Pseudomonas aerugionsa BUP2 – a dual producer of lipase and pyoverdine Thesis submitted to the University of Calicut in partial fulflment of the requirement for the award of the degree of DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY By Unni, K. N. Prof. Sailas Benjamin Research Supervisor Enzyme Technology Laboratory Biotechnology Division Department of Botany University of Calicut Kerala- 673 635 April, 2015 Sailas Benjamin,M.Phil., PGDPRJ, PhD., FISBT [PDFs: DFG (Germany), STA (Japan)] Professor of Biotechnology Enzyme Technology Laboratory Biotechnology Division Department of Botany University of Calicut Kerala - 673 635 INDIA Phone: +91-494-2407495 Extn.7406, 7407 (M): +91-94955-48315 Fax: +91-494-2400269 Email: benjamin@uoc.ac.in; Web: www.universityofcalicut.info CERTIFICATE Certified that the Ph.D. thesis entitled Pseudomonas aeruginosa BUP2- a dual producer of lipase and pyoverdine is an authentic record of the original research work accomplished by Mr. Unni, K.N. under my supervisionat the Enzyme Technology Laboratory, Biotechnology Division in the Department of Botany, University of Calicut, and that no part thereof has been presented earlier for the award of any other degree or diploma. Also certified that the contents in the thesis is subjected to Plagiarism Check using the software, iThenticate®, and that no text or data is reproduced from other’s work. Prof. Sailas Benjamin (Research Supervisor) DECLARATION I, Unni, K. N. do hereby declare that this thesis entitled “Pseudomonas aeruginosa BUP2– a dual producer of lipase and pyoverdine” is the summary of the research work carried out by me under the supervision of Dr. Sailas Benjamin, Professor, Department of Botany, University of Calicut in partial fulfilment of the requirement for the award of Ph.D. degree in Biotechnology, and also declare that no part of this thesis has been submitted by me for the award of any other degree or diploma. Calicut University UNNI,K. N. ACKNOWLEDGEMENT I am indebted to numerous persons from the beginning of this work and without all those timely help I could not have completed this work. I accord my profound gratitude to my research supervisor Dr. Sailas Benjamin, Professor, Enzyme Technology Laboratory, Department of Botany, University of Calicut, for the expert guidance, criticisms, constant support and assiduous eforts to bring out the work to its present zenith. I am deeply indebted to Professor John E.Thoppil, Head of Department and Professor K. M. Jayaram, Professor M. Sabu, Professor K.V. Mohanan, former Heads, Department of Botany, University of Calicut and for their whole hearted help ofered throughout the period and for creating conducive environment in the department for the successful completion of my work. I convey my immense sense of gratitude to my labmates Dr. Pradeep, Mr. M. K. Sarath Josh,Mr. S. Sajith and Mrs. Priji Prakasan, Mrs. S. Sreedevi, Dr. V. N. Jisha, Mrs. P. Princy, Ms. Neethu Kannan, Mr. Nideesh, Mr. E. S. Hareesh, Mr.A. Faisal in the Enzyme Technology Laboratory for being very helpful, suggestive and supportive during my research. I gratefully extend my sincere thanks to Professor Abraham Joseph, Dept. of Chemistry AssociateProfessorY. Shibuvardhanan, Dept . of Zoology, Professor P. Manimohan, AssistantProfessor C. C. Harilal, AssociateProfessorRadhakrishnan, AssistantProfessor A. K. Pardeep all other Teaching and Non-teaching Staf , Dept. of Botany for providing needful help during the research period. I convey my immense sense of gratitude to Professor M. V. Joseph,ProfessorManish Kumar and Professor K.K.Elyas, Dept. of Biotechnology, University of Calicut. I am indebted to other departments of Calicut University for providing various instrumentation facilities. I convey my gratitude to Professor Valerie Geofroy, Professor Herbert Budzikiewicz and Professor D. N. Kamrafor studying in molecular characterization of pyoverdine and for providing me a book on ‘Techniques in Rumen Microbiology’ which helped me a lot in the early isolation process. I express sincere thanks to Dr. BinodParameswaran, Scientist, NIIST, Thiruvananthapuram for helping me in RSM analysis. Dept. of Chemistry, University of Calicut for FT-IR analysis, Xcelris Labs, Ahmedabad for molecular identifcation of bacteria. I also convey gratitude to National Institute of Technology, Kozhikodefor taking fuorescent spectroscopy analyses. I extend my thanks to Mr. N. B. Shaji, Mr. K. Ajayakumar and Mr. Santhosh Mithra, Art and Photography Unit, University of Calicut for their assistance in the accomplishment of the photographs for the present study. I also express my thanks to Mr. P. M. Prakashan, Librarian, Department of Botanyfor the help rendered. I also express my thanks to Mr. K. Rajesh and co-workers, Bina Photostat, Villunniyalfor their tireless support in preparing this manuscript. I express my heartfelt gratitude to all my friends especially Santhosh, Anlu, Sajeesh, Bijesh, Sajith, Noufal, Sanjay, Jithin, Deepesh, Sanith, Jhon Bosco, Rahul, Rajan, Prabhu, Madhu, Manu, Vimal, Prasad, Shiney, Aparna, Sajith for their loving support and encouragement. My special words of thanks to all those who directly and indirectly helped me during this period. Words cannot adequately express my deep sense gratitude to my family, especially to my wife Smitha for her love, personal support and great inspiration at all times. My parents,Mr. Mohanapanicker andMrs.P. J. Shyla, for their unconditional love, support and care throughout my life, especially to achieve this milestone in my academic career. My brothers Mr. Sreekanth, Sanoop and Rajeesh for their love and support. My father-in-law Mr. Chandran Nair and mother-in-law C. Soumini, for looking after my son Mahadevan and for all their support to complete this study. Above all these, I owe to the Almighty for giving me the strength and health for the successful completion of the work. Unni, K. N. Dedicated to My Family, Teachers and Friends EQUIPMENTS USED Item Brand Country Chromatography column Magnum India Compound microscope Magnus India Cooling centrifuge Remi India Digital pH meter MK-VI Systronics India Double distillation Unit Borosil India Electrophoresis Unit Biotech India Environmental shaker Orbitek India FT- IR Heating Mantle Jasco Kemi Japan India Gel-documentation system BioRad Italy Image analyser UV trans-illuminator Towa Opticals Biotech R&D Laboratories Japan India Incubator Technico India Laboratory Oven Labline India Laminar air flow cabinet Kemi India Refrigerated centrifuge Remi India Refrigerator Godrej India Sonicator UV-Visible spectrophtometer QSONICA, LLC, XL-2000 Shimadzu USA Japan Spectrofluometer Magnetic Stirrer (KMS – 400) Micropipettes (0.5 -1000 μL) PerkinElmer LS-45 Kemi Accupipete USA India India Vortex mixture Kemi India Water bath Scigenics Biotech India Weighing balance Shimadzu Japan ABBREVIATIONS ddH2O : double distilled water EDTA : Ethylene Diamine Tetraacetic Acid g : gram g/l : :gram per litre gds : dry weight in grams l : litre mg : milligram mg/ml : milligram per millilitre ml : milli litre MW : molecular weight RSM : response surface methodology SDS-PAGE : Sodium dodecyl sulphate-poly acrylamide gel electrophoresis SmF : Submerged Fermentation SSF : Solid State Fermentation TEMED : N,N,N’,N’-tetra methyl ethylene diamine U/g : Units per gram U/mg : Units per milligram w/v : Weight per volume α : alpha β : beta ε : epsilon μg : microgram μl : microlitre μM : micromolar Contents Chapte r Title Page No. 1 Introduction 1 2 Review of Literature 5 3 Isolation and characterisation of microbes from the rumen of Malabari goat 38 4 Utility of rubber seed as potent solid substrates for the production of lipase by Pseudomonas aeruginosa strain BUP2 56 5 Production, optimisation, purification and chracterisation of lipase produced by Pseudomonas aeruginosa strain BUP2 69 6 Pseudomonas aeruginosa strain BUP2 produces type 2 pyoverdine 106 7 Type 2 pyoverdine as turn-off biosensor for the rapid detection of iron and copper in contaminated water 132 8 Summary and Conclusions 156 9 Bibliography 165 10 Appendix I : List of Publications 11 Appendix II : Culture Details Introduction Chapter 1 Introduction Overwhelming demand for a clean and safe environment has led to the discovery of many eco-friendly and biodegradable microbial products to satisfactorily address the public concerns regarding the deleterious effects of various pollutants on nature. Enzymes are the prominent class of microbial products, the use of which can be tracedback to ancient civilisations. The history of enzyme technology began in 1874 when the Danish chemist,Christian Hansen produced the crude rennet by extracting calves' dried stomachs (inner lining of the stomach of newborn or young calves is the rich