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International Biodeterioration & Biodegradation 57 (2006) 190–194

Accelerated biodegradation of n-alkanes in aqueous solution by the addition of fermented whey
¨ Tomas L. Ostberga,Ã, Anders P. Jonssonb, Ulla S. Lundstromc ¨

¨ Department of Natural Science, Mid Sweden University, SE-83125 Ostersund, Sweden ¨ Department of Engineering, Physics and Mathematics, Mid Sweden University, SE-83125 Ostersund, Sweden c Department of Natural Science, Mid Sweden University, SE-83170 Sundsvall, Sweden

Received 9 December 2005; accepted 31 January 2006 Available online 20 March 2006

Abstract The effect of fermented whey on the aerobic degradation of n-alkanes by a microbial consortium was investigated in an aqueous system. Microbial degradation of 100 mg n-alkanes lÀ1 (C12, C14, C16 and C18) in mineral nutrient medium was assessed by measuring the decrease in n-alkanes, production of CO2 and increase in biomass. The addition of fermented whey at a concentration of 5 mg dry weight lÀ1 to a nutrient medium receiving a small-sized inoculum (103.4 CFU mlÀ1) shortened the lag phase from 8 to 3 days, but the degradation rate during the degradation phase was not enhanced. The shortened lag phase at low initial concentration of microorganisms indicates that the fermented whey stimulates growth in the initial phase, without reducing the consortium’s capacity for n-alkane degradation. r 2006 Elsevier Ltd. All rights reserved.
Keywords: Fermented whey; Degradation; n-Alkanes; Petroleum hydrocarbons; Mineralization

1. Introduction Milk whey is a by-product of the dairy industry, and causes great disposal problems owing to the large volumes produced, and thus a large biological oxygen demand in sewage treatment plants. Sustainable resource utilization calls for new solutions and new applications for large-scale waste products. By fermenting whey with Lactobacillus, the main part of the lactose is metabolized to lactic acid and proteins are hydrolyzed to free amino acids. Fermented whey is a potential source of easily accessible carbon and micronutrients, which could be used to enhance the microbial degradation of pollutants. Addition of organic materials or individual chemicals to natural environments can stimulate degradation. When an added chemical is structurally analogous to the contaminant, it can stimulate the growth of degradative microorganisms that produce enzymes capable of transforming the analogous molecules, thus enhancing the degradative
ÃCorresponding author. Tel.: +46 63 165300; fax: +46 63 165500.

¨ E-mail address: (T.L. Ostberg). 0964-8305/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2006.01.006

capacity (Alexander, 1999). However, when a stimulatory organic amendment is not structurally analogous to the compound being metabolized, the benefit is nonspecific, for example, in increasing the biomass of organisms that only coincidentally carry out a co-metabolic reaction (Alexander, 1999). This increase in biomass can be an effect of adding an easily accessible carbon source and/or other nutrients. There is little information in the literature concerning the stimulatory effect of whey on microbial degradation of pollutants. Whey has been used as an oxygen consumer in order to create a reductive barrier for the in situ remediation of methyl tert-butyl ether contaminated soil (Barcelona and Xie, 2001) or as a carbon-source/electrondonor in a selenium bioremediation reactor system (Bledsoe et al., 1999). Other industrial by-products containing easily accessible carbon and micronutrients are yeast extract, a by-product of brewery industries, and molasses, a by-product of the sugar industry, both of these having been used as growth supplements in a number of biodegradation studies.

