This review is a synopsis of different bioprocess engineering approaches adopted for the production of marine enzymes. Three major modes of operation: batch, fed-batch and continuous have been used for production of enzymes (such as protease, chitinase, agarase, peroxidase) mainly from marine bacteria and fungi on a laboratory bioreactor and pilot plant scales. Submerged, immobilized and solid-state processes in batch mode were widely employed. The fed-batch process was also applied in several bioprocesses. Continuous processes with suspended cells as well as with immobilized cells have been used. Investigations in shake flasks were conducted with the prospect of large-scale processing in reactors.

A novel reactor system, the rotating disk bioreactor (RDBR), was used to mimic the niche environmental conditions of three salt-tolerant estuarine actinobacteria isolated from the Sundarbans region off the Bay of Bengal, designated MS310 (99% similar in its 16S rRNA gene sequence to Streptomyces parvallus), MS3/20 and MS1/7. The RDBR, operated at a rotational speed of one revolution per day, 50% submergence of discs, aeration rate of 1.0 vvm, and with a sucrose-containing medium, faithfully mimicked the intertidal estuarine habitat of these marine isolates, and supported biofilm formation and production of antimicrobial metabolites-in particular, actinomycin D by MS310. Onset of antibiotic production by MS310 occurs at 20 h in the RDBR compared to 55 h in a conventional stirred-tank bioreactor (STBR). Furthermore, peak antimicrobial activity is attained much earlier in the RDBR with MS310 (at 45 h) than that reported with a terrestrial strain of S. parvallus grown in a STBR (at 144 h). Peak antimicrobial activity of metabolites produced by MS1/7 and MS3/20 were also attained earlier in the RDBR (at 25 and 12 h, respectively) than in a STBR (at 80 and 28 h, respectively). Antibiotic synthesis in the three isolates, in general, appears to be associated with their growth. Overall, the RDBR may be considered the preferred alternative to the STBR for production of antimicrobials by biofilm-forming estuarine bacteria for its much higher surface/volume ratio, lower costs, and easy operability.

After initial small-scale experiments, a 25.0 l rotating disk bioreactor (RDBR) was investigated for the cultivation of a biofilm-forming salt-tolerant Streptomyces sp. MS1/7, producing an antimicrobial compound. Peak activity attainment rate, PAAR (ratio of the peak antimicrobial activity, PAMA and the time taken to attain PAMA) was determined. Of the three pH values examined (8.0, 9.0 and 10.0) maximum PAAR (1.82 mm/h) was attained at pH 9.0. Three aeration rates (9.0, 6.0 and 3.0 l/min) were considered at three levels (25, 50 and 75%) of disk submergence. At the highest aeration rate and 50% submergence level, PAMA (41 mm), PAAR (1.86 mm/h) and biofilm density (BD, 0.91 g/ml) attained their highest values. At any given aeration rate, PAMA was always higher at 50% submergence level. This supports our earlier premise that ideal intertidal conditions, 12 h periods of immersion and emersion, promote maximum BD and antimicrobial production in the niche-mimic RDBR.

A polymethylmethacrylate (PMMA) conico-cylindrical flask (CCF) with an inner arrangement consisting of eight equidistantly spaced rectangular strips mounted radially on a circular disk to provide additional surface area for microbial attachment and ports to allow air supply was employed for melanin production by Shewanella colwelliana and antibiotic production by Pseudoalteromonas rubra. The design allowed comparison of production between (1) CCF with hydrophobic surface (PMMA-CCF), (2) CCF with hydrophilic glass surface (GS-CCF), and (3) standard unbaffled Erlenmeyer flask (EF). Melanin production in the PMMA-CCF was higher by at most 33.5% and growth of S. colwelliana by at most 309.2% compared to the other vessels. Melanin synthesis was positively correlated with reactor surface area and hydrophobicity, suspended cell growth, and biofilm formation. Antibiotic production in the EF was higher by at most 83.3%, but growth of P. rubra was higher in the PMMA-CCF by at most 54.5% compared to the other vessels. A hydrophilic vessel surface, abundant air supply, but low shear stress enhanced antibiotic production. The CCF together with the EF allowed identification of the crucial parameters (vessel surface characteristics, growth, biofilm formation, and aeration) influencing productivity, knowledge of which in the initial stages of process development will facilitate informed decisions at the later phases.

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Actinomycin-D production by a biofilm-forming estuarine isolate îáòK píêÉéíçãóÅÉë sp. MS 310 is studied in smallscale shaken cultures, as well as in a 25 L rotating disk bioreactor, (RDBR) which, when operated at a disk rotational speed of 1 revolution/day with 50% disk submergence, mimics the intertidal conditions of the microbe's niche estuarine habitat-alternating 12 h periods of inundation and exposure. The ideal pH and temperature for antibiotic production are determined (pH 10, 30 o C) through a designed experimental study using shaken flasks. Subsequently, operating conditions in the RDBR are investigated employing a 3 n experimental design wherein each of two (n = 2) parameters îáòK aeration and disk submergence are considered at three levels îáòK high, medium, and low: 9.0, 6.0, and 3.0 L/min for aeration rate; and 75, 50, and 25% for disk submergence, (while maintaining the rotational speed at 1.0 rev/day). The niche-mimic condition along with maximum permissible aeration is found to be most favorable for antibiotic production -peak antibiotic activity (PAA) and peak activity attainment rate (PAAR) simultaneously attaining their highest values: 40 mm and 2.13 mm/h, respectively. Both PAA and PAAR increase with increasing aeration at all operating conditions examined -particularly, at the niche-mimic condition, a threefold increase in aeration rate (3~9 L/min) causes PAA to increase by 33%, whereas PAAR increases by 2.5 times, thus pointing to the strong aeration dependence of this actinomycin-D producer. Again, compared to the best values obtained in the 500 mL shaken flask experiments, corresponding RDBR values are 16% higher for PAA and more than 5 times higher for PAAR -strong evidence for employing these novel bioreactors for cultivation of antibiotic-producer marine microbes. © KSBB hÉóïçêÇëW=~ÅíáåçãóÅáåJaI=åáÅÜÉJãáãáÅI=êçí~íáåÖ=Çáëâ=ÄáçêÉ~ÅíçêI=éêçÇìÅíáîáíó= = = = =

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