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International Biodeterioration & Biodegradation 57 (2006) 190194 www.elsevier.com/locate/ibiod

Accelerated biodegradation of n-alkanes in aqueous solution by the addition of fermented whey

Tomas L. Ostberga,, Anders P. Jonssonb, Ulla S. Lundstromc
b

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
a

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 l1 (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 l1 to a nutrient medium receiving a small-sized inoculum (103.4 CFU ml1) 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 consortiums 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
Corresponding author. Tel.: +46 63 165300; fax: +46 63 165500.

E-mail address: tomas.ostberg@miun.se (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 benet is nonspecic, 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.

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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, 1997). Yeast extract also increased the aerobic dechlorination rate of chlorobenzoic acids (Fava et al., 1995) and eliminated substrate inhibition from chlorobenzoic acids at high concentrations (Armenante et al., 1995). Bacterial growth and aerobic degradation of bromophenol in desert soil was enhanced (Ronen et al., 2000), and during degradation of n-hexadecane by a mixed culture, the microbial biomass was also increased by addition of yeast extract (Espeche et al., 1994). Molasses has been shown to serve as an effective cosubstrate for aerobic degradation of the explosive 2,4,6trinitrotoluene (TNT) in liquid medium (Boopathy et al., 1994a, b), in soil (Boopathy et al., 1997; Widrig et al., 1997) and in a soil slurry reactor (Boopathy and Manning, 1999), and for aerobic/anoxic degradation of TNT (Boopathy et al., 1998) and the explosive hexahydro1,3,5-trinitro-1,3,5-triazine (RDX) in soil slurries (Boopathy and Manning, 2000). 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., 1996, 1997). In some of the studies mentioned above, the positive effects of an additional complex carbon source are due to co-metabolism (Riser-Roberts, 1998; Alexander, 1999). On the other hand when the microorganisms are able to metabolize the pollutant without a co-substrate, the addition of an easily accessible carbon source both stimulates the degradation as shown above or inhibits the degradation owing to diauxie, i.e. sequential use of substrate where the most accessible carbon source is degraded rst (Alexander, 1999). Several studies have shown that a wide range of microorganisms are capable of degrading diesel fuel (Margesin and Schinner, 1997a, 1999; Richard and Vogel, 1999; Marquez-Rocha et al., 2001) and n-alkanes (AlHadhrami et al., 1995; Olivera et al., 1997; Bej et al., 2000) without a co-substrate. 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, 1997b; Wrabel and Peckol, 2000; Rahman et al., 2003; Walworth et al., 2003). However, only a few studies have been made on the effects of organic growth supplements on the degradation of n-alkanes (Espeche et al., 1994; AlHadhrami et al., 1996, 1997). 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, especially petroleum hydrocarbons. The experiments were designed to study the effect of fermented whey on the aerobic degradation of n-alkanes in an aqueous environment, by measuring the n-alkane concentration, carbon dioxide production and increase in biomass.

2. Materials and methods 2.1. Fermented whey

The fermented whey (Biogen ActiveTM, Invekta Green AB) used throughout the study is a Lactobacillus sp. fermentation product of sweet milk whey with a pH of 3.2. The dry mass of the fermented whey was 36 g l1 and the main components of the dry matter were: lactic acid, 62%; proteins, 12% and free amino acids, 6.2%. The macronutrient content was (l1) 17 g carbon, 0.89 g nitrogen and 0.31 g phosphorus. This corresponds to an approximate C:N:P molar ratio of 141:6.3:1. The fermented whey was autoclaved for 20 min at 121 1C before use.

2.2. n-alkanes
The n-alkanes, C12, C14, C16 and C18 (Merck synthesis grade) representative of the main fuel constituents in diesel fuel oil, were used as model substances (Riser-Roberts, 1998). A stock solution containing 4 g l1 of each n-alkane in n-hexane (Merck, pa) was used.

2.3. Inoculum
A commercial microbial consortium (PDM-7 HC, PHaseIII Inc., Gilbert, Arizona, USA) suitable for degradation of petroleum hydrocarbons in general was used as inoculum and stored at 4 1C according to manufacturers recommendations. 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 concentration of culturable bacteria in the inoculum was 107.7 colony forming units (CFU) ml1, and an appropriate volume was added to each batch of mineral nutrient medium to give 103.4 and 104.4 CFU ml1, respectively.

