated. In these assays, dialyzed RPS hydrolyzate was used in an initial glucose concentration of 10 g l 1, being supplemented using different nutrient formulations as stated in Section 2.2.3.
Fig. 1. Time course of G. alkanivorans strain 1B cultivation in SFM medium using as carbon source: Panel a, non-dialyzed hydrolyzate; Panel b, dialyzed hydrolyzate. d, strain 1B growth; j, 2-HBP concentration; m, glucose concentration.
be more easily assimilated than DBT by bacterial cells. The absence of DBT desulfurization in the non-dialyzed hydrolyzate (Fig. 1a) might be explained by the presence of other S-sources of faster assimilation than DBT. It has been reported (Takada et al., 2005) that the presence of other sulfur sources in the culture medium, even in small concentrations, inhibits the desulfurization of DBT. The dialysis of the enzyme mixture is therefore essential for the occurrence of DBT desulfurization, unless a recombinant strain (lacking dsz substrate repression) is used. As shown in Fig. 1b, the detection of 2-HBP only occurs after 50 h of cultivation with the dialyzed hydrolyzate, probably due to the presence in the culture medium of a residual concentration of sulfur contaminants that were not completely removed by dialysis. In this cultivation, 250 lM of DBT was consumed after 96 h of culture (data not shown), but less than 125 lM of 2-HBP was determined for this time, which means that only 50% of the 2HBP produced was detected, possibly due to the volatile characteristics of this compound. This result is in agree-
Fig. 2. Time course of growth (Panel a) and 2-HBP production (Panel b) for all the formulations tested in the supplementation assays using RPS hydrolyzate as culture medium: , formulation 1 (RPS hydrolyzate); m, formulation 2 (RPS hydrolyzate and phosphates); s, formulation 3 (RPS hydrolyzate and ammonia); j, formulation 4 (RPS hydrolyzate, phosphates and ammonia); h, formulation 5 (RPS hydrolyzate, phosphates and magnesium); +, formulation 6 (RPS hydrolyzate, phosphates, ammonia and magnesium); d, formulation 7 (RPS hydrolyzate, phosphates, ammonia, magnesium and zinc); ·, formulation 8 (RPS hydrolyzate, phosphates, ammonia, magnesium and trace elements solution).
L. Alves et al. / Chemosphere 70 (2008) 967–973
Fig. 2 shows the time course of growth and production of 2-HBP by strain 1B obtained for each medium formulation. RPS hydrolyzate without additional nutrients (formulation 1), with phosphates (formulation 2) or with phosphates + magnesium (formulation 5) did not allow a significant growth neither 2-HBP production. Although formulation 3 (RPS hydrolyzate with ammonia) has allowed a high initial bacterial growth, this ended after the first 72 h of culture, probably due to the absence of some nutrients (Fig. 2a). The same behavior occurred with 2-HBP production after 96 h of cultivation. A complete consumption of the glucose and DBT present in culture medium (data not shown) was observed when formulations 4, 6, 7 and 8 were used. This indicates that these formulations can be used as complete culture medium for DBT desulfurization by G. alkanivorans strain 1B. Growth occurred throughout 120 h and a lmax value of 0.031 h 1 was achieved for formulation 7 and 0.027 h 1 for formulations 4, 6 and 8. The desulfurization patterns, in terms of 2-HBP productivity, were also very similar for formulations 4, 6, 7 and 8. However, formulation 7 seems to be more favorable to a faster desulfurization since the 2-HBP maximum productivity obtained was 14% higher (5.7 lM h 1) comparatively to the value of about 5 lM h 1 obtained for formulations 4, 6 and 8 (Fig. 2b). Formulation 7 consists of the composition of SFM medium supplemented with zinc. In a previous work it was found that this metal ion has an important role for the DBT desulfurization ability of G. alkanivorans strain 1B (data not shown).
971
Fig. 3. Time course of G. alkanivorans strain 1B cultivation with model oil: d, strain 1B growth; j, total sulfur concentration; m, glucose concentration.
3.3. Desulfurization of model oil Desulfurization of DBT, 4-mDBT and 4,6-dmDBT in a model oil system (containing 2 mM of each sulfur source dissolved in n-heptane) was carried out with G. alkanivorans strain 1B used as culture medium dialyzed RPS hydrolyzate supplemented with phosphates, ammonia, magnesium and zinc, corresponding to formulation 7. The volume ratio of oil-to-water was 0.1. Strain 1B cannot use n-heptane as carbon source (data not shown). Fig. 3. shows the bacterial growth and the sulfur and glucose consumption for 192 h of cultivation. The results show that strain 1B consumed almost all the glucose present in the culture medium after 96 h of culture, with a lmax of 0.062 h 1 and total sulfur was decreased 2.7-fold, to 2.23 mM after 168 h of cultivation. The specific desulfurization rates after 24, 48 and 72 h were 22.2, 11.1 and 4.8 lmol g 1 (DCW) h 1, respectively. Two alkylated DBTs, which are recalcitrant to hydrodesulfurization technology, were used in this work. In fact, DBTs substituted in positions 4 and 6 are the most recalcitrant to hydrodesulfurization (Okada et al., 2002). In Fig. 4, the time course of each individual sulfur source degradation is shown. Strain 1B simultaneously utilized all sulfur sources present in model oil desulfurizing, although DBT is preferentially utilized relatively to the alkylated DBTs. On the contrary, Prince and Grossman
Fig. 4. Time course of sulfur sources degradation during G. alkanivorans strain 1B cultivation with model oil: j, 4-mDBT concentration; m, 4,6dmDBT concentration; , DBT concentration.