source of rennet) with saline solution, apparently the first enzyme used for industrial purposes. Today, nearly 4000 enzymes are known to humans; of these, about 200 are in commercial use. Until the 1960s, the total sale of enzymes was only a few million US dollars annually, but by 2015 the global market for industrial enzymes is expected rise to 4.4 billion US dollar business (Binod et al., 2011). Though animals, plants and microbes are the rich source of enzymes, microbial enzymes are preferred to animal and plant sources, because: generally cheaper, easy to control the process parameters, easy to arrange the raw materials and potentially not harmful for use. Of the commercial enzymes, over half is contributed by fungi and yeast, while over a third is from bacteria, and the remaining (10-15%) is from plants and animals (Adrio and Demain, 2014). Carbohydrases (amylase, cellulase, lactase, and pectinase, etc.), proteases and lipases are the chief players on enzyme market. After proteases and carbohydrases, lipases are considered to be the third largest group, based on the total sale of enzymes. Microbial lipases 1 (triacylglycerol acylhydrolase, E.C. 3.1.1.3) are ubiquitous enzymes in nature that catalyse a broad range of reactions such as hydrolysis, inter-esterification, alcoholysis, acidolysis, esterification and aminolysis (Benjamin and Pandey, 1998; Pandey et al., 1999) depending on its aqueous-nonaqueous environment. Commerciallylipases are a billion-dollar business that comprises a wide variety of different applications in detergency, tannery, perfumery, cosmetics, foods, and pharmaceutics (Jaeger et al., 1999). When grown in nutrient medium, microbes usually produce non-ribosomal peptideseither extra- or intra-cellularly which include proteinaceous secondary metabolites such astoxins (e.g., δ-endotoxin (Smitha et al., 2013), siderophores (Saha et al., 2013), pigments (Venil et al., 2013;Pradeep et al., 2014) or biopolymers (Sreedevi et al., 2014). They are not vital for the microbial growth, but play crucial role in pathogenesis and as stored energy molecules. Most of them can be utilised ascandidates for improving health, nutrition and economics of humans by serving as immunomodulators, antitumor agents, receptor antagonists and agonists, pesticides, enzyme inhibitors and growth promoters of animals and plants, and colourants (Demain, 1998). Siderophores are one of the major groups of microbial secondary metabolites produced under iron limiting environment (Meyer, 2000). They are low molecular weight organic compounds embodied with high affinity for Fe (i.e., chelating agent); to solubilise them, which is otherwise unavailable to the microbial cells. These heterogeneous molecules such as pyoverdine, ferrichrome, ornibactin, enterobactin, azotobactin, etc., are secreted by certain bacteria, fungi and grasses (Saha et al., 2013). During the last few decades, siderophores have received much attention owing to their potential roles and 2 http://en.wikipedia.org/wiki/Siderophore http://en.wikipedia.org/wiki/Pigment applications in various areas of environmental as well as pharmaceutical research, owing to their non-toxicity, biodegradability and easy for isolation. Rationale: Microbes show cosmopolitan distribution in air, water, soil, alimentary canal, on skin, leave, etc.Rumen is the first and largest part of the alimentary system in cattle, which serves as the rich habitat for innumerable microbiota, represented by bacteria, fungi and protozoa (Jami and Mizrahi, 2012). Most of the microbiota dwelling in rumen are considered as non- pathogenic with no ill effects on humansi.e., generally regarded as safe (GRAS) status, (Cuting 2011); compared to other microorganisms growing under the harsh environments such as polluted soil, effluents, sewage or industrial wastes. In the light of the aforesaid background, this study focuses on the rumen microorganisms for the production of industrially significant biomolecules with emphasis on lipase and siderophore. Thus, the specific objectives of the present study are: 1. To isolateand cultivate useful bacteriainhabiting the rumen of Malabari goat. 2. To investigate whether the isolates could be used for the production of industrially-significant biomolecules such as lipase and pyoverdine, a siderophore. 3. Statistical optimisation of the production conditions for lipase and pyoverdine. 4. Purification and characterisation of lipase and pyoverdine. 5. Application of pyoverdine as turn-off biosensor 3 Review of Literature Chapter 2 Review of literature Background andrationale Rumen of ruminants is a treasury of many bacteria, fungi and protozoa. Species of many bacterial genera: Bifidobacterium, Butyrivibrio, Lactobacillus, Lactococcus, Propionibacterium and Prevetella (Cotta, 1992; Ogawa et al., 2001; Coakley et al., 2003; Lin et al., 2005; Lin, 2006; Liu et al., 2011); fungal genera: Piromonas, Neocallimastix, Orpinomyces and Sphaeromonas (Wubah and Fuller, 1991; Ando et al., 2009); and protozoans like Dasytricha ruminantium, Isotricha prostoma, Eremoplastron dilobum, Entodinium caudatum, Ophryoscolex purkinjei and Polyplastron multivesiculatum (Williams and Coleman, 1997; Or-Rashid et al., 2008; Khiaosa-Ard et al., 2009, Wright, 2009) were reported from the rumen of various ruminants. Most of the microbial strains have a wide range of genetic and metabolic diversity, which enable them for the production of industrially significant bio-molecules such as enzymes and secondary metabolites. Bacterial strains isolated from the rumen were found thriving on lipids (Hobson and Mann, 1961); cellulose (Stewartet al., 1979); hemicelluloses (Cotta, 1992); starch (Latham et al., 1979); pectin (Paster and Canale-Parola, 1985); sugar (Caldwell and Bryant, 1996) and protein (Hobson and Howard, 1969). Only a few studies were reported on the pigment producing rumen bacteria; a yellow-red coloured pigment was reported from the cellulose and protein degrading bacterial strains isolated from the rumen (Hungate, 1957; Blackburn and Hobson, 1960). Duncan et al. (1999) reported two pigments: a pyocyanin (blue-green pigment) and pyoverdine (yellowish- green pigment) produced by Pseudomonas aeruginosa isolated from sheep rumen. Literature shows that no microflora in the rumen of goat, especially of Malabari goat (Capra hircus L.) is explored so far. Since this study explores the rumen bacteria capable of producing lipase and siderophore, literature is reviewed giving emphasis to: Part I. Bacterial lipases, and Part II. Bacterial siderophores. Part I: Bacterial lipase Lipases or triacylglycerol acylhydrolases (EC 3.1.1.3) catalise lipolytic activities such as hydrolysis, acidolysis, alcoholysis, esterification, trans- esterification, racemic solution, stereo selectivity and chiral synthesis (Reis et al., 2009; Khare and Nakajima, 2000). Stability and selectivity of microbial lipases make it a versatile biocatalyst for various industrial applications (Griebeler et al., 2009). Most of the commercial lipases are of bacterial or fungal origin. The major fungal lipase producing genera include: Aspergillus, Penicillium, Mucor, Rhizopus and Fusarium. Bacillus, Pseudomonas, Achromobacter, Alcaligenes, Burkholderia and Staphylococcus are the predominant bacterial genera producing lipases. Of species of Pseudomonas; aeruginosa, cepacia, fragi and have commercially been exploited for the production of lipase (Kaieda et al., 2001; Alquati et al., 2002; Hazaa et al., 2009; Cesarin et al., 2014). Owing to the unique properties of lipases like stability, specificity and their action over a wide range of pH and temperature, the scientific community is now focused on the large scale production of lipases for use in food, pharmaceutical, cosmetics, leather, detergent and textile industries. The major limitation in the commercial use of lipases owes to their high production cost and lack of effective downstream processing. Hence, consumption of cheap agro-industrial residues as substrates for lipases could reduce the production cost to a considerable level. Detailed discussion presented in the succeeding part of this review would communicate an ample insight into the versatilities of microbial lipases. Structure of Lipases and their mechanism of action Lipases are serine hydrolases acting on the carboxyl ester bonds present in acylglycerols to release fatty acids and glycerol. Their active site consists of a Ser-His-Asp/Glu catalytic triad consisting of a nucleophillic serine located in a highly conserved Gly-X-Ser-X-Gly pentapeptide (Jaeger et al., 1999). The three dimensional structure of lipases revealed a characteristic α/β hydrolase fold (Nardini and Dijkstra, 1999). The catalytic core of lipase is composed of a central β-sheet consisting