جداسازی،غربالگری و کارایی جدایه‌های سودوموناس در تشکیل بیوفیلم بر حامل‌های آلی و معدنی و تجزیه فنانترن
محورهای موضوعی : زیست فناوری میکروبیمریم ترکاشوند 1 , امیر لکزیان 2 , امیر فتوت 3 , مهدی محمدی 4
1 - گروه علوم خاک،دانشکده کشاورزی،دانشگاه فردوسی مشهد، مشهد، ایران
2 - گروه علوم خاک،دانشکده کشاورزی،دانشگاه فردوسی مشهد،مشهد، ایران
3 - گروه علوم خاک، دانشکده کشاورزی، دانشگاه فردوسی مشهد، مشهد، ایران
4 - گروه زیست فناوری،پژوهشکده خلیج فارس،دانشگاه خلیج فارس، بوشهر، ایران
کلید واژه: بیوفیلم, فنانترن, سودوموناس آئروجینوزا, بیوچار, لاکاز,
چکیده مقاله :
سابقه و هدف: بیوفیلم های میکروبی یکی از مهمترین فرم های حیات میکروارگانیسم ها در محیط هستند. هدف از این پژوهش استفاده از بیوفیلم باکتری سودوموناس بومی خاک‌های ایران به منظور تجزیه فنانترن بود.مواد و روش ها: از بین 63 سودوموناس جداشده از خاک، جدایه باکتریایی با توان بالا در تولید بیوفیلم و تجزیه نفت خام انتخاب و شناسایی شد. سپس تاثیر حامل‌های معدنی (پومیس خام، پومیس فرآوری شده و پرلیت) و آلی (بیوچار و باگاس) بر تولید بیوفیلم میکروبی بررسی شد و ساختار بیوفیلم ها بوسیله میکروسکوپ الکترونی روبشی بررسی گردید. همچنین تاثیر تیمارهای باکتری سودوموناس آئروجینوزا در دو فرم رشدی پلانکتونی و بیوفیلم بر روی حامل بیوچار بر تولید آنزیم لاکاز و تجزیه فنانترن در محیط مایع بررسی شد.یافته ها: جدایه های خالص سازی شده از نظر تولید بیوفیلم و توان تجزیه نفت خام غربالگری شدند. جدایه B59 (سودوموناس آئروجینوزا) با 56/62% بیشترین درصد تجزیه نفت خام را نشان داد. بیشترین میزان لگاریتم واحد تشکیل کلنی مربوط به بیوفیلم تشکیل شده بر حامل بیوچار و برابر 8/8 و کمترین در حامل پومیس فراوری شده و برابر 9/2 بود. میزان فعالیت آنزیم لاکاز در فرم بیوفیلمی 8/39 و در فرم پلاکتونی1/33 واحد در دقیقه بود. همچنین فرم بیوفیلمی و پلانکتونی به ترتیب موجب تجزیه 17/97% و 37/91 % از فنانترون موجود در محیط شدند.نتیجه گیری: نتایج نشان داد که فرم بیوفیلمی باکتری در مقایسه با فرم پلانکتونی از طریق افزایش معنی‌دار واحد تشکیل کلنی و تولید بیشتر آنزیم لاکاز، موجب تجزیه بیشتر فنانترون در محیط مایع می گردد.
Background & Objectives: Microbial biofilms are one of the most important microorganism’s forms of life in the environment. The aim of this study was to use native Pseudomonas aeruginosa in biofilm form to clean up phenanthrene. Material & Methods: Among 63 Pseudomonas isolates from Iranian soil, bacterial isolate with high potency in biofilm formation and crude oil degradation was selected and genetically identified. The effect of inorganic (raw pumice, processed pumice and perlite) and organic (biochar and sugarcanes bagasse) carriers on biofilm formation was investigated. Biofilm structure was studied by scanning electron microscopy. Also, the effects of two growth form (biofilm, planktonic) on biochar carrier on laccase production and phenanthrene degradation in liquid media was investigated. Results: Purified Pseudomonas isolates were screened based on biofilm formation and crude oil degradation. B59 (Pseudomonas aeruginosa) with 56.62% showed the highest percentage of crude oil degradation. Highest and lowest CFU belonged to biochar and processed pumice with 8.8 and 2.9 respectively. Laccase activity was measured in biofilm form and planktonic form 39.8 and 33.1 Um-1 respectively. Biofilm form of Pseudomonas aeruginosa led to 97.17% phenanthrene degradation where planktonic form reached 91.37%. Conclusion: Results showed that biofilm form of Pseudomonas aeruginosa compared to planktonic form by significant increase in CFU and laccase activity result in more phenanthrene degradation in liquid media.
