The effect of addition of a microbial consortium (A) with 22 micro-organisms on the large scale (400 m3 ) bioremediation of municipal waste water contaminated with petroleum hydrocarbons was evaluated. Our results showed that addition of Consortium A was effective in reducing the level of contamination of total petroleum hydrocarbons (TPH) from 1086 mg L-1 to 56 mg L-1after just 21 days and to 18 mg L-1 in 104 days. Phytotoxicity assays using Brassica rapa and bacterial viable counts confirmed the efficacy of the bioaugmentation agent. Overall, these results represent a promising and cost effective approach to the remediation of contaminated wastewater.

•    Bioaugmentation
•    Bioreactor
•    Municipal waste water
•    Petroleum
•    GC analysis

Poi G, Shahsavari E, Aburto-Medina A, Mok PC, Ball AS (2016) Large Scale Bioaugmentation of Municipal Waste Water Contaminated with Petroleum Hydrocarbons. JSM Environ Sci Ecol 4(3): 1036.

Industrial effluents from the petroleum related industry contain a mixture of chemicals such as phenolic compounds, polycyclic aromatic hydrocarbons, heterocyclic compounds and aliphatic hydrocarbons [1-3]. Most of these compounds are toxic and threaten both human and environmental health. Among the cleanup methods employed to remove the contaminants, bioremediation or the use of microbes to degrade petroleum hydrocarbons represents a promising technology.

Bioaugmentation is one bioremediation approach that, in this case involves the addition of hydrocarbon degrading microorganisms to the contaminated effluents. The rationale for using bioaugmentation relies on the introduction of organisms capable of degrading the contaminants thereby enhancing the remediation But despite its apparent simplicity there have been some failures and these have been well detailed and reviewed [4- 8]. In contrast, reports of the successful application of a microbial consortia for bioremediation are limited [9], resulting in the conclusion that bioaugmentation is an unproven, inconsistent technology [6-8,10]. Despite the fact that pure microbial cultures have been shown to be effective in laboratory conditions, it is recognized that a mixed community of microbes would be needed for the complete mineralization of the various hydrocarbons found in wastewaters. In addition, it has been argued that real life conditions cannot be perfectly mimicked in the laboratory and consequently a gap exists between reported success in the laboratory and the reported lack of success in field work [11-13]. What is required are specific scale-up experiments specifically designed to meet the needs of the industry, carried out in situ in order to truly access the potential of this technology [14].

Therefore the aim of this study was to evaluate the efficacy of bioaugmentation in a large scale project (up to 400 m3 ) in situ using petroleum industry wastewaters and a microbial consortium with mixed species capable of degrading a wide range of hydrocarbons.

Bioaugmentation agents

The hydrocarbon degraders, 22 bacterial isolates from different species (mostly Bacilli sp.) were used as the bioaugmentation treatment (Consortium A) (Table 1). The bacteria were previously isolated from a municipal wastewater treatment plant and their ability to degrade hydrocarbons was confirmed in a previous study [15].

Laboratory experiments

A preliminary set of experiments was conducted to assess the efficacy of Consortium A for the biodegradation of petroleum hydrocarbons prior to field experiments. A sample (1.0 L) of high TPH (about 250,000 mg L-1) wastewater (WW) was filtered using Whatman No. 2 filter paper and adjusted to pH 7.0; aliquots (50 mL) were placed into sterile glass tubes maintained at 37ºC without stirring or aeration. The bioaugmentation agent was prepared as previously described [15].

A single inoculation (3.0% v/v) of Consortium A, containing around 108 bacteria mL-1 was performed at time 0, with no further inoculation in subsequent weeks. A control (C) was included using a single 3.0% (v/v) addition of RO water at time 0 to monitor changes in phenol concentration due to evaporation. The experiment was conducted in duplicate over a period of 57 days. The mean TPH levels at the start were 246,542 mg L-1 for Consortium A, and 247,108 mg L-1 for Control C.