of up to eight different β-strands connected to six α-helices (Jaeger and Reetz, 1998) (Figure 1). Figure 1. (A) Diagrammatic representation of lipase (Hamam, 2013); (B) X- ray structure of Pseudomonas areuginosa lipase (Jaeger et al., 1999) Figure 2. Schematicdiagram representing the mechanism of lipase action Unlike esterases, lipases are activated only when they are adsorbed to an oil- water interface (Martinelle et al., 1995). Lipase initiates hydrolysis of ester via an attack by the oxygen atom of the hydroxyl group of the nucleophilic serine residue on the activated carbonyl carbon of the susceptible lipid ester bond. As a result, a transient tetrahedral intermediate is formed with the oxyanion stabilised by two or three hydrogen bonds, the so-called oxyanion hole; The ester bond is cleaved and the alcohol moiety leaves the enzyme. The nucleophilic attack by the catalytic serine is mediated by the catalytic histidine and aspartic (or glutamic) acid (Cygler et al., 1994; Schrag et al., 1997). A schematic illustration of general action of lipase is given in Figure 2. Bacterial lipase Production of extracellular bacterial lipases has significant commercial importance, as their bulk production is very easy. Microbial lipases have gained special attention due to their versatile biochemical properties, simple extraction procedures and availability (Macrae and Hammond, 1985; Ghosh et al., 1996). Generally, vegetable oil processing factories, dairies, oil contaminated soil, coal tips, decaying food particles, hot springs and compost heaps are the natural habitats of lipase producing microorganisms (Wang et al.,1995) (Table 1). Bacterial lipases are classified into 8 different families; of this, family I is the largest, which houses 6 subfamilies (Arpigny and Jaeger, 1999). The lipases from Pseudomonas spp. are classified under the Families I.1 and I.2. Pseudomonas lipase Pseudomonas represents the heterogenous group of Gram-negative bacteriasuch as Burkholderia cepacia, Burkholderia multivorans, Pseudomonas aeruginosa, etc., the well known producers of lipase.Pseudomonas lipases exhibit interesting properties such as thermo resistance, coupled with activity at alkaline pH, which make them potential candidates for various biotechnological applications. Another important character of Pseudomonas lipases is its enantio-/ stereoselective nature, in which they have the capability to distinguish between the enantiomers in a racemic mixture (Jaeger and Reetz, 2000), and this unique property, is now widely exploited in pharmaceutical and agricultural sectors. Thermostable Pseudomonas lipases were reported to withstand 100 oC or even beyond to 150 oC with a short span of a few seconds (Andersson et al., 1979; Swaisgood and Bozoglul, 1984; Rathi et al., 2001). An alkaline lipase from P. alcaligenes M-1 was found better for eliminating fatty stains from clothes under machine wash conditions (Gerritse et al., 1998). Lipase from Pseudomonas cepacia acts as a most effective catalytic agent for the ethanolysis and methanolysis of grease (Hsu et al., 2002). Table 1. Prominent bacteria producing lipases. Bacteria Reference Achromobacter sp. Mitsuda et al., 1990 Acinetobacter sp. Wakelin and Forster, 1997 Alcaligenes sp. Ngooi et al., 1990 A. denitrificans Odera et al., 1986 Arthrobacter sp. Hirohara et al., 1985 Bacillus laterosporus Toyo-Jozo, 1988 B. sphericus Toyo-Jozo, 1988 B. thermocatenulatus Rua et al., 1997 B. thiaminolyticus Toyo-Jozo, 1988 Chromobacterium sp Mitsuda et al., 1992 C. viscosum Wu et al., 1996 Cryptococcus laurentii Yasohara et al., 1995 Flavobacterium ferruginem Toyo-Jozo, 1988 Geotrichum candidum Ngooi et al., 1990 Glomus versiforme Gaspar et al., 1997 Hansenula anomala Ionita et al., 1997 Humicola lanuginose Macris et al., 1996 Mycobacterium chelonae Chen et al., 1997 Neurospora sitophila Beuchat, 1982 Nocardia amarae Wakelin and Forster, 1997 Protaminobacter alboflavus Toyo-Jozo, 1988 Pseudomonas sp. Buisman et al., 1998 P. aeruginosa Odera et al., 1986 P. cepacia Ziemann et al., 1994 P. fluorescens Wu et al., 1996 P. fragi Santarossa et al., 2005 P. pseudoalcaligenes Lin et al., 1996 Staphylococcus hyicus Gotz et al., 1998 S. warneri Talon et al., 1996 S. xylosus Talon et al., 1996 Substrates for lipase production Most of the microbial lipases are extracellular and their production is highly influenced by the composition of the medium, besides physico-chemical factors such as temperature, pH, and dissolved oxygen. Lipases are inducible enzymes, therefore, enhancement in production generally occurs in the presence of a lipids or lipid-like substrates such as hydrolysable esters, oil industry wastes, vegetable oils, surfactants, fatty acids, triacylglycerols, bile salts, glycerol and tweens (Sharma et al., 2001; Damaso et al., 2008). Several abundant and cheap agro-residues like brans, oil cakes, bagasse, cottonseed and soybean sludge have been reported as effective for lipase production. Owing to high nutritional content, the agro-industrial residues are considered as very fine substrates for enzyme production; which may help to overcome the agricultural waste management problem, especially via solid state fermentation (Mahanta et al., 2008). In addition to this, various nitrogen sources like peptone, yeast extract, tryptone, meat peptone, ammonium sulfate, potassium nitrate, etc. were found to enhance lipase production (Lima et al., 2003). Lipase production by fermentation Submerged fermentation (SmF) has been defined as fermentation in the presence of excess water and it is very easy to monitor. Bacillus thermoleovorans ID-1 produced a thermophilic lipase in a medium containing 1.5 % olive oil; whose activity was 520 U/ml at pH of 7.5 and 70 oC (Lee et al., 1999). Pseudomonas aeruginosa strain Pse A produced lipase (4580 U/ml) in a medium containing gum arabic as inducer, which was found to be tolerant to organic solvents (Ruchi et al., 2008). Solid state fermentation (SSF) is a microbial process in which a solid material is used as the base substrate, on which microorganisms grow well and produce higher quantities of extracellular enzymes and other metabolites than they do in SmF. Mahanta et al. (2008) used deoiled jatropha seed cake as carbon source for the cultivation of Pseudomonas aeruginosa Pse A, and 625 U/gds lipase activity was reported. Purification strategies for bacterial lipases Bacterial lipases are mostly secreted in the medium, purification from the culture medium is the major task faced by industries. In many cases, enzymes used in commercial applications need not require high purity (e.g., detergent industry), but in medical, cosmetic, food, analytical chemistry, and for elucidating the protein structure; high purity enzyme is required (Taipa et al., 1992; Aires-Barros et al., 1994; Saxena et al., 2003). Ideal purification strategies adopted in industries should be of low-cost, quick, high-yielding and agreeable to large-scale operations. In addition, it must have the capability for continuous product recovery, with a relatively high selectivity for the desired product. About 80% of the purification schemes attempted thus far have used a precipitation step; followed by gel filtration, and ion exchange chromatography (Table 2). Recently emerged purification strategies include: immune purification, aqueous two-phase systems, reversed micellar system, membrane processes, hydrophobic interaction chromatography employing an epoxy activated spacer arm as a ligand, column chromatography with PEG (polyethylene glycol)/ sepharose gel or poly(vinyl alcohol) polymers as stationary phase, and aqueous two-phase systems (Saxena et al., 2003). Table 2. Purification strategies for bacterial lipases Bacterium Purification strategy Reference Acinetobacter calcoaceticus AAC323-1 Triton X-114-based aqueous two- phase partition Bompensier i et al., 1996 A. radioresistens CMC-1 Ammonium sulfate, PD-10 column, Mono Q, phenyl-Sepharose CL-4B column chromatography Hong and Chang, 1998 Acinetobacter sp. RAG-1 Mono Q, butyl Sepharose column, elution with Triton-X 100 Snellman et al., 2002 Bacillus sp. Ammonium sulfate, acrinol treatment, DEAE-Sephadex A-50,Toyopearl HW-55F, butyl Toyopearl 650 M Sugihara et al., 1991; Palekar et al., 2000 B. alcalophilus 50% ammonium sulfate, Sephadex G- 100 Ghanem et al., 2000 B. pumilus Ammonium sulfate fractionation, gel filtration on Sephadex G-100 Jose and Kurup, 1999 B.stearothermophilu s (recombinant lipase) CM-Sepharose, DEAE Sepharose Kim et al., 2000 B. thermocatenulatus Calcium soap, hexane extraction, methanol precipitation, Q-Sepharose (ion exchange) Schmidt- Dannert et al., 1994 Chromobacterium viscosum Alginate (macroaffinity ligand), elution by NaCl, 0.5 K Sharma and Gupta, 2001 Pseudomonas