(2003) stated that these alkylated compounds were removed in preference relatively to their unalkylated parent. The specific desulfurization rates in the model oil for DBT, 4-mDBT and 4,6-dmDBT were 0.78, 0.68 and 0.41 lmol g 1 (DCW) h 1, respectively. 4-mDBT has also been removed more extensively than 4,6-dmDBT, in the work performed by Prince and Grossman (2003). Okada et al. (2002) have also verified that desulfurization activities against alkyl DBTs decreased with increasing molecular weight of DBT derivatives. Fig. 5. shows the chromatograms obtained by GC analysis for an initial (Fig. 5a) and a final (Fig. 5b) sample of a model oil desulfurization with G. alkanivorans strain 1B. The peak with a retention time of 19.1 min corresponds to the internal standard used (anthracene). The peaks at 18.1, 20.7 and 23.2 min correspond to DBT, 4-mDBT and 4,6-dmDBT, respectively. The peak with retention time of 11.8 min corresponds to 2-HBP resulting from DBT desul-
972
L. Alves et al. / Chemosphere 70 (2008) 967–973
of 4-mDBT by strain 1B occurs by a different mechanism than the one for 4,6-dmDBT, since 2 compounds were produced, in a proportion of 1:5, detected at retention times of 13.9 and 14.5 min. This could suggest the cleavage of some carbon–sulfur bonds of 4-mDBT by strain 1B. 4. Conclusions Altogether, these results clearly show that RPS hydrolyzates can be employed as nutrients for DBT desulfurization by G. alkanivorans strain 1B (even with a low enrichment of the medium), providing process efficiencies similar to those achieved when using conventional medium containing commercial grade sugars. This procedure has a double profit because a harmful environmental waste is removed and RPS can be used as an alternative carbon source for a biotechnological process, as already reported for other industrial applications such as lactic acid (Lin et al., 2005) and ethanol production (Lark et al., 1997). Realistically, for a potential industrial application, an inexpensive culture medium would have to be employed in a large-scale process. Therefore, the cost of commercial enzyme formulations might economically hamper the present process, unless the enzymes added in the hydrolysis step are recovered and reused. This might be achieved by developing a process based on an enzymatic membrane reactor, in which a semi-permeable ultrafiltration membrane is used to retain the enzymes in the reactor while preserving their activity. Acknowledgement This work has been partially supported by Fundac¸a˜o para a Cieˆncia e Tecnologia (Contract POCTI/AMB/ 59108/04). References
Fig. 5. GC chromatograms obtained for the initial (Panel a) and final (Panel b) samples taken from G. alkanivorans strain 1B cultivation with model oil.
furization. The desulfurization of 4,6-dmDBT produces only one compound, detected at retention time of 16.7 min. The desulfurization of 4,6-dmDBT by Nocardia globerula R-9 also produces only one product that was identified by HPLC/GC–MS as monohydroxy dimethyl biphenyl (Luo et al., 2003). A different result was described for 4,6-dmDBT desulfurization by Sphingomonas paucimobilis strain TZS-7, which produces 3 compounds, suggesting that this compound is degraded through a ring-destructive pathway (Lu et al., 1999). In G. alkanivorans strain 1B, the degradation of 4,6-dmDBT was assumed to occur through the 4S pathway, which involves the carbon–sulfur bond cleavage reaction and generates hydroxybiphenyl compounds derived from the substrate. The desulfurization
