Sayani Mitra

A novel conico-cylindrical flask aids easy identification of critical process parameters for cultivation of marine bacteria

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.

Induced Biofilm Cultivation Enhances Riboflavin Production by an Intertidally Derived Candida famata

The aim of the investigation was to ascertain if surface attachment of Candida famata and aeration enhanced riboflavin production. A newly designed polymethylmethacrylate (PMMA) conico-cylindrical flask (CCF) holding eight equidistantly spaced rectangular strips mounted radially on a circular disk allowed comparison of riboflavin production between CCFs with hydrophobic surface (PMMA-CCF), hydrophilic glass surface (GS-CCF), and 500-ml Erlenmeyer flask (EF). Riboflavin production (mg/l) increased from 12.79 to 289.96, from 54.44 to 238.14, and from 36.98 to 158.71 in the GS-CCF, EF, and PMMA-CCF, respectively, when C. famata was grown as biofilm-induced cultures in contrast to traditional planktonic culture. Production was correlated with biofilm formation and planktonic growth was suppressed in cultivations that allowed higher biofilm formation. Enhanced aeration increased riboflavin production in hydrophilic vessels. Temporal pattern of biofilm progression based on two-channel fluorescence detection of extracellular polymeric substances and whole cells in a confocal laser scanning microscope followed by application of PHLIP and ImageJ volume viewer software demonstrated early maturity of a well-developed, stable biofilm on glass in contrast to PMMA surface. A strong correlation between hydrophilic reactor surface, aeration, biofilm formation, and riboflavin production was established in C. famata. Biofilm culture is a new-found means to improve riboflavin production by C. famata.

Ecological Roles and Biotechnological Applications of Marine and Intertidal Microbial Biofilms

This review is a retrospective of ecological effects of bioactivities produced by biofilms of surface-dwelling marine/intertidal microbes as well as of the industrial and environmental biotechnologies developed exploiting the knowledge of biofilm formation. Some examples of significant interest pertaining to the ecological aspects of biofilm-forming species belonging to the Roseobacter clade include autochthonous bacteria from turbot larvae-rearing units with potential application as a probiotic as well as production of tropodithietic acid and indigoidine. Species of the Pseudoalteromonas genus are important examples of successful surface colonizers through elaboration of the AlpP protein and antimicrobial agents possessing broad-spectrum antagonistic activity against medical and environmental isolates. Further examples of significance comprise antiprotozoan activity of Pseudoalteromonas tunicata elicited by violacein, inhibition of fungal colonization, antifouling activities, inhibition of algal spore germination, and 2-n-pentyl-4-quinolinol production. Nitrous oxide, an important greenhouse gas, emanates from surface-attached microbial activity of marine animals. Marine and intertidal biofilms have been applied in the biotechnological production of violacein, phenylnannolones, and exopolysaccharides from marine and tropical intertidal environments. More examples of importance encompass production of protease, cellulase, and xylanase, melanin, and riboflavin. Antifouling activity of Bacillus sp. and application of anammox bacterial biofilms in bioremediation are described. Marine biofilms have been used as anodes and cathodes in microbial fuel cells. Some of the reaction vessels for biofilm cultivation reviewed are roller bottle, rotating disc bioreactor, polymethylmethacrylate conico-cylindrical flask, fixed bed reactor, artificial microbial mats, packed-bed bioreactors, and the Tanaka photobioreactor.

Enhanced Biotransformation of Fluoranthene by Intertidally Derived Cunninghamella elegans under Biofilm-Based and Niche-Mimicking Conditions