sp. G6 Silicone 21 defoamer, ammonium sulfate (60% saturation) fractionation Kanwar et al., 2002 P. aeruginosa Ammonium sulfate precipitation, hydroxyapatite column Chromatography Sharon et al., 1998 P. cepacia Polyoxyethylene detergent C14EO6- based aqueous two-phase partitioning Terstappen et al., 1992 P. fluorescens Ultrafiltration, ammonium sulfate precipitation, DEAE-Toyopearl 650 M, phenyl Toyopearl 650 M Kojima et al., 1994 P. Acetone precipitation, Sephadex G- Lin et al., Bacterium Purification strategy Reference pseudoalcaligenes F-111 100 chromatography, fractogel phenyl 650 M chromatography, Sephadex G- 100 Chromatography 1996 P. pseudomallei Ammonium sulfate, Sephadex G-150 Kanwar and Goswami, 2002 P. putida 3SK DEAE-Sephadex A-50, Sephadex G- 100 Lee and Rhee, 1993 Serratia marcescens Ion-exchange chromatography, gel filtration Abdou, 2003. Staphylococcus haemolyticus 80% ammonium sulfate, DEAE- Sepharose CL-6B column, CM- Sepharose CL-6B, resource S column (ion-exchange chromatography) Oh et al., 1999 S. warneri 863 Nickel–NTA affinity chromatography, hydroxyapatite column (HIC) Kampen et al.,2001 His6-S. aureus Protamine sulfate, ammonium sulfate, nickel nitrilotriacetate, hydroxyapatite Simons et al., 1998 Properties of bacterial lipases Desired properties of lipase decide its industrial value. Various properties of bacterial lipases such as pH, temperature, stability, effective of metal ions, substrate specificity are evaluated under the following sections. pH and temperature Usually, bacterial lipases shows neutral (Dharmsthiti et al., 1998; Lee et al., 1999) or alkaline pH optima (Schmidt-Dannert et al., 1994; Sidhu et al., 1998a, 1998b; Sunna et al., 2002), by the exemption of P. fluorescens SIK W1 lipase, which has an acidic optimum at pH 4.8 (Andersson et al., 1979). Lipases from Bacillus stearothermophilus SB-1, B. atrophaeus SB-2 and B. licheniformis SB-3 show activity in broad pH range (Bradoo et al., 1999). Bacterial lipases exhibit stability over a wide range, from pH 4 to 11 (Kojima et al., 1994; Wang et al., 1995; Khyami-Horani, 1996; Dong et al., 1999). In general, bacterial lipases possess temperature optima in the range 30 – 60°C (Lesuisse et al., 1993; Wang et al., 1995; Dharmsthiti et al., 1998; Litthauer et al., 2002). But, reports suggest that bacterial lipases exhibit temperature optima in lower as well as higher ranges (Dharmsthiti and Luchai, 1999; Lee et al., 1999; Oh et al., 1999; Sunna et al., 2002). Nawani and Kaur (2000) reported that the thermostability of lipase from Bacillus sp was improved by the addition of stabilisers i.e., glycerol, sorbitol, ethylene glycol which retained the enzyme activity at 70 °C even after 150 min incubation. A few species form Pseudomonas have been claimed that the enzymes are stable at 100 °C or even beyond 150°C (Andersson et al., 1979; Swaisgood and Bozoglu, 1984; Rathi et al., 2001). B. stearothermophilus lipase was highly thermotolerant with a half-life of 15 - 25 min at 100°C (Bradoo et al., 1999). Stability in organic solvents Lipase stability in organic solvents is desirable for using them in various chemical reactions. Schmidt-Dannert et al. (1994) reported that acetone, ethanol and methanol, etc. could the activity of B. thermocatenulatus lipase, but acetone and hexane inhibited the action of lipases fromP. aeruginosa YS- 7 and Bacillus sp. (Sugihara et al., 1991). Lipase from A. calcoaceticusLP009 showed unstability with various organic solvents (Dharmsthiti et al., 1998). Effect of metal ions Cofactors are normally not necessary for lipase activity, but Ca2+ (divalent cations) often enhance its activity. Metal ions such as Co, Hg and Sn were found to inhibit the activity of lipase enzyme (Patkar and Bjorkling, 1994). Calcium-activated lipase were reported from a number of bacteria such as P. aeruginosa EF2 (Gilbert et al., 1991), B. thermoleovorans ID- 1 (Lee et al., 1999), B. subtilis 168 (Lesuisse et al., 1993), S. aureus 226 (Muraoka et al., 1982). In contrast, activity of lipase from P. aeruginosa 10145 was inhibited in the presence of Ca2+ (Finkelstein et al., 1970); on the other hand, lipase from A.calcoaceticus LP009 was stimulated by Fe3+ (Dharmsthiti et al., 1998). Application of lipases Lipases in detergent industry The lipolytic activity of lipases is mainly used in detergency, especially thermophilic and alkalophilic are preferred in this area. Mostly, it should be capable of performing in the presence of the various components of washing powder formulations (Posorske, 1984; Cheetham, 1995). To increase the action of detergency, a combination of various enzymes such as lipase, amylase, protease and cellulase are used in modern heavy duty powder detergents and automatic dishwasher detergents (Ito et al., 1998). The main advantage of these bio-detergents is high biodegradability, lack of any harmful residues, no adverse effect on sewage treatment processes and do not cause a risk to aquatic life. Pseudomonas alcaligenes lipase showed elevated activity at washing conditions, such as alkaline pH (7 - 11) and at a high temperature up to 60°C (Misset et al., 1994). Novo group has introduced an alkaline and positionally non-specific lipase from Streptomyces sp., which could be used in a wide range of applications like laundry, dish-washing detergents and industrial cleaners (Pandey et al., 1999). Lipases in food technology Modification of fat and oil is one of the major areas in food processing industry, a large potential market in future. Lipases are added to food for improving flavor and taste by the production of esters involving short chain fatty acids and alcohols (Macedo et al., 2003). The lipases play a vital role in the fermentation process of sausage production and to regulate the changes in long-chain fatty acid addition in ripening. Lipase mediated food products in the market include bread, nutraceuticals, chocolates etc. Before, lipases of diverse microbial origin have been used for cleansing rice flavor, altering soybean milk and for progress the aroma and increase the fermentation of apple wine, preparation of Koji (fermented cooked rice and/or soya beans), etc. (Seitz, 1974). Pulp and paper industry Microbial lipases have some crucial role in pulp industries (Bajpai, 1999). Lipases are widely used for increasing the pulping rate of pulp, to augment the whiteness and intensity and deinking of wastepaper. So, it can help the decrease of chemical usage, prolong equipment life, decrease the risk of pollution level in water and reduce composite cost; and also used to remove hydrophobic components of wood known as ‘pitch’ (Irie et al., 1993). Use of lipase in textile industry Use of lipases in textile industries is mainly found in the removal of lubricants and to give a fabric with better absorbency for enhanced levelness in dyeing; in addition, it reduces the chance for frequency of line and break in denim scrape systems. Lipase enzymes commercially used for the preparation of the desizing of denim and other cotton fabrics. A commercial lipase from Amano Pharmaceutical KK was dissolved in solution with aliphatic polyester, which improved fabric texture without losing its strength (Dyson et al., 2006); lipase can improve the wetting ability and absorbance in polyester fabrics (Hsieh and Cram, 1998). Moisture regaining ability of polyethylene terephthalate fabrics was found to be improved upon using lipases from P. cepacia and P. fluorescens (Kim and Song, 2006). However, lipase from Pseudomonas spp. was shown to degrade polymers of aliphatic polyethylene (Muller et al., 2005). Part II. Bacterial siderophores Introduction Iron (Fe) is an essential micronutrient necessary for the growth and survival of bacteria, and it directly controls a wide range of metabolic and signaling functions of the cell (Third et al., 2000). In nature, iron exists mainly in two forms i.e., the reduced ferrous (Fe2+) and the oxidised ferric (Fe3+) forms, whose redox potentials fluctuate depending on the molecules to which the iron is bound. This special characteristic feature turned iron into a major redox mediator in biological systems. In living cells, Fe is associated with iron-sulfur clusters or heme, and it plays significant roles in catalytic mechanisms of many enzymes like dehydrogenases, reductases, nitrogenases, etc. (Chincholkaret al., 2007). Though it is abundant on the earth’s crust, iron is not readily accessible in natural environment to many microorganisms owing to its insolubility in water. In order to circumvent this problem, many bacteria secrete chelating molecules called siderophores that solubilse iron to an easily assimilatory form (Meyer et al., 2002). Siderophores are low molecular weight, high affinity iron chelating compound comprised with a