59:415-28.
2. El-Azizi MA, Starks SE, hardori N. Interactions of Candida albicans with other Candida spp.
and bacteria in the biofilms. Journal of applied microbiology. 2004; 96(5):1067-73.
3. Shakibaie MR. Bacterial biofilm and its clinical implications. Annals of Microbiology and
Research. 2018; 2: 2(1).
4. Flemming HC, Wingender J, Szewzyk U, Steinberg P, Rice SA, jelleberg S. Biofilms: an
emergent form of bacterial life. Nature Reviews Microbiology. 2016;14(9): 563.
5. arn S , Duan J, Jenkinson IR. Book review: Role of biofilms in bioremediation. Frontiers in
Environmental Science. 2017; 15: 5:22.
6. Ramos JL, Marqués S, van Dillewijn P, Espinosa-Urgel M, Segura A, Duque E, rell T,
Ramos-González MI, Bursakov S, Roca A, Solano J. Laboratory research aimed at closing the
gaps in microbial bioremediation. Trends in biotechnology. 2011; 29(12): 641-7.
7. Chorom M, Hosseini SS. Bioremediation of Crude Oil-Polluted Soil by Sewage Sludge
(Symposium 3.5. 2 Risk Assessment and Risk Based Remediation, < Special Issue>
International Symposium: Soil Degradation Control, Remediation, and Reclamation, Tokyo
Metropolitan University Symposium Series No. 2, 2010). Pedologist. 2011; 54(3): 294-301.
8. ebria DY, hodadadi A, Ganjidoust H, Badkoubi A, Amoozegar MA. Isolation and
characterization of a novel native Bacillus strain capable of degrading diesel fuel. International
Journal of Environmental Science & Technology. 2009; 6(3):435-42.
9. Demeter MA, Lemire J, Yue G, Ceri H, Turner RJ. Culturing oil sands microbes as mixed
species communities enhances ex situ model naphthenic acid degradation. Frontiers in
microbiology. 2015; 4(6):936.
10. Tremaroli , acchi Suzzi C, Fedi S, Ceri H, annoni D, Turner RJ. Tolerance of
Pseudomonas pseudoalcaligenes F707 to metals, polychlorobiphenyls and chlorobenzoates:
effects on chemotaxis-, biofilm-and planktonic-grown cells. FEMS microbiology ecology.
2010;74(2): 291-301.
11. hang Y, Wang F, hu X, eng J, hao , Jiang X. Extracellular polymeric substances
govern the development of biofilm and mass transfer of polycyclic aromatic hydrocarbons for
improved biodegradation. Bioresource technology. 2015; 193: 274-80.
12. hoei NS, Andreolli M, Lampis S, allini G, Turner RJ. A comparison of the response of two
Burkholderia fungorum strains grown as planktonic cells versus biofilm to dibenzothiophene
and select polycyclic aromatic hydrocarbons. Canadian journal of microbiology. 2016; 62(10):
851-60.
13. Halan B, Schmid A, Buehler . Real-time solvent tolerance analysis of Pseudomonas sp.
strain LB120ΔC catalytic biofilms. Applied Environmental Microbiology, 2011; 77(5): 1563
-1571.
14. ingaro A, Nicolaou SA, Papoutsakis ET. Dissecting the assays to assess microbial
tolerance to toxic chemicals in bioprocessing. Trends in biotechnology. 2013; 31(11): 643-53.
