Seasonal and wastewater stream variation of trace organic compounds in a dairy processing plant aerobic bioreactor

Bioresource Technology 102 (2011) 7727–7736

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Seasonal and wastewater stream variation of trace organic compounds in a dairy processing plant aerobic bioreactor Michael W. Heaven a, Karl Wild b, Vincent Verheyen c, Alicia Cruickshank c, Mark Watkins a, David Nash a,⇑ a

Future Farming Systems Research Division, Department of Primary Industries, 1301 Hazeldean Road, Ellinbank, Victoria 3821, Australia Burra Foods Australia Pty. Ltd., 47 Station Street, Korumburra, Victoria 3950, Australia c School of Applied Science and Engineering, Bldg. 2W, Gippsland Campus, Monash University, Churchill, Victoria 3842, Australia b

a r t i c l e

i n f o

Article history: Received 7 March 2011 Received in revised form 13 May 2011 Accepted 1 June 2011 Available online 6 June 2011 Keywords: Dairy factory Wastewater Bioreactor Fatty acids GC–MS

a b s t r a c t Bioreactors are often an integral part of dairy factory efforts to reduce the biological oxygen demand of their wastewater. In this study, infeed, mixed liquor and supernatant samples of an aerobic bioreactor used by a dairy factory in South-Eastern Australia were analyzed for nutrients and organic compounds using gas chromatography–mass spectrometry and physicochemical analyses. Despite different concentrations of organic inputs into the bioreactor, nutrients and trace organic compounds were reduced significantly (i.e. average concentration of trace organic compounds: infeed = 1681 lg/L; mixed liquor = 257 lg/L; supernatant = 23 lg/L). However, during one sampling period the bioreactor was adversely affected by the organic loading. Trace organic compounds in the samples were predominantly fatty acids associated with animal products. The analyses suggest that it is possible to trace a disruptive input (i.e. infeed with high organic carbon concentrations) into an aerobic bioreactor by measuring concentrations of fatty acids or ammonia. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction The food industry often uses bioreactors to reduce biological oxygen demand (BOD) of wastewaters prior to discharge to the sewer or into other receiving environments (Cirja et al., 2008). Dairy factory wastewaters are some of the most polluted wastewaters in the food industry with up to 10 L of effluent wastewater produced per liter of processed milk (Vourch et al., 2008). The BOD concentrations for dairy factory wastewaters can vary widely depending on the season or product cycle. For instance, it has been reported that cheese factories have BODs ranging from 588 to 5000 mg/L whereas factories producing cream have BODs in the range of 1200–4000 mg/L (Demirel et al., 2005). Organic compounds in milk include proteins, carbohydrates and fats. These compounds account for a large proportion of the chemical oxygen demand (COD) found in dairy factory wastewaters and can have a serious impact on municipal waste treatment plants (Demirel et al., 2005). Protein in milk is made up primarily of the phosphoprotein salt casein (approximately 3% composition of milk) and whey proteins (Jensen et al., 1991). Carbohydrates in milk are mostly in the form of lactose (approximately 5% composition of milk). Milk fats (approximately 4% composition of milk) are almost solely composed of triglycerides, compounds consisting of a ⇑ Corresponding author. Tel.: +61 3 5624 2253; fax: +61 3 5624 2248. E-mail address: [email protected] (D. Nash). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.06.002

glycerol backbone and three fatty acids (FAs). FAs in milk are mainly C14, C16 and two types of C18 (i.e. linear alkyl chains with 14, 16, and 18 carbon atoms, respectively). Milk proteins, carbohydrates and fats are usually only found as residual material in wastewater due to their commercial value. For example, milk fat concentrations of 35–500 mg/L were detected in the wastewater of dairy waste treatment plants after milk processing (Perle et al., 1995). Most dairy factories use bioreactors or lagoons to reduce BOD and COD concentrations prior to discharge to municipal treatment systems, land or water (Cirja et al., 2008). In addition to BOD or COD, nitrate, phosphate or total suspended solids concentrations are usually monitored in effluents since an excess of phosphorus or nitrogen can lead to eutrophication of receiving waters (Gibson and Meyer, 2007). The analyses of nutrients, suspended solids, BOD and COD does not detect trace organic compounds such as endocrine disruptors that are typically found at low concentrations (Clara et al., 2005) and may be present in wastewaters (Cirja et al., 2008). Endocrine disruptors, typically plastics, pesticides or pharmaceuticals, are compounds suspected of altering the hormonal system of animals and humans and are often not removed by biological treatment of wastewater (Cirja et al., 2008). For instance, endocrine disruptors have been found in underground aquifers after chlorinated effluent was used to irrigate grain and dairy farms in Mexico (Durán-Alvarez et al., 2009). In Chile, pulp mills use various techniques such as