Field experiments

The preparation for translation and scale-up required the production of large volumes which requiring a modified protocol to produce Consortium A. Each pure culture was prepared using a loop of pure culture taken from a sub-culture plate of Nutrient Agar and inoculated into 50 mL of PB media that was incubated overnight at 37°C and shaken at 150 rpm. These were then used to inoculate 500 mL of production broth medium contained glucose (10 g), yeast extract (8 g) and NaCl (5 g) and incubated in a shaker incubator at 500 rpm at 37°C for 24 to 48 hours depending on which cultures were grown. These cultures were then stored in 4 L carboys at 4°C. Each carboy of pure culture was then placed in a pre-sterilized 20 L Braun™ fermenter which was stirred at 150 rpm and aerated for better mixing. A 500 mL of sterile phosphate buffer was added to the resulting mixture and allowed to mix for 5 minutes. This was then harvested to produce 4 L carboys of Consortium A which was then stored at 4°C until required.

In regards to the scale-up experiment, a biocluster bioreactor (750 litres) and a treatment tank were used. The treatment tank was a holding tank made of steel, concrete and fibreglass with a holding capacity of 400 m3 and an aeration grid to provide mixing and aeration at minimal cost. The consortium was prepared as described above but for the pilot scale experiment at the wastewater treatment plant(WWTP)due to scale limitations the inoculum was approximately 0.03% (v/v), added once a week into the biocluster as seed for the treatment tank. After addition of Consortium A, wastewater was then pumped from the biocluster into the treatment tank. Samples were collected at two weekly intervals from the treatment tank for further analyses

Total petroleum hydrocarbon (TPH) analysis

A modified standard protocol US EPA method (8015a) based on gas chromatography (GC) analysis was used for TPH measurement in both laboratory and field experiments. The GC system used was an HP 5890 Series II coupled with Agilent GC Chemstation software. The GC was equipped with a Flame Ionisation Detector (FID) for the detection of solids and liquids with boiling points below 2,500°C with a limit of detection of 1 mg L-1. The standards used were Alphagaz PIANO Calibration Standards from Supelco ®. Standard QC parameters were included throughout.

Phytotoxicity assay and enumeration of total viable bacteria: Phytotoxicity assays were conducted using Brassica rapa according to the methods of Khan, Waqas et al. (2015) [16], for each sampling time point (end day 104). The plate count technique was used to enumerate total viable bacteria using Plate Count Agar medium (Oxoid LTD, UK).

Data analysis

Data were analyzed using T test methods using IBM SPSS (Version 21). Standard error was shown where required.

Table 1: List of bacteria used in this study (Consortium A).

The TPH concentrations decreased to 0 from an initial 240,000 mg L-1 in just 43 days in the laboratory (Figure 1). TPH analysis was performed to establish the treatment with the highest efficacy to reduce the petroleum hydrocarbon content in the wastewater to below 1,000 mg L-1 without any agitation or aeration. A paired T-test confirmed a significant difference between the remaining TPH concentration in the control wastewater and that inoculated with Consortium A (P value = 0.03). Following successful laboratory trials, translational work was carried in situ in a field study. The addition of Consortium A into wastewater led to a significant and rapid reduction in the levels of TPH, from 1056 mg L-1 to 56 mg L-1 in only 21 days. Overall, the degradation of hydrocarbons decreased from 1086 to 18 mg L-1 in 104 days (Figure 2).

Plate count results showed that the number of total viable bacteria increased during the bioremediation process from 8.8 log10CFU/dry g soil at day 0 to 9.4 log10 CFU/dry g soil at day 21 (~ 4 fold increase) before reducing to 7.6 log10CFU/dry g soil at the end of experiment. These results confirm the efficacy of Consortium A in terms of both survival and contaminant degradation in situ at a large scale.