chromophore, an acyl moiety and variable peptide chain (Unni et al., 2004). Siderophores are produced by plants, bacteria, fungi and actinomycetes. Various types of siderophores are identified and characterised from the bacteria such as Shigella sp., Salmonella sp., Escherichia coli, Yersinia sp., Vibrio sp., Bordetella sp., Pseudomonas spp., Mycobacterium tuberculosis, Staphylococcus sp. and Bacillus anthracis (Table 3). Siderophores are used to trap, mobilise and transport iron to the microbial cells and play a crucial role in the pathogenesis and biofilm formation. Moreover, they tune the microflora of the surrounding environment by regulating the iron availability and protect themselves from heavy metal toxicities (Neilands, 1981). With the advent of novel technologies, many efforts have been accomplished to utilise these extracellular proteinaceous compounds for the welfare of human kind. Predominantly, they found potential applications in environmental as well as pharmaceutical research. Upon this background, this review critically evaluates the sources, types and applications of microbial siderophores. Table 3. List of some bacteria producing siderophores Microorganism Siderophore Molecular weight Ligand type Reference Acinetobactor baumanni Acinetobactin 346.3 Da Phenolic / Hydroxamate Yamamoto et al., 1994 Agrobacteruium sp. Agrobactin 403 Da Catechol 2-hydroxy phenyloxazoline Sonoda et al., 2002 Alteromonas luteoriolaces Alterobactin 927.9 Da Phenol/α- hydroxycarboxylate Reid et al., 1993 Azotobactor vinelandii Azotobactin 1138 Da Hexadentate hydroxamate / Phenolate Tindale et al., 2000 Bacillus subtilis Bacillibactin 882.8 Da Catecholate May et al., 2001 Bordetella sp. Alcaligin 404.4 g/mol Hydroxamate Hou et al., 1998 Burkholderia cepacia Ornibactin 734 g/mol Hydroxamate/ Hydroxycarboxylate Skol et al., 1999 Erwinia chrysathemi Chrysobactin 369.4 g/mol Bidentate Neema et al., 1993 Escherichia coli Enterobactin 669.6 g/mol Catechol Pollack and Neilands, 1970 Halomonas aquamarina strain DS40M3 Aquachelin Hydroxamte Martinez et al., 2002 Marinobactor hydrocarbonoclasticu s Petrobactin 719.8 Da Hydroxy carboxylate Barbeau, et al., 2002 Mycobacterium tuberculosis Mycobactin 828.0 g/mol Hydroxamate / Phenolate Snow, 1970 Pseudomonas aeruginosa Pyoverdine 1,365.4 g/ mol Hydroxamate / Phenolate Vossen et al., 1999 Pseudomonas cepacia Cepabactin 155.2 Da Bidentate Meyer etal., 1989 Rhodococcus erythropolis IGTS8 Heterobactin 598.9 Da Hexadentate hydroxamate / Phenolate Carran et al., 2001 Salmonella sp. Salmochelin 993,8 Da Catecholates and Phenolates Valdebenit o et al., 2006 Shigella sp. Aerobactin 564.5 g/mol Hexadentate hydroxamate / α- Hydroxycarboxylate Le Roy et al., 1993 Streptomyces pilosus Desferrioxam ine B 560.7 g/mol Hexadentate hydroxamate Gordeuk et al., 1992 Streptomycocus sp. Staphylo- 448.1 Da α-Hydroxy- Meiwes et Microorganism Siderophore Molecular weight Ligand type Reference ferrin B carboxylate / α- aminocarboxylate al., 1990 Vibrio cholera Vibriobactin 705.3 Da Catechol Grifith et al., 1984 Yersinia pestis Yersinibactin 479.1 Da Catecholate Perry et al., 1999 Types of siderophores Siderophores typically appeared as constant and stable complexes, and exhibit high affinity towards Fe rather than other metal ions present in nature (Hofte, 1993). The efficiency of a siderophore to chelate iron depends on the stability of the metal complex it forms (Wittenwiler, 2007). Generally, siderophores are synthesised by an array of complex multi-enzyme units, and most of them have a peptidic backbone with side chain of amino acid derivatives, which facilitates binding sites for iron. Siderophores are generally classified into three types, based on the moiety that donate oxygen (binding moiety) for coordinating with iron (Miethke, 2007; Saha et al., 2013). The binding groups of siderophores may include catecholate (phenolates),hydroxa- mates, carboxylates or mixed ligands (Figure 3 and 4). All the binding sites possess two coordinating sites for iron via lone pair of oxygen atoms, thereby forming a pentagonal ring; and most of the siderophores exist as hexadentate or octahedral complexes (Wittenwiler, 2007). Catecholate siderophores Catechol type siderophores possess catecholateC6H4(OH)2or 2,3- dihydroxybenzoate iron binding groups with two oxygen atoms for chelation. They may be linear or cyclic molecules. Enterobactin produced by E. coli, Aerobacter aerogenes and Salmonella typhimurium is a typical example for cyclic catecholate type siderophores, which is made of a cyclic triester of 2,3- dihydroxybenzoylserine (Saha et al., 2013, 2015). Agrobactin produced by Agrobacterium tumifaciens is a linear catecholate sideriphore with three residues of 2,3-dihydroxybenzoic acids, a spermidine chain and a oxazolin ring (Ong et al., 1979). Presence of all the catecholate siderophores could be detected by Neilands spectrophotometric assay, in which the siderophores reacts with FeCl3 to form a wine coloured complex having absorption maximum at λ495 (Neilands, 1981). Hydroxamate siderophores Gram-positive bacteria like Pseudomonas fluorescens, Neisseria gonorrhoeae, N. meningitids,etc., and actinomycetes are the major producers of hydroxamate siderophore, which possesses a characteristic structure [C(=O)N-(OH)-R], where R is an amino acid or its derivative (Saha et al., 2015). Ferrichrome is one of the largest family of hydroxamate siderophores mainly produced by fungi such as Fusarium and Trichoderma spp. Ferribactin is another hydroxamate siderophore reported from Pseudomonas fluorescens (Philson and Llinas, 1982). These compounds show the maximum absorbance between λ425-500 when bound to iron (Ali and Vidhale, 2013). The ferrioxamines and Desferrioxamine B are yet other important class of siderophores secreted by Streptomynces pilosus (Muller and Raymond, 1984). Carboxylate siderophores Carboxylate siderophores possess carboxyl or hydroxyl donor groups for iron ligation. ‘Rhizobactin’, produced by Rhizobium meliloti is one of the best characterised carboxylate types of siderophore(Smith et al., 1985). Staphyloferrin A provides two tridentate pendant ligands, comprising of a beta-hydroxy and a beta-carboxy-substituted carboxylic acid derivative, for octahedral metal chelation (Konetschny‐Rapp et al., 1990). Siderophores with mixed ligands Pyoverdine (PVD) is one of the best examples of siderophore with mixed ligands. The fluorescent Pseudomonas spp. such as P. aeruginosa, P. chlororaphis, P. fluorescens, P. putida and P. syringae are well-known producers of PVDs (Meyer, 2000). Of them, P. aeruginosa is the predominant producer of PVD. Reportedly, three distinct PVD types are being produced by strains of P. aeruginosa, viz., PVD 1, PVD 2 and PVD 3 - each subtype has characteristic peptide chain (Meyer et al., 1997). Of them, PVD 1 and PVD 2 together contribute 84% of PVDs, i.e., and the remaining 16% is represented by only PVD 3 subtype (Meyer et al., 1997). Mycobactins, produced by Gram-positive Mycobacterium tuberculosis is another mixed ligand siderophore, which has two hydroxamates, a phenolate, and oxazoline groups for ligation. The deduced structure of type PVDs are shown in the following Figure 5 (Visca et al., 2007). Group Chelation Hydroxamate Obj100 Catecholate Obj101 Hydroxyl- carboxylate Obj102 Figure 3. Major types of siderophore ligands and their co-ordination pattern Obj103 Fi gure 4. Ligands of bacterial siderophore Figure 5. Types of PVD (Visca et al., 2007) Role of siderophores in bacterial survival Siderophores play a crucial role in microbial pathogenecity. In humans, iron is bound to many protein complexes present both in intracellular as well as extracellular fluids, haeme in the haemoglobin with central Fe is a typical example. But the strictly regulated homeostasis does not allow the availability of free iron for pathogens in the human body. Hence, the bacterial siderophores contribute significantly to its virulence, i.e., by stealing Fe from the host for its survival (Wooldridge and Williams, 1993). The siderophores act as chelating agents with an extremely high affinity for Fe3+ with stability constants of around1030/M (Albrecht-Gary et al., 1994). 