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activated sludge treatment to attempt to remove pollutants from their wastewater. However, despite removing >90% of wood compounds containing resin acids and phytosterols, known to act as endocrine disruptors, fish downstream have been found to be affected by the effluent and the endocrine disruptors detected in the receiving environment (Orrego et al., 2010). A recent review found endocrine disruptors and pharmaceuticals enter municipal treatment plants through human or animal usage via indiscriminate disposal of the drug or wastewater into receiving waters (Caliman and Gavrilescu, 2009). The review highlights that bioreactors are still the main technology used to remove endocrine disruptors but that the results are compound dependent (i.e. the anti-inflammatory drug ibuprofen is efficiently degraded, whereas the epilepsy drug carbamazepine is not). Burra Foods Pty. Ltd., located in the Gippsland region of Victoria, Australia is a dairy factory that uses bioreactors to reduce the BOD of their wastewater prior to discharge to the local municipal treatment facility. Burra Foods produce customized fresh and frozen dairy ingredients for the food manufacturing sector with over 60% going to export markets. Using milk from farms in the region, the factory at Korumburra processes more than 10,000 kilotonnes of milk solids annually. Over the last 3 years, Burra Foods have reduced potable water use from 28 kL per tonne of milk solids to 13 kL per tonne of milk solids. This reduction has been in part through segregation of the clean wastewater stream, composed of milk condensate and rinse water, from the more organically rich trade wastewater stream. We previously analyzed the wastewater streams from Burra Foods using gas chromatography–mass spectrometry (GC–MS) for potentially hazardous trace organic nitrogen and phenol containing compounds that may limit reuse (Verheyen et al., 2009). The wastewater outputs of the factory (i.e. effluent wastewater, milk condensate, cleaning-in-place potable water, boiler blowdown and pasteurized wastewater) were analyzed and compared with other local water streams (i.e. potable water and creek water). The analyses suggested that the compounds in the clean wastewater streams were benign. However, the effluent wastewater contained the endocrine disruptor bisphenol A and the hazardous compound p-cresol. It is of environmental interest to determine if there are ways to further reduce the organic loading and/or the hazardous nature of the wastewater in the bioreactor (Clara et al., 2005). Conditions present in bioreactors have been shown to affect their performance (Cirja et al., 2008). For example, the review by Cirja found that sludge retention time of greater than 8 days is required to remove hazardous pollutants. Research into dairy factory bioreactors has included changing the temperature within the bioreactor, changing milk/whey ratios as they enter the wastewater streams, performing heat treatments on the wastewater (Vourch et al., 2008), or changing the hydraulic retention time of the bioreactor (Demirel et al., 2005). However, there has been little research into how seasonal factors and feedstock quality affect dairy factory wastewaters processed by a bioreactor (Tawfik et al., 2008). In this baseline study, we investigate the trace organic compounds detected during normal operation of a bioreactor at Burra Foods during the spring lactation cycle (August through to January) of cows in South-Eastern Australia. Wastewater samples were taken from the infeed prior to the wastewater entering the bioreactor, from the mixed liquor within the bioreactor, and from the supernatant wastewater as it was leaving the bioreactor for the final effluent tank. These samples are different from the previously analyzed effluent wastewater stream sampled in April 2007 which was taken from the final holding tank prior to it being sent to the Korumburra municipal wastewater plant and contained a variety of clean and dirty wastewater streams (Verheyen et al., 2009).

2. Methods Samples were collected from Burra Foods Australia Pty. Ltd., (38° 250 4200 S, 145° 490 0500 E) in South-Eastern Victoria, Australia. The treatment plant at Burra Foods treats and disposes of wastewater that is separated into different streams depending on the organic loads. Wastewater with an expected COD in the range of 400–700 mg/L is buffered in the up-front tank (260 kL capacity). The wastewater is pH neutralized with sodium hydroxide or nitric acid on its way to the equalization tank (50 kL capacity) where the pH is adjusted for bioreactor conditions using sodium hydroxide or carbon dioxide. The wastewater is then fed to one of three sequenced batch bioreactors (495 kL capacity). The sequence batch bioreactors are biological, aerobic treatment vessels with activated sludge. Aeration takes approximately 8 h followed by settling stages (approximately 2 h per stage) before the partially settled liquid passes to four decant tanks (50 kL capacity each). The decant tanks act in parallel to further gravity settle the solids from the liquid (4–6 h). The supernatant passes to the final effluent tank, where it is buffered and discharged to sewer. The biological sludge from the decant tanks is either recycled back to the bioreactors or sent to digesters (2  260 kL) depending on the concentration and settling rate of mixed liquor suspended solids. The digesters are also biological, aerobic treatment vessels. These usually run a much slower cycle (2–5 days) than the bioreactors to treat the wastewater more completely. Liquid from the digesters goes to either a bioreactor or the final effluent tank. Samples of the infeed, mixed liquor and supernatant were taken on 11 August 2008 (i.e. Winter), 29 October 2008 (i.e. Spring), 15 December 2008 and 29 January 2009 (i.e. both Summer). Each set of samples was structured to capture an entire cycle of input and output of a bioreactor. Infeed samples were taken from a tap as the wastewater was being transferred from the upfront tank to the bioreactor. Mixed liquor samples were taken after the bioreactor was 100% full and had been aerated for approximately 1 h. The samples were taken from a tap approximately 30 cm from the bottom of the bioreactor. Supernatant samples consisted of wastewater taken off the top of the settled solids after settling of the mixture had occurred in the bioreactor. The supernatant samples were taken from a tap as the wastewater was en-route to the final effluent tank. Wastewater samples (20 L) were collected in 20 L polypropylene containers and stored at <4 °C until analyzed. All materials (e.g. hosing and valves) in contact with the samples were prerinsed with 1% Extran MA03 (Merck, Kilsyth, Australia), 10% HCl (AR Grade, Ajax Chemicals, Taren Point, Australia), deionized water and finally excess sample prior to use. Water samples were analyzed for Total Organic Carbon (TOC), Total Solids (TS), Electrical Conductivity (EC), Dissolved Reactive Phosphorus (DRP), Total Phosphorus (TP), Total Dissolved Phosphorus (TDP), Total Nitrogen (TN), Total Dissolved Nitrogen (TDN), nitrate (NO3) and ammonia (NH3) using standard methods (Eaton et al., 2005). EC was measured using a Model 900C Conductivity meter (TPS Pty. Ltd., Brisbane, Australia). The samples were tested for TOC with a Shimadzu TOC-V (Shimadzu Scientific Instruments (Oceania) Pty. Ltd., Sydney, Australia). A Lachat Quickchem Series 8000 Flow Injection Analyzer (DKSH Australia Pty. Ltd., Hallam, Australia) was used for the analyses of: DRP (Method Number 10-115-01-1-A); TDP and TP (Method Number 10-115-01-1-E); TDN and TN (Method Number 10-107-04-1-A); NO3 (Method Number 10-107-04-1-C); and NH3 (Method Number 10-107-062-C). For GC–MS analyses, the wastewater samples were pre-concentrated using solid phase extraction (SPE) cartridges. Filtering of undissolved material to stop blockages in the SPE cartridges was