The historical prevalence of hydrocarbons in the environment has inevitably led to the acclimation and adaptation of microorganisms that appear to have substantial hydrocarbonoclastic activity [17]. This has been supported by the large numbers of organisms that have been reported to be involved in the bioremediation of crude [17-20], polycyclic aromatic hydrocarbons (PAHs) [21,22], diesel [11,23], diesel and fuel oil [24] and soil contaminated with weathered hydrocarbons [25], aircraft fuel [26], waste engine oil [27], waste sludge [28], weathered crude oil [29], oily sludge [30] and oil tank bottom sludge (OTBS) [31,32]. However unlike this study, many of the publications previously cited have included reports of the effectiveness of only pure cultures isolated and identified as part of the investigations with much of the work carried out at a laboratory scale. There is a need for reliable and predictable microbial consortium rather than individual organisms to handle the fluctuations commonly observed in wastewater with high concentrations of toxic components that can be effectively translated and scaled-up [33]. It has been argued that there was a gap between reported successes in the laboratory and the seemingly lack of reported success in field work [34]. This translation and scale-up closed this perceived gap in this study [14].

The use of chemical analyses does not measure the overall toxicity of effluents; the use of biological toxicity testing is intended to compensate for limitations arising from traditional chemical analyses [35]. In the current study, the percentage of B. rapa germination increased from 1 to 39% in wastewater sampled at time 0 and after 104 days, indicating that the degradation of the hydrocarbons decreased the toxicity (Figure 3). Plants are a key component in the terrestrial ecosystem and are a useful monitoring tool for evaluating adverse environmental impact by pollutants that are not evident by chemical measurements alone [36,37]. Quantitative measurements based on seed germination and seedling growth have been used as the basis of plant bioassays to evaluate the biotoxicity of hydrocarbons in the environment [38-44].

In the present study, the in situ, large scale remediation of wastewater contaminated with petroleum hydrocarbons bioremediation was achieved, with a 98% reduction in the contaminant and a much reduced toxicity. In this instance bioaugmentation using a microbial consortium represents an appropriate technology for use in a full scale treatment facility. This work is among the first to demonstrate its application at a biological wastewater treatment plant

1. Unell M, Nordin K, Jernberg C, Stenström J, Jansson JK. Degradation of mixtures of phenolic compounds by Arthrobacter chlorophenolicus A6. Biodegradation. 2008; 19: 495-505.

2. Yu Z, Chen Y, Feng D, Qian Y. Process development, simulation, and industrial implementation of a new coal-gasification wastewater treatment installation for phenol and ammonia removal. Industrial & Engineering Chemistry Research. 2010; 49: 2874-2881.

3. Chan H. Biodegradation of petroleum oil achieved by bacteria and nematodes in contaminated water. Separation and Purification Technology. 2011; 80: 459-466.

4. Goldstein RM, Mallory LM, Alexander M. Reasons for possible failure of inoculation to enhance biodegradation. Appl Environ Microbiol. 1985; 50: 977-983.

5. Stephenson D, Stephenson T. Bioaugmentation for enhancing biological wastewater treatment. Biotechnol Adv. 1992; 10: 549-559.

6. Bouchez T, Patureau D, Dabert P, Juretschko S, Doré J, Delgenès P, et al. Ecological study of a bioaugmentation failure. Environ Microbiol.2000; 2: 179-190. 

7. Vogel T, Walter M. Bioaugmentation. Manual of environmental microbiology. ASM Press, Washington, DC: 2001; 952-959.

8. Wagner-Dobler I. Microbial inoculants: snake oil or panacea. Bioremediation: A critical review. 2003; 259-289.

9. Shong J, Jimenez Diaz MR, Collins CH. Towards synthetic microbial consortia for bioprocessing. Curr Opin Biotechnol. 2012; 23: 798-802.

10. Iwamoto T, Nasu M. Current bioremediation practice and perspective. J Biosci Bioeng. 2001; 92: 1-8.

11. Bento FM, Camargo FA, Okeke BC, Frankenberger WT. Comparative bioremediation of soils contaminated with diesel oil by natural attenuation, biostimulation and bioaugmentation. Bioresour Technol. 2005; 96: 1049-1055.