15. Mandal A , Sarma PM, Singh B, Jeyaseelan CP, Channashettar A, Lal B, Datta J.
Bioremediation: an environment friendly sustainable biotechnological solution for remediation
of petroleum hydrocarbon contaminated waste. ARPN Journal of Science and Technology.
2012; 2(8): 1-2.
16. Seo JS, eum YS, Li X. Bacterial degradation of aromatic compounds. International journal
of environmental research and public health. 2009 Jan;6(1):278-309.
17. O Toole GA. Microtiter dish biofilm formation assay. Journal of isualized Experiments,
2011; 30(47):e2437.
18. Rahman S, Thahira-Rahman J, Lakshmanaperumalsamy P, Banat IM. Towards efficient
crude oil degradation by a mixed bacterial consortium. Bioresource technology. 2002; 85(3):
257-261.
19. Marmur J. A procedure for the isolation of deoxyribonucleic acid from micro-organisms.
Journal of molecular biology. 1961; 3(2):208-IN1.
20. Nie M, Nie H, He M, Lin Y, Wang L, Jin P, hang S. Immobilization of biofilms of
Pseudomonas aeruginosa NY3 and their application in the removal of hydrocarbons from
highly concentrated oil-containing wastewater on the laboratory scale. Journal of
environmental management. 2016;173: 34-40.
21. Weber , Delben J, Bromage TG, Duarte S. Comparison of SEM and PSEM imaging
techniques with respect to Streptococcus mutans biofilm topography. FEMS microbiology
letters. 2014; 350(2): 175-179.
22. im E , Ahn IS, LW L, ML S. Enhanced In-situ Mobilization and Biodegradation of
Phenanthrens from Soil by a Solvent/Surfactant System. Journal of microbiology and
biotechnology. 2001; 11(4): 716-719.
23. Moody JD, Freeman JP, Doerge DR, Cerniglia CE. Degradation of phenanthrene and
anthracene by cell suspensions of Mycobacterium sp. strain PYR-1. Applied Environmental
Microbiology, 2001; 67(4): 1476-83.
24. ahng H, Nam . Molecular characteristics of Pseudomonas rhodesiae strain 1 in
response to phenanthrene. 2002.
25. Xiong , Pang , Wang B, Ji X, Liu Y, Yang G, Dong C, Chen J, Lucia L. Biomechanical
Pulping of Poplar with Crude Enzyme Secreted from Trametes sp. lg-9. BioResources. 2018;
13(2): 3420-8.
26. Nelson P. Index to EPA test methods. United States Environmental Protection Agency, Region I; 2003.
27. Wasi S, Tabrez S, Ahmad M. Use of Pseudomonas spp. for the bioremediation of
environmental pollutants: a review. Environmental monitoring and assessment. 2013; 185(10):
8147-55.
28. De ievit TR. uorum sensing in Pseudomonas aeruginosa biofilms. Environmental
microbiology. 2009; 11(2): 279-88.
29. Ha DG, O Toole GA. c‐di‐GMP and its effects on biofilm formation and dispersion: a
Pseudomonas aeruginosa review. Microbial Biofilms. 2015; 7: 301-317.
30. Høiby N, Ciofu O, Bjarnsholt T. Pseudomonas aeruginosa biofilms in cystic fibrosis. Future
microbiology. 2010; 5(11): 1663-1674.
31. Bjarnsholt T. The role of bacterial biofilms in chronic infections. Apmis. 2013; 121:1-58.
32. Rasamiravaka T, Labtani , Duez P, El Jaziri M. The formation of biofilms by Pseudomonas
aeruginosa: a review of the natural and synthetic compounds interfering with control
mechanisms. BioMed research international. 2015.
33. Emtiazi G, Shakarami H, Nahvi I, Mirdamadian SH. Utilization of petroleum hydrocarbons by
Pseudomonas sp. and transformed E. coli. African Journal of Biotechnology. 2005 ;4(2):172-6.