27 When the bacterium enters the host where the availability of iron is low, they secrete siderophores to the external environment to compete with the host proteins (like transferrin and hemoglobin) for capturing Fe ion. For instance, Staphylococcus aureus, a human pathogen tactically chelates iron for its invasion establishment in the host. During the staphylococcal infection, bacterium secretes the toxin called hemolysins, which rupture the red blood cells and release hemoglobin, followed by degradation into heme and free Fe ion (Tong and Guo, 2009). The siderophore-Fe3+ complexes then return back to the specific cell surface receptors, which in turn is dissociated intracellularly to release Fe2+ to be incorporated into the bacterial metalloenzymes; and the excess of Fe ions are stored in bacterioferritins and other related proteins (Chiancone et al., 2004; Chu et al., 2010). Upon assimilating Fe in sufficient quantities, further secretion of siderophore is suppressed by a repressor protein called Fur by organising itself into the DNA sequence that regulates the biosynthesis of siderophores (Chu et al., 2010). It is found that metals other than Fe can be sequestered by siderophores in some bacteria. For instance, azotochelin produced by Azotobacter vinelandii sequesters molebdate rather than iron due to the high stability of azotobacter- Mo complexes, which in turn reduces the availability of free siderophores to sequester Fe. Thus, the iron deficiency induces the bacterium to produce a more efficient iron chelator, the protochelin (Duheme et al., 1998). Similarly, pyoverdine and pyochelin, the two major siderophores produced by P. aeruginosa were able to chelate Ag+, Al3+, Cd2+, Co2+, Cr2+, Cu2+, Eu3+, Ga3+, Hg2+, Mn2+, Ni2+, Pb2+, Sn2+, Tb3+, Ti+ and Zn2+ present in the growth medium at varying intensities, apart from Fe. Interestingly, the siderophores only sequester the elements but not allow them to enter the bacterial cells, and thus 28 protect the microbes from heavy metal contaminations (Braud et al., 2009 a,b). Usually, these heavy metals diffuse into the cells via porins present in the cell membrane, but their diffusion into the cell dependent on the size of the sequestered form, i.e., larger ones will be restricted from entry (Schalk et al., 2011). Siderophore iron complex transport mechanism in bacterial cell Ferri-siderophores, the chelated Fe3+ siderophore complexes are transported across the double membrane system of Gram-negative bacteria through the specific receptors on the outer membrane, whereas they are transported by membrane anchored binding proteins in Gram-positive bacteria (Koster, 2001; Braun and Braun, 2002). The transportation of ferri-siderophores is well explored in E. coli, which is mediated by three multi-component protein systems of the outer membrane, periplasm and cytoplasmic membrane (Ali and Vidhale, 2013). In E. coli, the ferri-siderophores binds to the outer membrane protein called Fep A, which is a barrel shaped protein made up of 22 beta strands with a cork or plug like extension made of approximately 160 aminoacid residues. In order to transport the metal conjugate through Fep A, the cork has to be dislocated by utilising the energy from the gradient across the inner membrane with the help of two sets of proteins, Ton B (span the entire periplasm) as well as the cytoplasmic membrane anchor proteins called ExbB and ExbD. In the periplasmic space, the ferri-siderophores are transported to the ABC-transporter system which is composed of 2 proteins, permease that spans the entire inner membrane and ATPase that hydrolyse ATP to release energy, required for the transportation into the cytoplasm. The mechanism by which iron is released from the siderophore is not elucidated completely to date; however, the bound Fe3+ of siderophore complexes are 29 reduced enzymatically to Fe2+; which does not have a high affinity for siderophore, so that it gets dissociated from the complex (Figure 6). Figure 6. Transport of siderophores into the bacterial cell In Gram-positive bacteria too, transportation of siderophores is mediated by the ABC-transporter proteins as that of Gram-negative bacteria (Grigg et al., 2010). Biotechnological application of siderophores Now-a-days, the heavy metal sequestering coupled with the coordinating properties of siderophores is exploited in various field of biotechnology. It is found that the siderophore are successfully used as a drug for the treatment of 30 iron over loaded therapy, drug delivery systems against multidrug resistant bacteria, bioremediation and biosensor development (Figure 7). Obj104 Figure 7. Major applications of siderophores Medicinal applications Metal overload toxicity is one of the major deadly conditions, frequently observed in chronically dialysed patients as they have lost the ability to eliminate the metals via renal excretion. For instance, β-thalassemia is characterised by the overload of iron in the blood stream, which may lead to the damage of liver through hemochromatosis and hemosiderosis; heart and endocrine systems. In such cases, siderophore-mediated treatment is found to be effective. Desferrioxamine, a siderophore produced by Streptomyces 31 pilosus is the only commercially available chelating agent for the treatment of diseases due to Fe and Al overload; though it has got several disadvantages such as low sequestering, lipophilicity and oral inactivity (Gabutti et al., 1996; Ackrill et al., 1980). Clinical investigations conducted so far, have confirmed that iron supports the growth of tumor cells in human body. The catalytic effects of iron generate free radicals in an uncontrollable manner, which inactivate the host defense mechanism against neoplastic cells (Lijec Vjesn, 2000). Hence, it is believed that the elimination of free metal iron from the blood stream can prevent the proliferation of malignant cell effectively. Combination therapy of desferrioxamine with recombinant α-2-interferon is found effective in the treatment of hepatocellular carcinoma (Kontouras et al., 1995). The synergetic effect of deferoxamine and an IgG monoclonal anti-transferrin receptor antibody could be used to control the proliferation of murine lymphoid tumors (Kemp et al., 1995). Siderophores such as pyridinone, desferrioxamine, deferiprone were found effective for the removal of non- transferrin bound iron from blood stream after chemotherapy (Miller, 1989). As siderophores cause iron depletion in cells, it can also be used for the treatment of many parasitic diseases. For instance, desferrioxamine B restricts the growth of Trypanosoma brucei, a protozoic parasite causing sleeping sickness (Breidbach et al., 2002). Similarly, aerobactin produced by Klebsiella pneumonia, acts as antimalarial agent (Gysin et al., 1993). Siderophore mediated drug delivery Now-a-days bacteria acquire antibiotic resistance at an alarming rate. Reports revealed that almost all approved antibacterial drugs used for clinical 32 practices as facing bacterial resistance at different intensities; which indicates that no existing antibiotic will be effective in coming one or two decades. The seriousness of this problem is becoming obvious as resistant infections once limited to hospitals are dispersed to the community as well (Marshall, 2008). Siderophore-mediated iron gaining is necessary for the virulence of most pathogenic bacteria, which bear specific cell surface receptors for the purpose (Miethke and Marahiel, 2007). Hence, the siderophore-mediated delivery of antibiotics can selectively target the specific receptors on antibiotic resistant bacteria causing disease and the treatment is now known as Trojan horse strategy. Trojan horse strategy utilises the coordination abilities of siderophores to carry drugs specifically to the target cells. The siderophore- antibiotic conjugate can overcome the membrane associated resistance mechanisms and augment the effectiveness of drugs up to 100-folds relative to passive diffusion (Braun, 1999). In this strategy, the target-specific siderophore is bound to the drug with the help of a spacer, and all together enters the cell where the spacer is hydrolysed enzymatically to release the drug thereby causing death of the cell. Many natural and synthetic antibiotic conjugates are used for the antimicrobial therapy. The naturally occurring siderophore-antibiotic complexes are known as sideromycins (Braun et al., 2009). For instance, albomycin, produced by Actinomyces subtropicus is composed of an antibiotic peptide moiety linked to a ferrichrome like siderophore moiety, called the thioribosyl pyrimidine (a nucleoside-analogue) via a serine spacer. When albomycin enters the microbial cells via ferrichrome uptake system, the serine spacer gets hydrolysed to exert antimicrobial activity (Pramanik and Braun, 2006; Ali and Vidhale, 2013). Salimycin produced by Streptomyces 33 violaceus is another sideromycin, a trishydroxamate siderophore; and the aminoglycoside antibiotic linked by a dicarboxylic spacer. It is found effective against many Gram-positive bacteria especially, Staphylococci and Streptococci(Miethke and Marahiel, 2007). Ferrimycin A1 is another natural siderophore produced by Streptomycesgriseoflavus, which is composed of ferrioxamine B and an antibiotically active group; which is active against Gram-positive