Alves, L., Melo, M., Mendonc¸a, D., Simo˜es, F., Matos, J., Tenreiro, R., Gı´rio, F.M., 2007. Sequencing, cloning and expression of the dsz genes required for dibenzothiophene sulfone desulfurization from Gordonia alkanivorans strain 1B. Enzyme Microb. Technol. 40, 1598–1603. Alves, L., Salgueiro, R., Rodrigues, C., Mesquita, E., Matos, J., Gı´rio, F.M., 2005. Desulfurization of dibenzothiophene, benzothiophene and other thiophene analogues by a newly isolated bacterium, Gordonia alkanivorans strain 1B. Appl. Biochem. Biotechnol. 120, 199–208. Bae, S.O., Shoda, M., 2005. Production of bacterial cellulose by Acetobacter xylinum BPR2001 using molasses medium in a jar fermentor. Appl. Microbiol. Biotechnol. 67, 45–51. Bustos, G., Moldes, A.B., Cruz, J.M., Domı´nguez, J.M., 2005. Production of lactic acid from vine-trimming wastes and viticulture lees using a simultaneous saccharification fermentation method. J. Sci. Food Agri. 85, 466–472. Gupta, N., Roychoudhury, P.K., Deb, J.K., 2005. Biotechnology of desulfurization of diesel: prospects and challenges. Appl. Microbiol. Biotechnol. 66, 356–366. Hu, P.H., Chua, H., Huang, A.L., Ho, K.P., 1999. Conversion of industrial food wastes by Alcaligenes latus into polyhydroxyalkanoates. Appl. Biochem. Biotechnol. 77, 445–454. Israilides, C.J., Smith, A., Harthill, J.E., Barnett, C., Bambalov, G., Scanlon, B., 1998. Pullulan content of the ethanol precipitate from
L. Alves et al. / Chemosphere 70 (2008) 967–973 fermented agro-industrial wastes. Appl. Microbiol. Biotechnol. 49, 613–617. Karimi, K., Emtiazi, G., Taherzadeh, M.J., 2006. Ethanol production from dilute-acid pretreated rice straw by simultaneous saccharification and fermentation with Mucor indicus, Rhizopus oryzae, and Saccharomyces cerevisiae. Enzyme Microb. Technol. 40, 138–144. Lark, N., Xia, Y.K., Qin, C.G., Gong, C.S., Tsao, G.T., 1997. Production of ethanol from recycled paper sludge using cellulase and yeast, Kluveromyces marxianus. Biomass Bioenerg. 12, 135–143. Li, M.Z., Squires, C.H., Monticello, D.J., Childs, J.D., 1996. Genetic analysis of the dsz promoter and associated regulatory regions of Rhodococcus erythropolis IGTS8. J. Bacteriol. 178, 6409–6418. Lin, J.Q., Lee, S.M., Koo, Y.M., 2005. Modeling and simulation of simultaneous saccharification and fermentation of paper mill sludge to lactic acid. J. Microbiol. Biotechnol. 15, 40–47. Lu, J., Nakajima-Kambe, T., Shigeno, T., Ohbo, A., Nomura, N., Nakahara, T., 1999. Biodegradation of dibenzothiophene and 4,6dimethyldibenzothiophene by Sphingomonas paucimobilis strain TZS7. J. Biosci. Bioeng. 88, 293–299. Luo, M.F., Gou, Z.X., Xing, J.M., Liu, H.H., Chen, J.Y., 2003. Microbial desulfurization of model and straight-run diesel oils. J. Chem. Technol. Biot. 78, 873–876. Ma, C.Q., Feng, J.H., Zeng, Y.Y., Cai, X.F., Sun, B.P., Zhang, Z.B., Blankespoor, H.D., Xu, P., 2006. Methods for the preparation of a biodesulfurization biocatalyst using Rhodococcus sp.. Chemosphere 65, 165–169. Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31, 426–428.
973
Nascimento, R.P., Coelho, R.R.R., Marques, S., Alves, L., Girio, F.M., bom, E.P.S., Amaral-Collaco, M.T., 2003. Production and partial characterisation of xylanase from Streptomyces sp. strain AMT-3 isolated from Brazilian cerrado soil. Enzyme Microb. Technol. 31, 549–555. Okada, H., Nomura, N., Nakahara, T., Maruhashi, K., 2002. Analyses of substrate specificity of the desulfurizing bacterium Mycobacterium sp. G3. J. Biosci. Bioeng. 93, 228–233. Oral, J., Sikula, J., Puchyr, R., Hajny, Z., Stehlik, P., Bebar, L., 2005. Processing of waste from pulp and paper plant. J. Clean. Prod. 13, 509–515. Prince, R.C., Grossman, M.J., 2003. Substrate preferences in biodesulfurization of diesel range fuels by Rhodococcus sp. strain ECRD-1. Appl. Environ. Microb. 69, 5833–5838. Takada, M., Nomura, N., Okada, H., Nakajima-Kambe, T., Nakahara, T., Uchiyama, H., 2005. De-repression and comparison of oil–water separation activity of the dibenzothiophene desulfurizing bacterium, Mycobacterium sp G3. Biotechnol. Lett. 27, 871–874. Thomas, S., 2000. Production of lactic acid from pulp mill solid waste and xylose using Lactobacillus delbrueckii (NRRL B445). Appl. Biochem. Biotechnol., 455–468. Van Wyk, J.P.H., Mohulatsi, M., 2003. Biodegradation of wastepaper by cellulase from Trichoderma viride. Bioresource Technol. 86, 21–23. Wen, Z.Y., Liao, W., Chen, S.L., 2004. Hydrolysis of animal manure lignocellulosics for reducing sugar production. Bioresource Technol. 91, 31–39. Yoo, S.D., Harcum, S.W., 1999. Xanthan gum production from waste sugar beet pulp. Bioresource Technol. 70, 105–109.