M.W. Heaven et al. / Bioresource Technology 102 (2011) 7727–7736

carried out using muffled (450 °C) Schleicher and Schuell GF6 glass fiber filter paper (PerkinElmer, Rowville, Australia). The filtrate was then passed via a siphon onto preconditioned (10 mL methylene chloride then 10 mL methanol (HPLC Grade, Merck, Kilsyth, Australia)) 6 mL Bond Elut 1 g polar PPL (a styrene divinyl benzene type solid adsorbent phase with a nominal pore size of 150 Å) SPE cartridges (Varian Inc., Mulgrave, Australia) using a Vac Elut 20 extraction manifold (Varian Inc., Mulgrave, Australia). The cartridges were top-loaded with a plug of ChemTube-Hydromatix diatomaceous earth (Varian Inc., Mulgrave, Australia) to remove residual solids. Cartridges were replaced once the flow rate reduced to less than 0.1 mL/min. No more than 5 L was processed on any individual cartridge. Trace organic compounds were then extracted from the SPE cartridges. The loaded SPE cartridges were air dried for 30 min on a Vac Elut 20 extraction manifold under vacuum. Each cartridge was then eluted with 3 mL methylene chloride with internal standards added (100 ll of d5-phenol, 2-flurophenol, 2,4,6-tribromophenol and d14-chrysene (all concentrations 100 lg/ml), Accustandard, New Haven, CT, USA). For each sample, aliquots of the methylene chloride extract were combined and the composites dried with anhydrous sodium sulfate. The cleanup procedure involved acid–base separation of compounds using 5 M sodium hydroxide for the base and 1:1 v:v sulfuric acid in water. The acidified fraction was used for further analyses. The extract was divided in two with the other sample kept for future reference. The first sample was analyzed both before and after derivatization. The derivatization process involved methylation of samples using diazomethane dissolved in diethyl ether. Methylated products were then reacted with bis-(trimethylsilyl)-trifluoroacetamide plus 1% trimethyl chlorosilane to synthesize trimethylsilyl ethers of the products prior to injection into the GC–MS. The GC–MS (CP8400 GC and Saturn 2200 ITMS; Varian Inc., Middelburg, The Netherlands and Walnut Creek, CA, USA) was equipped with a 8400 autosampler and 1079 split–splitless injector operated at 290 °C. The split vent was closed during injection, opened to 1:80 after 0.20 min and reduced to 1:15 after 1 min. A Varian Factor Four capillary column VF-5 ms 30 m  0.25 mm ID and 0.25 lm film thickness was used for separation using helium carrier gas pressure programmed to a constant flow (1 mL/min.). The column oven was programmed to hold 75 °C for 2 min, to increase to 320 °C at 8 °C/min and to hold for a further 14 min. The transfer line to the mass spectrometer was heated to 170 °C and the trap was operated at 150 °C. In MS mode, the scan range was 35–650 amu with a 0.61 s/scan. Tentative identities were assigned to compounds based on their retention time and mass spectral data. Mass spectra were compared to the NIST/EPA/NIH 2005 library (Gaithersburg, MD, USA) with all computer spectral matches checked manually. Peak structural assignments were further validated by comparing their retention time and mass spectra with trimethylsilylated and nontrimethylsilylated samples. Tentative concentrations were based on the methylated internal standard, 2,4,6-tribromophenol. Statistical analyses of the data were performed using GenStat. ANOVA (Analysis of Variance) was used to determine if there were any significant variances between sample dates for the total concentration of trace organic compounds, between sample types for the total concentration of trace organic compounds (i.e. infeed, mixed liquor, supernatant), for date or sample type difference for the linear FAs detected, and between different physicochemical measurements (p < 0.05). Regression and correlation analyses were also conducted on and between the physicochemical measurements and concentrations of trace organic compounds over different sample dates and types.

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3. Results and discussion After the infeed wastewater entered the bioreactor, the concentration of almost all nutrients and organic carbon was reduced before it reached the effluent tank for disposal to the municipal wastewater treatment plant (Table 1). Infeed samples at Burra Foods had Total Organic Carbon (TOC) concentrations of 110– 1900 mg/L C (Average TOC: 615 mg/L C) which is consistent with other studies suggesting that dairy factory infeed wastewater can vary by more than 100% of the average value of a factory production run (Demirel et al., 2005). The bioreactor was found to remove between 78% and 99% of the TOC from the infeed wastewater. Phosphorus and nitrogen concentrations were reduced by the bioreactor by an average of 52% and 56%, respectively. Average Total Phosphorus (TP) was unusual in that, rather than decreasing in concentration, it increased from an average of 18.3 mg/L in the infeed samples to 20.8 mg/L in the mixed liquor samples. This increase was due to the December 2008 (TP concentration: infeed = 11.7 mg/L; mixed liquor = 16.1 mg/L) and January 2009 (TP concentration: infeed = 12.6 mg/L; mixed liquor = 30.2 mg/L) samples and may indicate deterioration in the conditions in the bioreactor due to microbial lysis (i.e. cell death) which can increase phosphorus concentrations (Ersu et al., 2010). Electroconductivity (EC) was reduced the least of all analytes with an average of 66% remaining. There was a high degree of correlation between Dissolved Reactive Phosphorus (DRP) and Total Dissolved Phosphorus (TDP) (R2 = 0.97) indicating that most of the soluble phosphorus was probably orthophosphate (Eaton et al., 2005). There was also a high degree of correlation between TOC and DRP, and TOC and TDP (both R2 = 0.89), perhaps indicating that a large proportion of the organic carbon may be phosphoprotein from residual milk product (Verheyen et al., 2009). For all mixed liquor samples, the ammonia concentrations were higher than the nitrate concentrations. This result may indicate that the bioreactor was not optimized and nitrification (conversion of ammonia to nitrate), an aerobic process, was slower than denitrification (conversion of nitrate to nitrous oxides or nitrogen gas), an anaerobic process. The total concentration of trace organic compounds, as measured by GC–MS analyses, was statistically examined to see if there was any significant difference between the infeed, mixed liquor and supernatant samples on each date. Despite different concentrations of organic inputs into the bioreactor, trace organic compound concentrations were reduced significantly (i.e. average concentration of trace organic compounds: infeed = 1681 lg/L; mixed liquor = 257 lg/L; supernatant = 23 lg/L). The samples were significantly different for August 2008, December 2008 and January 2009. However, the samples were not significantly different for the October 2008 samples. This implies that in October 2008 the trace organic compounds were not adequately digested by the microbial population in the bioreactor. Along with the total trace organic compound concentration, the October infeed sample also recorded the highest TOC (1900 mg/L C, Avg.: 862 mg/L C), phosphorus (TP = 41.8 mg/L, Avg.: 18.3 mg/L) and Total Solids (TS) (5907 mg/L, Avg.: 1907 mg/L) concentrations. Ammonia concentrations were also at their highest concentration in the mixed liquor sample from October 2008 (4.5 mg/L N). Overall, the analyses suggest that the bioreactor was overloaded in nutrients during the October 2008 sampling that resulted in the bioreactor to degrade in performance. The total concentration of trace organic compounds was also statistically examined, by type of sample, to determine possible differences over the sampling period. There was no significant difference in the total concentration of trace organic compounds between sampling dates for either the mixed liquor or the