12. Head IM, Jones DM, Röling WF. Marine microorganisms make a meal of oil. Nat Rev Microbiol. 2006; 4: 173-182.

13. McKew BA, Coulon F, Yakimov MM, Denaro R, Genovese M, Smith CJ, et al. Efficacy of intervention strategies for bioremediation of crude oil in marine systems and effects on indigenous hydrocarbonoclastic bacteria. Environ Microbiol. 2007; 9: 1562-1571.

14. Macaulay BM, Rees D. Bioremediation of Oil Spills: A Review of Challenges for Research Advancement. Annals of Environmental Science. 2014; 8: 2.

15. Koshlaf E, Shahsavari E, Aburto-Medina A, Taha M, Haleyur N, Makadia TH, et al. Bioremediation potential of diesel-contaminated Libyan soil. Ecotoxicol Environ Saf. 2016; 133: 297-305.

16. Khan S, Waqas M, Ding F, Shamshad I, Arp HP, Li G. The influence of various biochars on the bioaccessibility and bioaccumulation of PAHs and potentially toxic elements to turnips (Brassica rapa L.). J Hazard Mater. 2015; 300: 243-253.

17. Prince RC, Lessard RR. Crude Oil Releases to the Environment: Natural Fate and Remediation Options. Encycl Energy. 2004; 1: 727-736.

18. Gogoi BK, Dutta NN, Goswami P, Krishna Mohan TR. A case study of bioremediation of petroleum-hydrocarbon contaminated soil at a crude oil spill site. Advances in Environmental Research. 2003; 7: 767-782.

19. Mnif S, Chamkha M, Labat M, Sayadi S. Simultaneous hydrocarbon biodegradation and biosurfactant production by oilfield-selected bacteria. J Appl Microbiol. 2011; 111: 525-536.

20. Zhao D, Liu C, Liu L, Zhang Y, Liu Q, Wei-Min Wu. Selection of functional consortium for crude oil-contaminated soil remediation. International Biodeterioration & Biodegradation. 2011; 65: 1244-1248.

21. Jacques RJ, Okeke BC, Bento FM, Teixeira AS, Peralba MC, Camargo FA. Microbial consortium bioaugmentation of a polycyclic aromatic hydrocarbons contaminated soil. Bioresour Technol. 2008; 99: 2637- 2643.

22. Lu XY, Zhang T, Fang HH. Bacteria-mediated PAH degradation in soil and sediment. Appl Microbiol Biotechnol. 2011; 89: 1357-1371.

23. Alisi C, Musella R, Tasso F, Ubaldi C, Manzo S, Cremisini C, et al. Bioremediation of diesel oil in a co-contaminated soil by bioaugmentation with a microbial formula tailored with native strains selected for heavy metals resistance. Sci Total Environ. 2009; 407: 3024-3032.

24. Lin TC, Pan PT, Cheng SS. Ex situ bioremediation of oil-contaminated soil. J Hazard Mater. 2010; 176: 27-34.

25. Sheppard PJ, Adetutu EM, Makadia TH, Ball AS. Microbial community and ecotoxicity analysis of bioremediated, weathered hydrocarboncontaminated soil. Soil Research. 2011; 49: 261-269.

26. Lebkowska M, Zborowska E, Karwowska E, Miaskiewicz-Peska E, Muszynski A, Tabernacka A, et al. Bioremediation of soil polluted with fuels by sequential multiple injection of native microorganisms: Fieldscale processes in Poland. Ecological Engineering. 2011; 37: 1895- 1900.

27. Aleer S, Adetutu EM, Makadia TH, Patil S, Ball AS. Harnessing the Hydrocarbon-Degrading Potential of Contaminated Soils for the Bioremediation of Waste Engine Oil. Water Air Soil Pollution. 2010; 218: 121-130.

