غربال‌گری باکتری‌های تثبیت‌کننده نیتروژن مبتنی بر روش رنگ‌سنجی نوین

نوع مقاله : مقاله پژوهشی

نویسندگان

1 گروه میکروبیولوژی، دانشکده علوم زیستی، دانشگاه الزهرا، تهران، ایران

2 بخش بیوتکنولوژی میکروبی- پژوهشگاه بیوتکنولوژِی کشاورزی ایران

10.30466/asr.2025.55844.1870

چکیده

تلاش برای کاهش مصرف کودهای شیمیایی نیتروژن‌دار با هدف توسعه کشاورزی پایدار منجر به اختصاص سهم 70 درصدی کودهای زیستی تثبیت‌کننده نیتروژن اتمسفری در بازار جهانی کودهای میکروبی شده است. پیچیدگی و هزینه بالای روش‌های مرسوم اندازه‌گیری میزان تثبیت زیستی نیتروژن یکی از چالش‌های مهم در غربال‌گری سویه‌های باکتریایی دیازوتروف است که ضرورت معرفی روش‌های ساده، دقیق و ارزان‌قیمت را محرز می‌سازد. هدف پژوهش حاضر توسعه روش جدید رنگ­سنجی مبتنی بر نرم‌افزار رایگان Color grab و گوشی هوشمند همراه بود. این روش تبدیل داده‌های حاصل از دستگاه اسپکتروفتومتری به شاخص‌های RGB و HSV و نیز اندازه‌گیری کمی نیتروژن را با هزینه‌ی کم و سهولت بالا امکان‌پذیر می‌سازد. غربال‌گری کیفی 12 جدایه باکتریایی Bacillus subtilis و سویه استاندارد Azotobacter chroococcum در محیط کشت عاری از نیتروژنNFB) ) و حاوی برموتیمول بلو با تغییر رنگ محیط انجام شد. اندازه‌گیری کمی میزان تثبیت زیستی نیتروژن به کمک منحنی استاندارد ترسیم‌شده با روش اسپکتروفتومتری صورت گرفت. با تبدیل داده‌های حاصل از طیف‌سنجی به شاخص رنگ‌سنجی در گوشی هوشمند همراه، امکان غربال سریع، دقیق و کم‌هزینه سویه‌های تثبیت‌کننده نیتروژن فراهم گردید. بر اساس نتایج حاصل از روش رنگ‌سنجی، شاخصS  (97/0(R2 ≤  در هر دو فضای باز و بسته با موفقیت مبنای ترسیم منحنی استاندارد مبتنی بر غلظت‌های مختلف نیتروژن در محدوده صفرتاµg ml-1  18 قرار گرفتند. بر اساس تغییر رنگ محیط کشت NFB، تمام جدایه‌ها به‌جز جدایه‌های  A15d و g33b قادر به تولید یون آمونیوم بودند. میزان کمی غلظت نیتروژن برای جدایه‌های مورد ارزیابی در محدوده µg ml-1 65/1 تا 32/13 برآورد گردید. بیشترین میزان تولید نیتروژن مربوط به جدایه‌های g19l وW بود که به ترتیب معادل µg ml 32/13 و µg ml-149/12 برآورد گردید. همخوانی بسیار خوب داده‌های حاصل از روش طیف‌سنجی و رنگ‌سنجی مؤید کارایی روش مذکور به‌منظور غربال‌گری دقیق و ساده سویه‌های باکتریایی دیازوتروف بود.

کلیدواژه‌ها

موضوعات


عنوان مقاله [English]

Screening of Nitrogen-Fixing Bacteria Based on a Novel Colorimetric Method

نویسندگان [English]

  • Maryam Layegh Haghighi, 1
  • Mohammadi, Ali 1
  • Maryam Mousivand 2
  • , Elmira Tizjang 2
1 Department of Microbiology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran
2 Microbial Biotechnology Department, Agricultural Biotechnology Research Institute of Iran, Agricultural Research, Education and Extension Organization, 3135933151, Karaj, Iran
چکیده [English]

Sustainable agricultural development has reduced nitrogen fertilizer consumption and resulted in nitrogen biofertilizers accounting for 70% of the global microbial market. The complexity and high cost of conventional methodologies remain major challenges for screening diazotrophic bacteria; therefore, simple, precise, and low-cost techniques should be introduced. In this research, a novel colorimetric method was developed by converting spectrophotometric data into RGB and HSV values using the smartphone Color Grab application to estimate biological nitrogen fixation. Twelve Bacillus subtilis isolates and Azotobacter chroococcum were screened for nitrogen fixation based on the color change of NFB medium containing bromothymolblue. Nitrogen concentration was quantified using a standard curve obtained from spectrophotometric data. Converting spectroscopic data into smartphone colorimetry values enables fast, accurate, and low-cost monitoring of nitrogen-fixing strains. According to the colorimetric results, the S index (R2 ≤ 0.97) in both open and closed systems was successfully used for standard curve drawing in the range of 0 to 18 µg ml-1 to estimate different nitrogen concentrations. Based on the NFB medium color shift, all bacterial isolates except A15d and g33b strains were able to produce ammonia. The nitrogen fixation capacity of the bacterial isolates ranged from 1.65 µg ml-1to 13.32 µg ml-1. Strains g19l and W exhibited the highest nitrogen concentrations, estimated at 13.32 and 12.49 µg ml-1, respectively. The high concordance between the spectroscopic and colorimetric data substantiated the efficacy of the employed method for precise and streamlined screening of diazotrophic bacterial strains.

کلیدواژه‌ها [English]

  • bio-fertilizers
  • colorimetric methods
  • nitrogen fixing bacteria
  • smartphone
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