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Table 1 Physicochemical measurements of samples taken from infeed (I), mixed liquor (ML) and supernatant (S) from bioreactor at Burra Foods Pty. Ltd. (11/08, 29/10 and 15/12/2008, 29/01/2009). 11/08/2008

% Fat TOCb(mg/L C) DRP (mg/L) TDP (mg/L) TP (mg/L) TDN (mg/L) TN (mg/L) NH3 (mg N/L) NO3 (mg N/L) TS (mg/L) EC (lS/cm) pH

29/10/2008

15/12/2008

29/01/2009

Ia

ML

S

I

ML

S

I

ML

S

I

ML

S

8.2 110 4.0 5.7 7.3 55.7 58.7 1.3 21.1 1680 2210 10.9

8.2 25 3.7 3.8 4.0 5.7 7.7 2.0 <0.01 1143 1740 9.3

8.2 24 0.7 0.7 1.1 3.2 4.0 1.5 0.01 1103 1572 9.0

12.6 1900 39.0 39.5 41.8 40.2 42.0 4.1 20.2 5907 1558 7.5

12.6 43 15.8 17.7 32.7 10.5 42.8 4.5 0.2 3075 1585 8.4

12.6 15 10.5 11.1 11.5 13.2 14.2 0.7 7.5 999 1540 8.5

26.0 320 4.2 7.4 11.7 10.7 24.9 2.6 6.1 2952 5170 12.2

26.0 84 3.8 10.7 16.1 12.5 59.4 3.4 0.1 3386 2279 9.5

26.0 60 7.5 7.7 7.9 11.4 12.3 1.9 6.6 1277 2038 8.6

10.9 129 3.3 9.5 12.6 93.9 94.0 1.0 10.1 2092 1871 10.2

10.9 28 4.2 7.5 30.2 21.6 23.9 3.7 2.1 2996 1797 8.3

10.9 13 8.5 10.5 11.0 22.1 23.8 2.4 12.2 1391 1986 8.4

a

Sample key: I = infeed, ML = mixed liquor, S = supernatant. Physicochemical acronyms: Total Organic Carbon (TOC), Dissolved Reactive Phosphorus (DRP), Total Dissolved Phosphorus (TDP), Total Phosphorus (TP), Total Dissolved Nitrogen (TDN), Total Nitrogen (TN), ammonia (NH3), nitrate (NO3), Total Solids (TS), Electrical Conductivity (EC). b

supernatant wastewater streams. This suggests that once the infeed reaches the bioreactor it is digested by the bacteria whatever the input. However, the total organic compound concentrations of the infeed samples were found to be significantly different from each other with the most concentrated sample in January 2009 (3875 mg/L) and the least concentrated sample in October 2008 (128 mg/L). This difference in the seasonal concentration of trace organic compounds in the infeed samples was not reflected in the TOC analyses indicating that the trace organics investigated in this study were not a fixed proportion of TOC. The changing product cycle at Burra Foods may contribute to the different infeed sample concentrations with some products producing greater organic loadings than others (Demirel et al., 2005; Vourch et al., 2008). Another possibility is that the chemical composition of the milk changed during the lactation season. For example, it is known that casein concentrations decrease from early to late lactation (Hickey et al., 2006). Therefore larger compounds like caseins that enter the dairy factory bioreactor via milk would be detected by TOC analyses but are too large to be detected by GC–MS. Over 120 compounds were detected by GC–MS analyses. By far the most common trace organic compounds detected were FAs, making up half of the total compounds found (Table 2). A large proportion of the FAs by concentration were linear FAs that consist of a single linear alkyl chain with a carboxylic acid group at one end. Linear FAs are often grouped into even-numbered (number of carbons on alkyl chain = even) FAs, that are typically animal and plant based FAs, and odd-numbered (number of carbons on alkyl chain = odd) FAs, that are often of a microbial origin (Christie, 1998). For the Burra Foods samples the ratio of FAs was in favor of even-numbered FAs suggesting the source of FAs in the wastewater streams is from an animal (i.e. milk) source (Fig. 1). The ratio of even:odd numbered FAs increased from approximately 2:1 in the infeed samples, through to approximately 3:1 in the mixed liquor samples, and finally 7:1 in the supernatant samples. The number of total even and odd FAs detected decreased over the same sample range. This suggests that the bioreactor microbes are preferentially consuming odd numbered FAs over even numbered FAs or the odd numbered FAs are being sequestered in the bioreactor. In the infeed samples, the number of even-numbered FAs peaked with six different linear FAs detected in the October sample before tailing off in January 2009 to only three FAs. The linear FAs octanoic (11/08/2008, C8, 824.1 lg/L) and decanoic acid (29/01/ 2009, C10, 849.7 lg/L) were the most concentrated of the trace organic compounds detected in all infeed samples. In general, linear FAs were reduced in both number and concentration when going from the infeed samples to the mixed liquor