34. Pazarlioğlu N , Telefoncu A. Biodegradation of phenol by Pseudomonas putida immobilized
on activated pumice particles. Process Biochemistry. 2005; 40(5):1807-14.
35. Radhakrishnan M , Sugumaran E. Efficacy of sugarcane bagasse to produce bacterial biofilm
in water for fish culture. Journal of Ecobiotechnology. 2010.
36. Frankel ML, Bhuiyan TI, eksha A, Demeter MA, Layzell DB, Helleur RJ, Hill JM, Turner
RJ. Removal and biodegradation of naphthenic acids by biochar and attached environmental
biofilms in the presence of co-contaminating metals. Bioresource technology. 2016; 216:
352-361.
37. Accinelli C, Saccà ML, Mencarelli M, icari A. Application of bioplastic moving bed biofilm
carriers for the removal of synthetic pollutants from wastewater. Bioresource technology. 2012;
120:180-186.
38. Sun D, Hale L, Crowley D. Nutrient supplementation of pinewood biochar for use as a
bacterial inoculum carrier. Biology and fertility of soils. 2016; 52(4): 515-522.
39. Ming J, Wang , Yoza BA, Liang J, Guo H, Li J, Guo S, Chen C. Bioreactor performance
using biochar and its effect on aerobic granulation. Bioresource Technology. 2020; 300:
122620.
40. Sun D, Hale L, Crowley D. Nutrient supplementation of pinewood biochar for use as a
bacterial inoculum carrier. Biology and fertility of soils. 2016; 52(4): 515-522.
41. Horemans B, Albers P, Springael D. The biofilm concept from a bioremediation
perspective.2015; 23-40
42. Demeter MA, Lemire JA, Turner RJ, Harrison JJ. Biofilm Survival Strategies in Polluted
Environments. Biofilms in Bioremediation: Current Research and Emerging Technologies.
2016; 15: 43-56.
43. Chandra R, Chowdhary P. Properties of bacterial laccases and their application in
bioremediation of industrial wastes. Environmental Science: Processes & Impacts. 2015; 17(2):
326-42.
44. Arunkumar T, Alex Anand D, Narendrakumar G. Production and partial purification of laccase from Pseudomonas aeruginosa ADN04. Journal of Pure & Applied Microbiology. 2014; 8
(2): 727-731.
45. Strong PJ, Claus H. Laccase: a review of its past and its future in bioremediation. Critical
Reviews in Environmental Science and Technology. 2011; 41(4): 373-434.
46. Han MJ, Choi HT, Song HG. Degradation of phenanthrene by Trametes versicolor and its
laccase. The Journal of Microbiology. 2004; 42(2): 94-8.
47. Di Lorenzo A, arcamonti M, Parascandola P, ignola R, Bernardi A, Sacceddu P, Sisto R, de
Alteriis E. Characterization and performance of a toluene-degrading biofilm developed on
pumice stones. Microbial Cell Factories. 2005; 4(1):4.
48. Liu J, Chen S, Ding J, Xiao Y, Han H, hong G. Sugarcane bagasse as support for
immobilization of Bacillus pumilus H -2 and its use in bioremediation of
mesotrione-contaminated soils. Applied microbiology and biotechnology. 2015; 99(24):
10839-51.
49. hang H, Tang J, Wang L, Liu J, Gurav RG, Sun . A novel bioremediation strategy for
petroleum hydrocarbon pollutants using salt tolerant Corynebacterium variabile HRJ4 and
biochar. Journal of Environmental Sciences. 2016; 47:7-13.
50. Lerch T , Chenu C, Dignac MF, Barriuso E, Mariotti A. Biofilm vs. Planktonic Lifestyle:
consequences for Pesticide 2, 4-D metabolism by Cupriavidus necator JMP134. Frontiers in
microbiology. 2017; 23: 8:904.
51. Johnsen AR, Wick LY, Harms H. Principles of microbial PAH-degradation in soil.
Environmental pollution. 2005; 133(1): 71-84.
52. Decho AW. Microbial biofilms in intertidal systems: an overview. Continental shelf research.
2000; 20(10-11): 1257-73.
_||_