ABSTRACT The aims of the investigation were to ascertain if surface attachment of Cunninghamella elegans and niche intertidal conditions provided in a bioreactor influenced biotransformation of fluoranthene by C. elegans. A newly designed polymethylmethacrylate (PMMA) conico-cylindrical flask (CCF) holding eight equidistantly spaced rectangular strips mounted radially on a circular disc allowed comparison of fluoranthene biotransformation between CCFs with a hydrophobic surface (PMMA-CCF) and a hydrophilic glass surface (GS-CCF) and a 500-ml Erlenmeyer flask (EF). Fluoranthene biotransformation was higher by 22-fold, biofilm growth was higher by 3-fold, and cytochrome P450 gene expression was higher by 2.1-fold when C. elegans was cultivated with 2% inoculum as biofilm culture in PMMA-CCF compared to planktonic culture in EF. Biotransformation was enhanced by 7-fold with 10% inoculum. The temporal pattern of biofilm progression based on three-channel fluorescence detection by confocal laser scanning microscopy demonstrated well-developed, stable biofilm with greater colocalization of fluoranthene within extracellular polymeric substances and filaments of the biofilm grown on PMMA in contrast to a glass surface. A bioreactor with discs rotating at 2 revolutions per day affording 6-hourly emersion and immersion mimicked the niche intertidal habitat of C. elegans and supported biofilm formation and transformation of fluoranthene. The amount of transformed metabolite was 3.5-fold, biofilm growth was 3-fold, and cytochrome P450 gene expression was 1.9-fold higher in the process mimicking the intertidal conditions than in a submerged process without disc rotation. In the CCF and reactor, where biofilm formation was comparatively greater, higher concentration of exopolysaccharides allowed increased mobilization of fluoranthene within the biofilm with consequential higher gene expression leading to enhanced volumetric productivity.

Enhanced biofilm formation and melanin synthesis by the oyster settlement-promoting Shewanella colwelliana is related to hydrophobic surface and simulated intertidal environment.

A direct relationship between biofilm formation and melanogenesis in Shewanella colwelliana with increased oyster recruitment is already established. Previously, S. colwelliana was grown in a newly patented biofilm-cultivation device, the conico-cylindrical flask (CCF), offering interchangeable hydrophobic/hydrophilic surfaces. Melanization was enhanced when S. colwelliana was cultivated in a hydrophobic vessel compared with a hydrophilic vessel. In the present study, melanogenesis in the CCF was positively correlated with increased architectural parameters of the biofilm (mean thickness and biovolume obtained by confocal laser scanning microscopy) and melanin gene (melA) expression observed by densitometry. Niche intertidal conditions were mimicked in a process operated in an ultra-low-speed rotating disk bioreactor, which demonstrated enhanced biofilm formation, melanogenesis, exopolysaccharide synthesis and melA gene expression compared with a process where 12-h periodic immersion and emersion was prevented. The wettability properties of the settling plane as well as intermittent wetting and drying, which influenced biofilm formation and melA expression, may affect oyster settlement in nature.

6 – Bioprocess engineering approaches for the production of marine enzymes

: This chapter focuses on the attempts made to translate novel marine enzymatic activities to commercial bioprocesses. Cultures can be suspended or immobilized in the production medium. The cylindrical tank is the most common reactor and alternatives to the stirred reactor include vessels with no mechanical agitation. There are three principal modes of bioreactor operation: batch, fed-batch and continuous. Solid-state fermentation denotes cultivation of microorganisms on solid, moist substrates. Bioreactors with novel design elements have been applied for studying and enriching marine microbes in bioreactors to attain good control of the environmental factors. Three different strategies can be distinguished for (i) mimicking the natural environment, (ii) stimulating the uncultured microbes or producing metabolites of interest and (iii) controlling redox conditions on the sediment/water interface. Some examples of laboratory reactor-scale production of marine enzymes are: protease produced by Antarctic Bacillus, immobilization of Teredinobacter turnirae and biofilm cultivation of an intertidal gamma-Proteobacterium. Xylanase was produced by the hyperthermophilic Pyrodictium abyssi and L-glutaminase by the marine fungus Beauveria bassiana in a packed-bed reactor. Continuous cultivation of Pyrococcus furiosus produced saccharification enzymes, while quinol oxidase was obtained from a barophilic Shewanella sp. grown in a pressurized vessel. Pyruvate carboxylase was obtained from hyperthermophilic Methanococcus jannaschii.