bacteria, particularly Staphylococcus aureus and Bacillus spp. (Ballouche et al., 2009; Górska et al., 2014). With the advent of novel technologies, many synthetic antibiotics have been used as potent candidates for antimicrobial therapies. These synthetic conjugates help to overcome the adaptations of pathogens responsible for antibiotic resistance, such as decreased outer membrane permeability, enzyme inactivation, and diffusion barriers (Górska et al., 2014). For instance, the catechol conjugates of the aminopenicillins were found effective against antibiotic resistant strains of P. aeruginosa (Page, 2013). When pyoverdine produced by P. aeruginosa is coupled with quinolone antibiotic, its effectiveness was found enhanced significantly, in comparison to to quinolone alone used for antibiotic therapy (Hennard et al., 2001; Rivault, 2007). A mixed ligand biscatecholate-monohydroxamate siderophore conjugated with carbacephalosporin exhibited remarkable specificity and high bactericidal potency against the Gram-negative pathogen, Acinetobacter baumannii (Wencewicz and Miller, 2013). Thus, the utilisation of the chelating property of the siderophores would open up new possibilities in clinical and pharmaceutical applications, when conventional antibiotics are phased out. Bioremediation 34 Uncontrollable accumulation of metal wastes in environment is one of the curses of industrialisation in modern world. Majority of the toxic contaminations include heavy (Al, Ni, Pb, Fe, Zn, Cd, Hg, Cu, Cr, etc.) and radioactive metals (Co, U, Th, Pu, etc.). The appropriate remediation of the toxic metal waste is one of the hectic tasks exposed before the scientific communities. Recent studies have revealed that microbial siderophore mediated metal waste eradication is one of the promising methods for addressing this problem satisfactorily. Siderophores can be used to mobilise metals from metal contaminated soil. For examples, the pyoverdine from Pseudomonas fluorescens chelated the metals such as Fe, Ni and Co from uranium mine waste (Edberg et al., 2010). Similarly siderophores (pyoverdine and pyochellin) produced by Pseudomonas aeruginosa could chelate a wide range of heavy metals such as Ag, Al, Cd, Co, Cr, Cu, Eu, Ga, Hg, Mn, Ni, Pb, Sn, Tb, Ti and Zn (Braud et al., 2009 a, b). Desferrioxamine sideophore from Streptomyces pilosus could coordinate with plutonium (V), in addition to the iron chelating property (Keberle, 1964); andAzotochelin, a catecholate siderophore secreted by Azotobacter vinelandii chelates molybdenum (Duhme et al., 1998). Biosensor Biological substances (tissues, microorganism, organelles, cell receptors, enzymes, antibodies, nucleic acids) coupled with electronic device is known as biosensors, and are widely used for the detection of toxic chemical substances in natural samples such as soil, water, etc. It is based on the fact that biomolecules exhibit better sensitivity and molecular specificity than inorganic and synthesised molecules. Many recent studies revealed that fluorescent siderophore-based biosensor is more suitable for the recognition 35 of heavy metals in water samples. For example, Barrero et al. (1993) immobilised pyoverdine, the fluorescent siderophore from P. fluorescence on a porous glass for detecting Fe3+ ion from water samples. Pyoverdine secreted by P. aeruginosa could chelate Fe3+, Fe2+ and Cu2+ ions, which quenched the fluorescence intensity (Yodar and Kissalita, 2006, 2011). Parabactin secreted by Paracoccus denitrificanscould effectively be employed for the detection of iron present in ocean (Chung et al., 2006). Pyoverdine could also be used for the identification of furazoidone in water samples, a broad spectrum antibiotic, highly suspected in carcinogenic, genotoxic and mutagenic effects; the detection mechanism was based on the fluorescence quenching occurred due to the electron transfer from pyoverdine to furazolidone (Yin et al., 2014). Conclusion Thus the latest trend in lipase research is the development of new and improved lipases, which have wide industrial applications and they play a crucial role in the turnover of water insoluble compounds, especially in the catalytic resolution of chemical molecules. Therefore, industry prefers lipases with higher activity available at economical rate. Bacterial siderophores are considered as a group of versatile biomoecules, which could be exploited in various fields of biotechnology for the sustainability and welfare of humans, animals and plants; and after all, for a healthy environment. To date, only a few studies have revealed the potential benefits of siderophores in highly demanding applications, especially in environmental and medical biotechnology. In tune with emerging technologies, more researches need to be focused to elucidate the specific role 36 and mechanism of action of each siderophore in various biological systems, i.e., effective utilisation of these natural biomolecules as the candidates for green technologies in biomedicine and in the management of environment. 37 Isolation and characterisation of microbes from the rumen of Malabari goat Chapter 3 Isolation and characterisation of microbes from the rumen of Malabari goat Aim and Rationale Isolation, screening and identification of microbes for the production of industrially significant biomolecules such as lipase and siderophore from the rumen content of Malabari goat. Rumen is one of the richsources of industrially significant microbial niche. On the basis of these facts, isolation and characterisation of bacteria from the rumen of Malabari goat are envisaged in this study. Introduction The inhabitants of the rumen microbial eco-system considered as a multifaceted consortium of diverse microbial genera such as bacteria (1010‒ 1011 cells/ml) representing more than 50 genera, anaerobic fungi (103‒105 zoospores/ml) representing five genera, protozoa (104‒105cells/ml) from 25 genera and bacteriophage (108‒109 cells/ml). Ruminants are usually fed on agricultural residues which contained cellulose, hemicellulose, starch, lignin, protein and a very small quantity of vegetable oils. The symbiotic association of the rumen microbes performs synergistically for the bioconversion of lignocellulosic feeds into volatile fatty acids (Kamra, 2005). The vast microbial diversity can ideally exploit the excellent source of industrially potent bio molecule production by scientific approaches. But so far in India, this is a low-priority area of research for microbiologists, biotechnologists and molecular biologists. Under these point of view, this study particularly focuses the isolation and selection of rumen microbes which having the tremendous production capability of two classic representatives of industrially significant bio-molecules i.e., lipase and siderophore. Materials and methods Sample preparation Rumen contents from both male and female Malabari goats were collected aseptically in screw-capped tubes from the slaughter house at Chelari (11.18189600 oN; 75.82206300 oE), Malappuram District of KeralaState, as described by (Prive et al., 2010). Briefly, 10 ml sterile double distilled water (ddH2O) was added to 10 g sample (rumen content) and centrifuged at 800 × g for 4 oC at 5 min. The supernatant (1 ml) obtained as above was serially diluted (up to 10-6) with pre-sterilised ddH2O (Priji et al., 2013). Isolation of microbes This diluted sample (100 µl) was aseptically transferred to semi-synthetic medium - designated as BUP medium (Table 4). The primary stage cultivation was done in anaerobic environment attained by the support of anaerobic chamber (KIM Microsystems, India) saturated with a mixture of gases (80% N2, 10% CO2and 10% H2). The microbial strains were steadily adapted to the aerobic system by frequent subcultures in a specially designed conical flask (Figure 8) by our laboratory, designated as ‘Benjamin flask’ (Priji et al., 2013). Table 4. Composition of BUP medium Ingredient Weight (g/l) Peptone 5 Beef extract 3 NH4NO3 5 NH4SO4 4 K2HPO4 2 NaCl 2 MgSO4 0.1 Cysteine-HCL 0.5 pH 6.7 ± 0.1 Screening for lipase production The isolated pure cultures were screened for lipase production by agar diffusion methods, as described below. Tributyrin agar method: Wells were bored on the tributyrin agar plates (1 ml tributyrin, 0.001g CaCl2, 2g agar) using sterile cork borer and 30 μl of the cultural supernatant withdrawn from a overnight (12 h) cultural medium and dispensed on the wells on Tributyrin agar plates. The plates were then incubated at 37 °C for 24 h (Pagu et al., 2013). Appearance of opaque halo around the colonies is the positive inference for the production of lipase. Chromogenic plate method:Chromogenic agar plates containing (in 100 ml) 1 ml tributyrin, 0.01g phenol red, 0.001g CaCl2, 2g agar