1997. Molasses has been shown to serve as an effective cosubstrate for aerobic degradation of the explosive 2.3 mg FeCl3 (all synthesis grade) lÀ1 carbon dioxide-free water was adjusted to pH 7..L.2:1. Arizona. A stock solution containing 4 g lÀ1 of each n-alkane in n-hexane (Merck. Materials and methods 2. Gilbert. Several studies have shown that a wide range of microorganisms are capable of degrading diesel fuel (Margesin and Schinner. 1995).5-trinitro-1.. The macronutrient content was (lÀ1) 17 g carbon. 1997) and in a soil slurry reactor (Boopathy and Manning. Yeast extract also increased the aerobic dechlorination rate of chlorobenzoic acids (Fava et al. respectively.5-triazine (RDX) in soil slurries (Boopathy and Manning. 62%. 1997a. 1999).5.3.1. and 100 ml nutrient medium containing the appropriate amount of microbial inoculum. b). Degradation experiment Aliquots of the n-alkane solution were added to 500-ml Erlenmeyer flasks to give a total n-alkane concentration of 100 mg lÀ1. 2003. Molasses has also been shown to increase the oxidation rate and degradation of n-alkanes by a single strain and a mixed culture of bacteria in synthetic seawater medium (Al-Hadhrami et al.. CO2 and biomass. 1997.e. 12% and free amino acids. The most common approach in enhancing the aerobic degradation of diesel fuel and nalkanes has been to add fertilizers in order to attain an appropriate C:N:P ratio (Margesin and Schinner.4. 0. and an appropriate volume was added to each batch of mineral nutrient medium to give 103. 2. Olivera et al. 1995) and eliminated substrate inhibition from chlorobenzoic acids at high concentrations (Armenante et al. 2000). the remaining hydroxide ions were determined by titration with 40 mM HCl using phenolphthalein as end-point indicator... AlHadhrami et al. 1996. 2000) without a co-substrate. 1999). by measuring the n-alkane concentration. 1999. the addition of an easily accessible carbon source both stimulates the degradation as shown above or inhibits the degradation owing to diauxie.25 mg CaCl2.. The change in biomass concentration was determined by photometry (Philips PU8650) by measuring optical density (OD) at 650 nm (A650) in a 4-cm cuvette. saturated BaCl2 was added to the vials to precipitate the carbonates. The experimental setup is given in Table 1. 1996.ARTICLE IN PRESS ¨ T. USA) suitable for degradation of petroleum hydrocarbons in general was used as inoculum and stored at 4 1C according to manufacturers’ recommendations. The ground-glass stopper on each flask was equipped with a glass loop designed to hold a 2-ml glass vial containing 1 ml 2 M NaOH to entrap evolved CO2. The n-hexane solvent was allowed to evaporate for 12 h at room temperature. For pH determination. Wrabel and Peckol. the C:N:P molar ratio was 22:2. All experiments were performed in triplicate.. Alexander. especially petroleum hydrocarbons. Marquez-Rocha et al. 1999.7 colony forming units (CFU) mlÀ1. The dry mass of the fermented whey was 36 g lÀ1 and the main components of the dry matter were: lactic acid. 1997). i... Medium The medium containing 2. Ostberg et al. 25 mg K2HPO4. 1998). 1994a. 1994). and after filtration. 1995. The aim of this study has been to investigate the possibility of using fermented whey as an organic supplement to enhance degradation of organic pollutants. Richard and Vogel. .4 and 104.2%. and during degradation of n-hexadecane by a mixed culture. carbon dioxide production and increase in biomass. The total number of culturable bacteria determined on yeast extract agar (Merck) after incubation for 2 days at 20 1C according to standard procedures (ISO 6222). The experiments were designed to study the effect of fermented whey on the aerobic degradation of n-alkanes in an aqueous environment. 2. 2000). was then added to the Erlenmeyer flasks. Widrig et al. This corresponds to an approximate C:N:P molar ratio of 141:6. C16 and C18 (Merck synthesis grade) representative of the main fuel constituents in diesel fuel oil. 25 mg KH2PO4. Fermented whey The fermented whey (Biogen ActiveTM. Invekta Green AB) used throughout the study is a Lactobacillus sp. In some of the studies mentioned above. were used as model substances (Riser-Roberts. in soil (Boopathy et al.31 g phosphorus. The concentration of culturable bacteria in the inoculum was 107.6 mg MgSO4. 6.3. n-alkanes The n-alkanes. PHaseIII Inc..4. and for aerobic/anoxic degradation of TNT (Boopathy et al. 1997).. Initially. a pH meter (Mettler Toledo MP225) was used.5 and finally autoclaved at 121 1C for 20 min 2. At each sampling. The fermented whey was autoclaved for 20 min at 121 1C before use. the entire contents of the experimental flasks were utilized for the determination of n-alkanes. C12. Rahman et al. 1998. All flasks were incubated at 2272 1C on a horizontal shaker operating at 80 rpm.. Bacterial growth and aerobic degradation of bromophenol in desert soil was enhanced (Ronen et al.. three flasks being used at each sampling time for each experimental condition. 1994. 2001) and n-alkanes (AlHadhrami et al. pa) was used. the positive effects of an additional complex carbon source are due to co-metabolism (Riser-Roberts. 2.3.89 g nitrogen and 0. Walworth et al.2. the microbial biomass was also increased by addition of yeast extract (Espeche et al. Prior to analysis. 2000. On the other hand when the microorganisms are able to metabolize the pollutant without a co-substrate. 1998) and the explosive hexahydro1. fermentation product of sweet milk whey with a pH of 3. sequential use of substrate where the most accessible carbon source is degraded first (Alexander. However. 29 mg NH4NO3 and 1. 2.. 1997).. / International Biodeterioration & Biodegradation 57 (2006) 190–194 191 An organic growth supplement such as yeast extract was required to stimulate growth of a single bacterial strain on aromatic hydrocarbons in seawater (Law and Teo.4 CFU mlÀ1.. with and without 5 mg fermented whey lÀ1.2. proteins. 2003).. Inoculum A commercial microbial consortium (PDM-7 HC. 1997b. C14. 0.. only a few studies have been made on the effects of organic growth supplements on the degradation of n-alkanes (Espeche et al. 1999).6trinitrotoluene (TNT) in liquid medium (Boopathy et al.3:1. Bej et al.