samples with further reductions by the time supernatant samples were taken (Fig. 1). However, there was an increase of the number of even numbered FAs from the mixed liquor sample compared to the supernatant sample taken on January 29. The digestion of fatty acids was most striking when sampling from the supernatant where some FA concentrations were only 1% of that detected in the mixed liquor (C9 on December 2008, C10 on October 2008). Only one odd-numbered linear FA, C9, was detected in the supernatant samples (December 2008, January 2009) suggesting that the supernatant comprised few microbes or microbial products. The detection of odd-numbered FA in the analyses would be a sign that microbial decomposition had occurred due to the microbes starving (Barker and Stuckey, 1999). However, the highest TOC in the supernatant samples was in December 2008 (60 mg/L C) when C9 was detected, suggesting that bioreactor microbes were not starving and that the source of these fatty acids was possibly undigested infeed FA. Besides linear FAs, there were also over 40 non-linear FAs detected (Fig. 1 and Table 3). Non-linear FAs include those with saturated bonds (i.e. 4-octenoic acid, retention time: 6.18 min), or side chains (i.e. 3-methyl-pentanoic acid, retention time: 3.19 min). Non-linear FAs are often metabolites from animal, plant and/or microbial sources (Christie, 1998). In the infeed samples, the total concentration of non-linear FAs followed the same trend as the total concentration of even-numbered FAs suggesting that the non-linear FAs from these samples also originated from milk (Fig. 1). The total concentration of even-numbered FAs and nonlinear FAs also followed the same trend in the mixed liquor samples suggesting similar uptake by the bioreactor colonies for both classes of FAs. A general increase in the concentration of evennumbered, odd-numbered and non-linear FAs was detected in the supernatant samples from August 2008 to January 2009. The concentration of FAs is usually reduced in a properly functioning bioreactor (Massé et al., 2006). Therefore this result provides more evidence that the large input of organic carbon as shown by the TOC in October 2008 had a deleterious effect on the operation of the bioreactor. A number of non-linear FAs detected were prominent due to their detection in most of the infeed samples at the highest concentrations relative to other FAs. One of these was 14-methyl-heptadecanoic acid (R.T. = 20.91 min) which is typically found in milk as a marker of ruminant bacteria (Vlaeminck et al., 2006). Its detection suggests that some milk product was in the infeed samples. Another non-linear FA was isopalmitic acid (14-methyl-pentadecanoic acid). This compound is a minor milk lipid that has been sourced to aerobic bacteria (Lin and Yokota, 2006). This compound

2.3b

0.2a

0.3b

0.1b

0.5a

3.2b 0.4b 7.8b

1.1c

0.5a

1.7a

58.6a 1.5a 2.2a

4.2a 133.4b 87.6c 5.3b 21.7a

31.8a 0.1a

3.9a

2.2a 55.0b 5.3c

13.8a 0.8a 0.3a 19.5a

27.7

a

c

b

2

a

44.0a

0.4b

1.0a

0.5b

19.1a 5.0b 5.0b

4.0a

R as follows: a P 80%; b = 70–79%; c = 60–69%. Cn where n = number of carbon atoms in alkyl chain. RT = retention time.

849.7b 16.0b 4.8a 27.2a

7.0 1.1b 5.4a

3.88 4.82 4.63 6.32 8.09 9.80 11.47 14.86 17.03 16.76 19.93 19.75 22.16 C5 C6 C7 C8 C9 C10 C11 C12 C14 C15 C16 C17 C18

0.7a

1.9c 10.8a 824.1b 1.5a 3.9b

a

15/12/2008 29/10/2008

3.6b

1.2c

0.3a 1.3a 3.9a

11/08/2008 29/01/2009 15/12/2008 11/08/2008

29/10/2008 Mixed liquor

11/08/2008

29/01/2009 Infeed

Tentative concentration (lg/L)a RTc (min) FAb

Table 2 Linear fatty acids (FA) found in bioreactor infeed, mixed liquor and supernatant samples from Burra Foods (11/08, 29/10 and 15/12/2008, 29/01/2009).