28. Makadia TH, Adetutu EM, Simons KL, Jardine D, Sheppard PJ, Ball AS. Re-use of remediated soils for the bioremediation of waste oil sludge. J Environ Manage. 2011; 92: 866-871.

29. Kadali KK, Simons KL, Skuza PP, Moore RB, Ball AS. A complementary approach to identifying and assessing the remediation potential of hydrocarbonoclastic bacteria. J Microbiol Methods. 2012; 88: 348- 355.

30. Cerqueira VS, Hollenbach EB, Maboni F, Vainstein MH, Camargo FA, do Carmo R Peralba M, et al. Biodegradation potential of oily sludge by pure and mixed bacterial cultures. Bioresour Technol. 2011; 102:11003-11010.

31. Matsui T, Yamamoto T, Shinzato N, Mitsuta T, Nakano K, Namihira T. Degradation of oil tank sludge using long-chain alkane-degrading bacteria. Ann Microbiol. 2014; 64: 391-395.

32. Adetutu E, Bird C, Kadali K, Bueti A, Shahsavari E, Taha M, et al. Exploiting the intrinsic hydrocarbon-degrading microbial capacities in oil tank bottom sludge and waste soil for sludge bioremediation. Int J Environ Sci Technol. 2014; 1-10.

33. Fang F, Han H, Zhao Q, Xu C, Zhang L. Bioaugmentation of biological contact oxidation reactor (BCOR) with phenol-degrading bacteria for coal gasification wastewater (CGW) treatment. Bioresour Technol. 2013; 150: 314-320.

34. Coulon F, McKew BA, Osborn AM, McGenity TJ, Timmis KN. Effects of temperature and biostimulation on oil-degrading microbial communities in temperate estuarine waters. Environ Microbiol. 2007; 9: 177-186.

35. Ma M, Li J, Wang Z. Assessing the detoxication efficiencies of wastewater treatment processes using a battery of bioassays/ biomarkers. Arch Environ Contam Toxicol. 2005; 49: 480-487.

36. Banks M, Schultz K. Comparison of plants for germination toxicity tests in petroleum-contaminated soils. Water, air, and soil pollution. 2005; 67: 211-219.

37. Oleszczuk P. Phytotoxicity of municipal sewage sludge composts related to physico-chemical properties, PAHs and heavy metals. Ecotoxicol Environ Saf. 2008; 69: 496-505.

38. Chouychai W, Thongkukiatkul A, Upatham S, Lee H, Pokethitiyook P, Kruatrachue M. Phytotoxicity assay of crop plants to phenanthrene and pyrene contaminants in acidic soil. Environ Toxicol. 2007; 22: 597-604.

39. Dawson J, Godsiffe E, Thompson I, Ralebitso-Senior TK, Killham K, Paton G. Application of biological indicators to assess recovery of hydrocarbon impacted soils. Soil Biology and Biochemistry. 2007; 39: 164-177.

40. Coulon F, Al Awadi M, Cowie W, Mardlin D, Pollard S, Cunningham C, et al. When is a soil remediated? Comparison of biopiled and windrowed soils contaminated with bunker-fuel in a full-scale trial. Environ Pollut. 2010; 158: 3032-3040.

41. Lors C, Ponge JF, Martínez Aldaya M, Damidot D. Comparison of solidphase bioassays and ecoscores to evaluate the toxicity of contaminated soils. Environ Pollut. 2010; 158: 2640-2647.

42. Tang J, Wang M, Wang F, Sun Q, Zhou Q. Eco-toxicity of petroleum hydrocarbon contaminated soil. J Environ Sci (China). 2011; 845-851.

43. Tang J, Lu X, Sun Q, Zhu W. Aging effect of petroleum hydrocarbons in soil under different attenuation conditions. Agriculture, Ecosystems & Environment. 2012; 149: 109-117.

44. Kummerová M, Zezulka Š, Babula P, Vá?ová L. Root response in Pisum sativum and Zea mays under fluoranthene stress: morphological and anatomical traits. Chemosphere. 2013; 90: 665-673