Extraction of Ferredoxin from Spinach leaves and its effect on growth of anaerobes

Abstract

Spinach leaves purchased from local markets of Khartoum State in winter season were tested as reducing agents in culture media (solid and liquid) for cultivation of anaerobic bacteria. In the present work, ferredoxin was extracted from the locally grown spinach leaves. The spinach ferredoxin was purified through the Diethyl amino ethyl (DEAE) – cellulose column chromatography at 4oC and 15oC and was sterilized by filtration. Spinach ferredoxin activity was determined by the absorbancy measuerment (UV. Spectrophotometer) in ultraviolet (UV)- visible region. Different concentrations of sterilized spinach ferredoxin were added to the Reinforced Clostridial Agar (RCA) and Reinforced Clostridial Medium (RCM) as substitutes for cysteine hydrochloride. The modified RCA and RCM media were then used for cultivation of Clostridium perfringens and Clostridium chauvoei strains. Absorbancy measurment and viable counts of some of the above mentioned anaerobic bacteria on spinach ferredoxin media gave better counts than the control media (RCA) and (RCM). The highest growth occurred with 1% spinach ferredoxin added to both (RCA) and (RCM). Moreover, spinach ferredoxin supported the anaerobic bacterial growth even in its lowest concentration (0.1%). Generally, some of the tested anaerobic bacteria produced larger colonies on media prepared from the spinach ferredoxin. The redox-potential (E0) was measured by platinum electrode during the growth of Clostridium chauvoei strain (Kad1) on modified RCM without cysteine hydrochloride +1% spinach ferredoxin, RCM (control) and RCM without cysteine hydrochloride or spinach ferredoxin. In this study, ferredoxin was extracted successfully and purified from Clostridium perfringens but not from Clostridium chauvoei strain (Kad1) using the DEAE-cellulose mini column chromatography at 4oC.