Supernatant

29/10/2008

0.2a 0.2b 2.0a

15/12/2008

1.3b

29/01/2009

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appeared to be successfully digested in the bioreactor as isopalmitic acid was the second most concentrated non-linear FA detected in the infeed (August 2008, 250.3 lg/L; January 2009, 201.9 lg/L) but was only detected at 0.5 lg/L in a single supernatant sample (December 2008). Besides FAs, 62 other compounds were detected in the 12 samples (Table 4). The majority of these were phenols or heterocyclic compounds (both 31% of total). Acidic compounds made up 18% while amines were only 8% of the detected compounds. Some compounds detected were common to the infeed samples and all sampling dates including benzenepropanoic acid. The concentration of benzenepropanoic acid varied considerably from October 2008 (1.2 lg/L) to January 2009 (150.3 lg/L). This compound is used as an antioxidant in plastics and may come from newly built parts of the bioreactor, some of which was only constructed and upgraded in April 2008. A more complex analogue of benzenepropanoic acid has been reported from GC–MS analyses of waste from a new anaerobic membrane reactor and was attributed to the plastics used in construction (Trzcinski and Stuckey, 2010). Concentrations of benzenepropanoic acid decreased from the August 2008 sample (10.8 lg/L) through to the October 2008 sample before increasing again through to January 2009 indicating that there may be intermittent leaching from factory machinery. As benzenepropanoic acid was not detected in other parts of the factory during previous analyses (Verheyen et al., 2009), it would appear that it is isolated to the infeed wastewater stream. Benzenepropanoic acid has been found at elevated concentrations in waste disposal sites so if this compound was detected in other wastewater streams or the immediate environment, it could be used as a marker of the release of polluted waters (Hallbourg et al., 1992). An oxidized analogue of benzenepropanoic acid, a-oxo-benzenepropanoic acid (0.4– 2.5 lg/L), was detected in the mixed liquor samples, perhaps indicating the metabolic fate of benzenepropanoic acid in the bioreactor. Though difficult to process by bacteria, once oxidized, benzenepropanoic acids are part of the metabolic pathway in cells for the biosynthesis of phenylalanine (Ward and Singh, 2000). Like benzenepropanoic acid, benzothiophenes are recalcitrant compounds requiring multiple species of bacteria to break them down to less toxic products (Gai et al., 2008). The compound 3phenyl-benzo[b]thiophene was detected in all the infeed samples (0.8–63.5 lg/L). It was also found in the January 2009 mixed liquor sample (13.5 lg/L). Since the January 2009 mixed liquor sample contained this compound but the August mixed liquor sample didn’t, it would appear that bacteria responsible for degrading 3-phenyl-benzo[b]thiophene were underperforming in January 2009 (47.8 lg/L) compared to August 2008 (63.5 lg/ L). 3-Phenyl-benzo[b]thiophene compound is a toxic compound found in tars, creosotes used to protect wood from degradation, and incomplete combustion of wood. 3-Phenyl-benzo[b]thiophene is found to bioaccumulate in animals, more so than other polyaromatic hydrocarbons, one of the many reasons the use of creosote is regulated (Hartnik et al., 2007). Burra Foods is situated on the edge of the town of Korumburra so it is possible the source of this compound would be from smoke drifting in via wood combustion heaters used in homes. Phenylalanine metabolites were detected in all wastewater streams. The common cellular metabolite phenylacetic acid (Edahiro and Seki, 2006) was found in all infeed samples as well as the October 2008 (260.0 lg/L) and December 2008 (2.5 lg/L) sampling periods of the mixed liquor samples and supernatant samples (0.2 lg/L). Phenylacetic acid was the most concentrated compound besides the linear FAs and benzoic acid in the infeed samples (493.0 lg/L), and the most concentrated compound found in the mixed liquor samples. In the

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M.W. Heaven et al. / Bioresource Technology 102 (2011) 7727–7736 Even No. FAs Odd No. FAs Even No. FAs Concentration (mg/L) Odd No. FAs Concentration (mg/L) Non Linear FAs (mg/L)

7

10000

6

No. of FAs detected

5

100 4

3 10

2

Total Concentration of FAs ( µg/L)

1000

1 1

0

0 11-Aug

29-Oct 15-Dec Infeed

29-Jan

11-Aug

29-Oct 15-Dec Mixed Liquor

29-Jan

11-Aug

29-Oct 15-Dec Supernatant

29-Jan

Sample Date/Type Fig. 1. Number and concentration of Fatty Acids (FA) detected from infeed, mixed liquor and supernatant samples from Burra Foods Pty. Ltd.

supernatant samples, the compound appears to have been consumed with only 0.2 lg/L detected. The hydroxyl analogue of phenylacetic acid has been found to be converted to p-cresol enzymatically (Barker, 1981). The isomers m-cresol (3-methylphenol) (mixed liquor concentrations: 11.5–28.5 lg/L) and o-cresol (2-methylphenol) (supernatant sample concentration: 0.1 lg/ L) were detected in this analyses. The third isomer, p-cresol, was found in our previous analyses of effluent wastewater steams of this factory (Verheyen et al., 2009). Therefore, the analyses suggest that phenylacetic acid may be metabolized microbially and that cresol is from bioreactor microbes rather than from animal sources as was speculated previously. The detection of phenylacetic acid in the supernatant suggests that the bacteria responsible for the conversion of this metabolite to cresol was not operating as efficiently during October or December 2008 compared to August 2008 or January 2009 where despite greater infeed concentrations, the compound was not detected in either the mixed liquor or supernatant samples. In the mixed liquor samples, the compound p-formylphenol, a possible precursor to the ubiquitous plant flavor molecule vanillin and a hydrolysis product of lignin (Jing et al., 2009), was the only non-FA compound detected on all dates. This compound is often detected together with sources of natural phenols including bacteria and plant material which would possibly make it difficult to break down in a bioreactor due to their antimicrobial properties (Bountagkidou et al., 2010). This recalcitrance can be seen in the analyses where p-formylphenol was most concentrated during the summer months (December sample: 6.8 lg/L; January sample: 2.4 lg/L) in the mixed liquor samples but was not completely digested (supernatant samples: December 2008: 0.2 lg/L; January 2009; 0.4 lg/L). Only 3-hydroxybenzoic acid and diethylene glycol were detected in all supernatant samples. The two compounds were not found in either the infeed or the mixed liquor samples indicating their source is somewhere between where the mixed liquor wastewater and supernatant wastewater were sampled. 3-Hydroxyben-