the whole sample was extracted directly in the experimental flasks. sterile control (m) and inoculum+fermented whey without n-alkanes (  ) according to the experimental setup (Table 1). Fig. 1a). The purified sample was concentrated using a rotary evaporator and further concentrated to a final volume of 2 ml with a stream of nitrogen.4 OD [A650] 0.3 0. carrier gas.4 104. 3. 2). reference with 103. 80–300 1C at 20 1C minÀ1 and 300 1C (10 min). C16 (m) and C18 (K). split ratio. resulting in a total starting concentration of n-alkanes of 90 mg lÀ1.2 0. Results and discussion Remaining n-Alkanes [%] 100 80 60 40 20 0 0 1 2 3 4 5 Time [days] 6 7 8 9 The abiotic loss of n-alkanes during the experiment was negligible according to the sterile control (Fig. 0.4 103. pa) was added to preclude formation of emulsions. 1.4 CFU mlÀ1 (K). pa) containing 2. The separation was performed with a specially constructed all-glass microseparator by adding sufficient water to the bottom of the flask to allow the organic phase layer to fill the microseparator. 250 1C.5 mg n-pentadecane (Merck. The n-alkanes of shorter chain length. The quantity of n-alkane was determined by GC-FID. Statistical analysis Data for n-alkane concentration. 0. 25 ml n-hexane (Merck. 1a–c. Experimental conditions were as follows: temperature program 80 1C (1 min). The symbols represent the n-alkane with the carbon chain length of C12 (E). using a Varian 3400CX fitted with an Alltech ECONO-CAPTM EC-1 capillary column (30 m  0. when the solvent hexane was evaporated there was also a loss of mainly n-dodecane due to evaporation. Ostberg et al.4 CFU mlÀ1 (’).4 — 103. pa).4 CO2 [mmol] 0. Before the start of the experiment.5 ml. versus time. SD bars are shown or contained within the symbol.0 0 (c) 1 2 3 4 5 6 7 8 Time [days] 9 10 11 12 13 0 1 2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 5 6 7 8 9 10 11 12 13 Fermented whey Reference High CFU control Sterile control Inoculum+fermented whey (a) 2. high CFU control with 104.6. C14 (’). 2. C14. SD bars are shown or contained within the symbol.L. The flask was closed and stirred vigorously for 45 min with a magnetic stirrer and was then allowed to rest for 15 min. injector temperature.1 0 (b) 0.4 CFU mlÀ1 inoculum. The degradation of the n-alkanes occurred sequentially (Fig. .4 CFU mlÀ1 (E).25 mm). nitrogen. n ¼ 3.ARTICLE IN PRESS 192 Table 1 Experimental setup Experiment n-Alkanes (mg lÀ1) 100 100 100 100 — Inoculum (CFU mlÀ1) 103. n ¼ 3. being more water-soluble. pa) as an internal standard was added to the flask. 1:50.4 Fermented whey (mg lÀ1) 5 — — 5 5 ¨ T. The degradation of n-alkanes. C16 and C18 nalkanes. production of CO2 and change in OD for the different experimental conditions are presented in Fig. The organic phase was then cleaned up via a small column filled with 2 g Florisil (SigmaAldrich) covered with a layer of 2 g anhydrous sodium sulfate (Merck.7. 300 1C. / International Biodeterioration & Biodegradation 57 (2006) 190–194 100 n-Alkanes [mg l-1] 80 60 40 20 0 0 0. The graphs show that the addition of 5 mg fermented whey lÀ1 at an inoculum concentration of 103. Graph (a) shows the decrease in total concentration of C12. The degradation pattern of each individual n-alkane for the high CFU control (Table 1) in the degradation study of 100 mg n-alkanes lÀ1 in mineral nutrient medium and 104. the fermented whey was totally degraded. 8 g MgSO4 (Merck.32 mm.1 0. indicated by 81% mineralization (data not shown). sample size. Degradation study of 100 mg n-alkanes lÀ1 in mineral nutrient medium with and without 5 mg (dry weight) fermented whey lÀ1.2 0. detector temperature. The symbols represent fermented whey with 103. Extraction and analysis of n-alkanes In order to avoid losses of n-alkanes during extraction. In addition.4 CFU mlÀ1 shortens the lag phase from 8 to 3 days Fig.3 0. were degraded preferentially. After acidifying with HCl to pH 2. All analyses were performed in Microsoft Excel. biomass and evolved CO2 were subjected to two-sided unequal variance Students t-test in order to test the significant differences between the treatments. graph (b) the accumulated CO2-production and graph (c) the increase in biomass expressed as optical density (OD) at 650 nm. During the experiment. 2.

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