2.4. Medium
The medium containing 2.6 mg MgSO4, 0.25 mg CaCl2, 25 mg KH2PO4, 25 mg K2HPO4, 29 mg NH4NO3 and 1.3 mg FeCl3 (all synthesis grade) l1 carbon dioxide-free water was adjusted to pH 7.5 and nally autoclaved at 121 1C for 20 min

2.5. Degradation experiment

Aliquots of the n-alkane solution were added to 500-ml Erlenmeyer asks to give a total n-alkane concentration of 100 mg l1. The n-hexane solvent was allowed to evaporate for 12 h at room temperature, and 100 ml nutrient medium containing the appropriate amount of microbial inoculum, with and without 5 mg fermented whey l1, was then added to the Erlenmeyer asks. All asks were incubated at 2272 1C on a horizontal shaker operating at 80 rpm. Initially, the C:N:P molar ratio was 22:2.2:1. The experimental setup is given in Table 1. The ground-glass stopper on each ask was equipped with a glass loop designed to hold a 2-ml glass vial containing 1 ml 2 M NaOH to entrap evolved CO2. Prior to analysis, saturated BaCl2 was added to the vials to precipitate the carbonates, and after ltration, the remaining hydroxide ions were determined by titration with 40 mM HCl using phenolphthalein as end-point indicator. At each sampling, the entire contents of the experimental asks were utilized for the determination of n-alkanes, CO2 and biomass. All experiments were performed in triplicate, three asks being used at each sampling time for each experimental condition. 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. For pH determination, a pH meter (Mettler Toledo MP225) was used.

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192 Table 1 Experimental setup Experiment n-Alkanes (mg l1) 100 100 100 100 Inoculum (CFU ml1) 103.4 103.4 104.4 103.4 Fermented whey (mg l1) 5 5 5 T.L. Ostberg et al. / International Biodeterioration & Biodegradation 57 (2006) 190194

100 n-Alkanes [mg l-1] 80 60 40 20 0 0 0.4 CO2 [mmol] 0.3 0.2 0.1 0 (b) 0.4 OD [A650] 0.3 0.2 0.1 0.0 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)

2.6. Extraction and analysis of n-alkanes

In order to avoid losses of n-alkanes during extraction, the whole sample was extracted directly in the experimental asks. After acidifying with HCl to pH 2, 8 g MgSO4 (Merck, pa) was added to preclude formation of emulsions. In addition, 25 ml n-hexane (Merck, pa) containing 2.5 mg n-pentadecane (Merck, pa) as an internal standard was added to the ask. The ask was closed and stirred vigorously for 45 min with a magnetic stirrer and was then allowed to rest for 15 min. The separation was performed with a specially constructed all-glass microseparator by adding sufcient water to the bottom of the ask to allow the organic phase layer to ll the microseparator. The organic phase was then cleaned up via a small column lled with 2 g Florisil (SigmaAldrich) covered with a layer of 2 g anhydrous sodium sulfate (Merck, pa). The puried sample was concentrated using a rotary evaporator and further concentrated to a nal volume of 2 ml with a stream of nitrogen. The quantity of n-alkane was determined by GC-FID, using a Varian 3400CX tted with an Alltech ECONO-CAPTM EC-1 capillary column (30 m 0.32 mm, 0.25 mm). Experimental conditions were as follows: temperature program 80 1C (1 min), 80300 1C at 20 1C min1 and 300 1C (10 min); injector temperature, 250 1C; detector temperature, 300 1C; carrier gas, nitrogen; split ratio, 1:50; sample size, 0.5 ml.

2.7. Statistical analysis

Data for n-alkane concentration, biomass and evolved CO2 were subjected to two-sided unequal variance Students t-test in order to test the signicant differences between the treatments. All analyses were performed in Microsoft Excel.