zoic acid is a common bacterial metabolite so its source may actually be from microbes within the pipes between the bioreactor and the effluent tank (Cueva et al., 2010). Several compounds were unusual in that they were detected only on a certain date. For instance, benzoic acid was found only in the December samples and was the most concentrated infeed sample compound after the FAs C8 and C12 in the January 2009 samples (587.8 lg/L). It is a common compound found in plants and animals as well as being a precursor for many manufacturing processes (Juárez-Jiménez et al., 2010). Another compound, Nmethyloxindole, was only detected in the August 2008 samples. This was one of the more unusual compounds detected as its concentration increased as the wastewater moved between the mixed liquor (9.3 lg/L) and supernatant (13.1 lg/L) stages. This suggests that the compound is probably a product of bacteria metabolism within the bioreactor. Evidence for this has been found by researchers in the USA where it was observed that N-methyloxindole is a possible methanogenic metabolite of indoles (Gu and Berry, 1991). Our previous research showed that the indole compounds were likely metabolites of microbial attack on the amino acid tryptophan (Verheyen et al., 2009). This current research further validates that indole compounds were some of the dominant trace organic compounds detected in the mixed liquor (e.g. 29 October 2008: 1H-indole-3-acetic acid (47.7 lg/L)) but were not detected in the infeed samples. Sebacic acid was found within the mixed liquor (0.8 lg/L) and supernatant samples (0.1 lg/L) in the January 2009 samples. Sebacic acid is extracted from castor oil plants and used as a plasticizer so it can be assumed that it came from the bioreactor construction material (Ogunniyi, 2006). Another component of castor oil was ricinoleic acid (Goswami et al., 2010), found only once (Infeed sample concentration: 57.3 lg/L) during January 2009. It has also been detected as a component of fatty acids in microbes so it is possible that the presence of this compound indicates some bacterial metabolite of lactic acid digestion in the infeed (Kishino et al., 2009).

Table 3 Tentative identities and concentrations of non-linear fatty acids (FA) found in bioreactor infeed, mixed liquor and supernatant samples from Burra Foods (11/08, 29/10 and 15/12/2008, 29/01/2009). Tentative identity

RTa (min)

Tentative concentration (lg/L)b Infeed 11/08/ 2008

a

9.51 9.66 10.25 10.65 11.83 13.02 13.41 14.86 15.15 15.71 15.73 15.91 16.49 16.76 16.83 16.96 17.35 17.59 18.27 18.28 18.48 18.57 19.40 19.62 20.07 20.56 20.62 20.69 20.91 21.75 22.57 23.11 23.80 23.93 24.02 24.11

RT = retention time. R2 as follows: a P 80%; b = 70–79%; c = 60–69%.

29/01/ 2009

11/08/ 2008

Supernatant 29/10/ 2008

15/12/ 2008

29/01/ 2009

11/08/ 2008

29/10/ 2008

15/12/ 2008

0.1a

0.2a 0.6a

29/01/ 2009

15.5a 6.7b 7.1a

2.5a

0.4a

0.3a 1.8a

0.2b 0.1b 0.7b 0.5a 0.2b 1.5a 49.7a 24.4b

44.9a

1.6a

7.2a

38.9a

59.4a

85.4a 24.2a 14.6a 217.7a 197.3a

7.4a

21.6a

14.0a

29.6a 0.8b

3.1a

0.1a

0.1a

0.8a

0.1b

2.7a 21.2c 3.2a 115.9a

0.9a 13.7a

14.5a 35.3a 1.4b

66.5a

0.5a

5.3a

1.4a 4.0a

1.5a

0.3a

0.3b 6.4a 7.4b

0.4c 23.4a

8.6a 0.5b

11.5a

1.0a

5.1a

14.2a

4.2b 14.9a

0.6b

9.1b 250.3a 3.4b

201.9a

4.3a

7.7a 1.2c

2.1a

0.5a

1.4a 0.5b

31.5b

5.3a

1.7a

7.5a 8.3a 118.7a

3.2a 5.7a

4.4a 6.0a

79.8b

0.2a

4.1a 2.0b 5.1a

41.1b 54.5a 57.3b

17.0c

7733

b

4.09 4.23 4.66 4.90 5.71 6.18 6.59 6.60 6.76 6.95 7.23

15/12/ 2008

M.W. Heaven et al. / Bioresource Technology 102 (2011) 7727–7736

5-Methyl-hexanoic acid 4,6-Dimethyl-octanoic acid 2-Methyl-heptanoic acid 2-Ethyl-hexanoic acid 6-Methyl-heptanoic acid 4-Octenoic acid b-Lactic acid 2-Methyl-2-propylhexanoic acid 2-Ethyl-2-methyl-eicosanoic acid 2-Ethyl-hexanoic acid 2,3-Dimethyl-2-(1-methylethyl)-butanoic acid 4-Decenoic acid 11-Hydroxy-8-dodecenoic acid 2-Hydroxy-glutaconic acid (E)-2-hexadecenoic acid Octanedioic acid 10-Methyl-undecanoic acid Nonanedioic acid Sebacic acid Methyl 3-hydroxydodecanoate 3-Hydroxy-octadecanoic acid Z-11-tetradecenoic acid 12-Methyl-tridecanoic acid 9,10-Dihydroxy-octadecandioic acid 9-Methyl-tetradecanoic acid Azelaic acid 3,6-Epoxy-tetradecanedioic acid 9-Tetradecenoic acid Dodecanedioic acid 11-Hexadecenoic acid (Z)-9-hexadecenoic acid hexadecyl ester 3-Hydroxy-tetradecanoic acid 14-Methyl-pentadecanoic acid 14-Methyl-hexadecanoic acid Palmitelaidic acid Tetradecanedioic acid Linoleic acid (E)-9-octadecenoic acid 11-Octadecenoic acid 14-Methyl-heptadecanoic acid 15-Hydroxy-hexadecanoic acid 1,14-Tetradecandioic acid 10-Hydroxy-octadecanoic acid 9,10-Dihydroxy-octadecanoic acid 9,10-Dihydroxy-octadecanedioic acid Ricinoleic acid 9,10,12,13,18-Pentahydroxyoctadecanoic acid

Mixed liquor 29/10/ 2008

Tentative identity

RTa (min)

Tentative concentration (lg/L)b Infeed 11/08/ 2008

5.89 5.95 6.41 6.78 7.06 7.19 7.31 7.68 7.88 8.34 8.82 9.06 9.23 9.28 9.58 9.80 10.27 10.70 10.87 11.02 11.03 11.09 11.68 11.83 11.89 12.02 12.23 12.28 12.62 12.79 13.63 14.06 14.39 14.67 14.67 14.73 14.93 15.10 15.31 16.41 16.52 16.54 17.34 17.80 17.97 18.28 18.56 18.61 19.67

Mixed liqour 29/10/ 2008

15/12/ 2008

29/01/ 2009

1.3a

20.3a

587.8a

11/08/ 2008

Supernatant 29/10/ 2008

15/12/ 2008

29/01/ 2009

7.7a

22.6a

34.2a

20.5a

28.5a 23.3a

11.5a

11/08/ 2008

29/10/ 2008

0.2a

15/12/ 2008

29/01/ 2009

0.2a 0.1a

25.2b 40.8a

6.1a

26.8a 2.1a

493.0a

260.0a

0.8b

2.5a 2.6a

0.2a

0.2a

0.1a

0.1a

0.1a

0.2a

0.4a

0.1c 0.1b 10.8a

0.2b 1.2a

4.9a

150.3a 0.1a 7.0a

2.7a 1.3b 396.9a 3.3a 7.5a 1.5a

51.0a 194.9a

4.1a

0.3a

0.9a 0.7a

6.8a

184.2a

0.4a 2.4a

0.8a

3.1a 31.0b

0.2a

0.4a

0.9a

0.2a

0.6a

0.2a

0.6a 9.3a

13.1a 0.4c 0.4a

0.2a 63.5a

0.8a

3.2a

74.9b 47.8a

0.1a 0.4b

2.5b

1.2b

0.2a

0.5b 13.5a 1.3a

1.1a 0.2b

0.7b

0.4a 0.5b 1.1a 0.7a 0.2c

5.2a

2.6a

1.1c 0.2c 2.3a 2.4a

1.3a 3.5a

6.8a

0.1a 1.6a

106.2a 47.7a 1.6a 0.9b 8.6b 2.8a 23.8c 0.3a

7.6a

11.4a

M.W. Heaven et al. / Bioresource Technology 102 (2011) 7727–7736

Octanol Benzoic acid 2-Methylphenol 3-Methylphenol Tert-butyl (dimethyl)silylheptanoate Ethyl a-butylacetoacetate Phenylacetic acid Cyclohexanecarboxylic acid n-Butylamine Diethylene glycol Glycerol Benzenepropanoic acid p-Hydroxyacetophenone 2-Methyl-2H-indazol-3-amine Bicyclo[4.4.0]dec-5-en-4-one-1-carboxylic acid 2,2’-(1,2-Ethanediyl)bis[2-methyl-1,3-dioxolane] p-Tert-butyl phenol p-Formylphenol 4-Cyanophenol Resorcinol Salicylic acid Trans-cinnamic acid 1H-indole N-Methyloxindole 3-Hydroxybenzoic acid 5-Methyl-2-nitrophenol 4-Hydroxyacetophenone a-Oxo-benzenepropanoic acid 3-Phenyl-benzo[b]thiophene 4-Nitrophenol Dodecanol 3-Methyl-3H-benzothiazol-2-one 3-Bromo-4-methoxyphenylacetonitrile Aniline 4-(1,1,3,3,-Tetramethylbutyl) phenol 7-Ethyl-6,8-dimethyl-7H-[1,2,3,4,5] pentathiepino[6,7c]pyrrole 3-Bromomethyl-indole-5-ol 2,6-Di-tertbutyl-4-methoxyphenol 2-Methyl-4-hydroxyquinoline 1H-indole-2-carboxylic acid 2-(2-Benzyldeca-hydroisoquinolin-3-yl) ethanol 4-Hydroxy-5-nitro benzoic acid 1H-indole-3-acetic acid 1H-indole-3-carboxaldehyde 2,20 -Bibenzothiazole (Z)-9-hexadecenoic acid hexadecyl ester (4-Tert-butylphenoxy)methyl acetate 1-(Tert-butyldimethylsilyl)-3-(O-methyloxime)-1Hindole-2,3-dione b-D-galactopyranoside, methyl 2,3,4-tris-O-

7734

Table 4 Tentative identities and concentrations of trace organic compounds detected in infeed, mixed liquor and supernatant samples taken from Burra Foods bioreactor (11/08, 29/10 and 15/12/2008, 29/01/2009).

7735

0.2a

One endocrine disruptor was found within the bioreactor. The endocrine disruptor bisphenol A was found in the August and December 2008 mixed liquor and supernatant samples. It had been found previously in the effluent wastewater stream (Verheyen et al., 2009). The new plastic lining of the bioreactor could possibly be a source of this compound as the concentration increased in August 2008 from 1.2 lg/L in the mixed liquor sample to 6.2 lg/L in the supernatant sample. The concentrations detected are comparable to influent concentrations detected in municipal treatment plants found in Australia and overseas (Clara et al., 2005).

4. Conclusion

0.1a 1.1a 6.2a

0.5a

M.W. Heaven et al. / Bioresource Technology 102 (2011) 7727–7736

0.9b

Analyses of the infeed, mixed liquor and supernatant samples of a dairy factory bioreactor have shown that seasonal variations of the milk do not significantly affect the bioreactor’s function to digest wastewater compounds for disposal downstream. FAs, known to inhibit the microbes within the bioreactor, can remain high if the bioreactor is unduly overloaded leading to increased FAs concentrations in the supernatant. GC–MS analyses can reveal in detail what types of compound (i.e. fatty acids) are being released into the sewage or environment which may allow for a more tailored response to resolving any issues within an aerobic bioreactor.

RT = retention time. R2 as follows: a P 80%; b = 70–79%; c = 60–69%. a

b

(trimethylsilyl)-acetate Juvabione 2,4-Dihydroxybenzoic acid 1-Tripropylsilyloxyundecane Bisphenol A 2,3-Dihydroxy propyl laurate 9,10-Dihydroxy octadecanoic acid, ethyl ester

19.72 20.03 20.48 21.18 21.70 22.95

42.9b

5.5b

73.9b

1.2a

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