Fig. 1. Degradation study of 100 mg n-alkanes l1 in mineral nutrient medium with and without 5 mg (dry weight) fermented whey l1. Graph (a) shows the decrease in total concentration of C12, C14, C16 and C18 nalkanes, graph (b) the accumulated CO2-production and graph (c) the increase in biomass expressed as optical density (OD) at 650 nm, versus time. The symbols represent fermented whey with 103.4 CFU ml1 (E), reference with 103.4 CFU ml1 (), high CFU control with 104.4 CFU ml1 (K), sterile control (m) and inoculum+fermented whey without n-alkanes ( ) according to the experimental setup (Table 1). n 3, SD bars are shown or contained within the symbol.

3. 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. 1a). Before the start of the experiment, when the solvent hexane was evaporated there was also a loss of mainly n-dodecane due to evaporation, resulting in a total starting concentration of n-alkanes of 90 mg l1. The degradation of the n-alkanes occurred sequentially (Fig. 2). The n-alkanes of shorter chain length, being more water-soluble, were degraded preferentially. During the experiment, the fermented whey was totally degraded, indicated by 81% mineralization (data not shown). The degradation of n-alkanes, production of CO2 and change in OD for the different experimental conditions are presented in Fig. 1ac. The graphs show that the addition of 5 mg fermented whey l1 at an inoculum concentration of 103.4 CFU ml1 shortens the lag phase from 8 to 3 days

Fig. 2. The degradation pattern of each individual n-alkane for the high CFU control (Table 1) in the degradation study of 100 mg n-alkanes l1 in mineral nutrient medium and 104.4 CFU ml1 inoculum. The symbols represent the n-alkane with the carbon chain length of C12 (E), C14 (), C16 (m) and C18 (K). n 3, SD bars are shown or contained within the symbol.

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compared with the control without whey. The maximum degradation rates were, respectively, 53 and 57 mg alkanes l1 day1 with and without fermented whey. This stimulation cannot have been due to the contribution of macronutrients in the fermented whey, because of its relatively low nitrogen and phosphorus content and also because the mineral nutrient medium contained nitrogen and phosphorus at non-limiting concentrations. Fig. 1ac also shows that increasing the initial inoculum concentration to 104.4 CFU ml1 (high CFU control) gives a 6-day shortening of the lag time without the addition of fermented whey. The increase in optical density during the degradation phase corresponded to a doubling time of approximately 9 h at low inoculum concentration and 13 h at high concentration (Fig. 1c). With a doubling time in this range, it would take 12 days for the bacterial population to increase 10-fold from 103.4 to 104.4 CFU ml1. This shows that the 6-day increase in lag phase at low inoculum concentration was not just a direct result of the bacterial concentration being one-tenth of the high concentration, but also an effect of some other mechanism controlling the lag phase, e.g. appearance of new genotypes more decisive for growth and/or acclimatization of a small population. Al-Hadhrami and colleagues (1996) showed that addition of an alternative carbon and nutrient source, such as molasses, increased respiration and n-alkane degradation in synthetic seawater. These authors suggested that the combination of easily accessible carbon and nutrients, such as vitamins, increased the metabolic activity, and thereby stimulated the production of enzymes effective in alkane degradation. Similarly, it is possible that fermented whey acts as a growth substrate in an early and critical phase of the culture to increase the biomass, without reducing the ability of the consortium to degrade n-alkanes. Alternatively, fermented whey might supply growth factors that are needed for rapid growth of the consortium, being a complex mixture of easily accessible carbon and growth-stimulating compounds such as vitamins and amino acids, which all have the potential to increase the biomass. However, it is not possible from this study to determine the mechanism by which fermented whey stimulates the growth of cultures initiated by a small inoculum.

Acknowledgments We thank Dr Dan Bylund for valuable comments on the manuscript. This work has been supported by the Swedish Knowledge Foundation (KK-stiftelsen).

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4. Conclusion In this study, it has been shown that fermented whey can shorten the lag time of bacterial degradation of n-alkanes in an aqueous solution. Such accelerated degradation might be possible when dealing with petroleum hydrocarbons or other types of pollutants in various environmental situations, and thereby open up new uses for a large-scale waste product such as whey, e.g. in soil bioremediation, bioreactors and treatment of industrial efuent.

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