were prepared and pH was adjusted between 7.3 and 7.4. The culture (24 h old) supernatant was then introduced into the well, bored at the centre of chromogenic agar plate, and incubated for 24 h at 37 oC. Heat-inactivated culture supernatant was kept as control. Subsequently, the plates were observed for color change from red to yellow, and indication for the production of lipase (Priji et al., 2014). Screening for siderophore production Chromasurol S (CAS) Assay CAS assay was employed for the detection of siderophore; both in liquid and on solid-agar media (Schwyn and Neilands, 1987). For the liquid CAS assay, the pure isolatewas grown in minimal medium supplemented with 3.2% piperazine-N, N’- bis (2- ethanesulphonoc acid) (MM9/PIPES), 3% casamino acids and 2% glucose (Schwyn and Neilands, 1987), which was incubated at 37 °C on a rotary shaker at 133 rpm for 48 h. Color change in the medium was scored visually to confirm the production of siderophore, i.e., the basic blue colour would change to brown. For solid CAS assay, about 1 mg cell pellet obtained after centrifuging (8000 × g) the culture (24 h old) in liquid CAS medium was spotted on to the centre of CAS-agar plates and incubated at 37 °C for 5 d, appearance of a clear orange halo around the spotted cell mass would be an indication for the production of siderophore. Identification and characterisation of bacteria Morphological characterisation The isolates were identified macro-morphologically by observing the colony characteristics such as color, texture and topography of the surface and edges; and also by micro-morphological evaluation on Gram staining. Identification up to species level has been accomplished based on these morpho-cytological as well as biochemical characteristics such as fermentation reactions in the medium containing various sugars (glucose, lactose, sucrose and maltose), IMViC, starch hydrolysis and nitrate reduction tests. Gram’s staining  The bacterial smear was heat fixed.  The smear was gently flooded with crystal violet and left it for 1 min.  Tilted the slide slightly and gently rinsed with tap water or distilled water using a wash bottle.  Gently flooded the smear with Gram’s iodine and left for 1 minute.  Tilted the slide slightly and gently rinsed with tap water.  Smear was decolorised using 95% ethyl alcohol and then rinsed with water.  Gently flooded the smear with safranin to counter-stain and let stand for 45 seconds.  Tilted the slide slightly and gently rinsed with tap water.  The slide was observed under binocular microscope (100X). The photographs were taken by image analyser fitted to a camera. Spore staining by malachite green  Bacterial smear was prepared in the usual manner.  The smear was allowed to air-dry and heat-fixed.  Smears were flooded with malachite green and placed on a warm hot plate allowing the preparation to steam for 10 min., then cooled and washed under running tap water.  Counter stained with safranin for 1 min.  Washed with running tap water and air-dried.  The slides were observed under the binocular microscope (100 X).  The photographs were taken by Image analyser fitted with digital camera. Biochemical Characterisation The biochemical characterisation included: indole production, methyl red, Voges-Proskauer, citrate utilisation, carbohydrate fermentation (glucose, lactose, sucrose and maltose), starch hydrolysis and nitrate reduction tests (Cappuccino and Sherman, 1996) (Table5). Indole production test To determine the indole production from tryptophan by bacterial catabolism. The cultures were grown in tryptophan broth for 24-48 h, and then a few drops of Kovac's reagent were added. Formation of a pink indole ring at the surface of culture was recorded as a positive reaction. Procedure  One per cent tryptone broth was prepared. It was sterilised by autoclaving at 15 psi, 121 oC for 15 min.  Using sterile techniques, the test organism was inoculated into the medium in appropriately labelled conical flasks, and incubated for 4 days in an incubating shaker.  The culture tubes prepared for indole production test were incubated at 35 oC for 48 h.  Kovac’s reagent was added to it and the tubes were gently shaken after intervals for 10-15 min.  The culture tubes were subsequently allowed to stand to permit the reagent to come to the top Methyl-red and Voges-Proskauer (MRVP) test To determine acid production during microbial metabolism of carbohydrates (Sreedevi et al., 2013) Procedure  MRVP broth (pH 6.9) was prepared in 10 ml tubes.  Five ml of the broth was poured in each of the tubes and sterilised by autoclaving at 15 psi, 121 oC for 20 min.  MRVP broths were inoculated and one tube kept as un-inoculated comparative control.  All culture tubes were incubated at 35 oC for 48 h.  Half of the tubes were used for methyl-red test, and the other half for Voges-Proskauertest.  In the tubes assigned for methyl-red test, 5 drops of methyl red indicator dye was added, the red persistence of red colour is an indication for positive test, and change in colour from red to yellow is negative test.  In the tubes assigned for Voges-Proskauer test, 12 drops of Voges- Proskauer solution A, and three drops of Voges-Proskauer solution B were added.  The culture tubes were shaken gently for 30 sec with the caps off, to expose the medium to oxygen.  The reaction was allowed to stand for 15 - 30 min, and observed for a change in colour from yellow to pinkish red. Citrate utilisation test To determine the ability of the bacterium to utilise/ferment citrate as the sole carbon source (Sreedevi et al., 2013) Procedure  Simmon’s citrate agar slants were prepared (pH 6.9).  All the ingredients, except phosphates, which were to be dissolved separately in 100 ml of water, were dissolved and volume made to 1l. The pH was set at 6.9.  The medium was poured in the culture tubes, and sterilised by autoclaving at 15 psi, 121 oC, 20 min and slants were prepared.  Simmon’s citrate agar slants were inoculated by means of a stab inoculation.  One blank tube (un-inoculated) was kept as control.  All the slants were incubated at 37 oC for 48 h Carbohydrate fermentation test To determine the fermentative degradation of various carbohydrates, phenol red carbohydrate broth with respective sugars like glucose, lactose, sucrose, and maltose were used. Starch hydrolysis test To determine the ability of microorganisms excreting hydrolytic extracellular enzymes capable of degrading the polysaccharide starch. Procedure  Starch-agar medium was melted and cooled to 45 oC, and poured into sterile petri dishes.  It was allowed to solidify.  Using sterile technique, a single streak inoculation was made at the center of the appropriately labelled plate.  Inoculated plates were incubated for 48 h at 37 oC in an inverted position.  The surface of the plates was flooded with iodine solution for 30 sec.  Excess iodine solution was poured off. Nitrate reduction test To determine the ability of the bacteria to reduce nitrates to nitrite. Procedure  The nitrate reduction broth supplemented with 0.1% potassium nitrate was prepared.  The culture was inoculated into the pre-sterilised medium in the conical flask by means of a loop inoculation.  Incubated all cultures for 24 - 48 h at 37 oC.  A piece of starch iodide paper was dipped in each culture, which was precipitated with 1N HCL and kept in hot air oven at 60 oC for 5 min and observed. Table 5. Summary of media constituents, reagents and inference in the present study Biochemical test Medium used Reagent Inference Indole production test (IPT) Tryptone broth Tryptone - 1.0 % NaCl - 1.0 % pH - 7.0 ±0.2 Kovac’s reagent p-Dimethyl aminobenzaldehyde (DMAB) – 5% Amyl alcohol - 75 ml Conc. HCL - 25 ml Catabolism of tryptophan Methyl-RedTest (MRT) MR-VP broth Glucose – 0.5 % Peptone – 0.7% K2HPO4 – 0.5% NaCl – 0.5% pH - 7.0 ±0.2 Methyl red - 0.02% Glucose oxidation Voges – Proskauer Test (VPT) MR-VP broth Voges - Proskauer reagent 12 drops of reagent A and 3 drops of reagent B Barritt’s Reagent A α- Naphthol- 5.0% Absolute alcohol- 100 ml Barritt’s Reagent B KOH –4.0% Distilled water - 100 ml Production of neutral end products Citrate utilisation test (CUT) Simmon’s citrate agar NaCl - 1.0% MgSO4 - 0.02% NH4H2PO4 - 0.1% KH2PO4 - 0.1% Bromothymol blue - 0.008% Citrate fermentation Biochemical test Medium used Reagent Inference Sodium citrate - 0.2% Agar - 2.0% pH - 7.0 ±0.2 Carbohydrate fermentation test Glucose fermentation(GF) Lactose fermentation(LF) Sucrose fermentation(SF) Maltose fermentation(MF) Peptone - 0.1% NaCl- 0.05% Glucose/lactose/ sucrose/ Maltose - 0.1% pH - 6.9 Phenol red-0.0012% Fermentatio n of sugars Starch hydrolysis test (SHT) Peptone - 0.1% NaCl - 0.05% Starch - 0.1% pH - 6.9 Phenol red- 0.0012% Secretion of extracellular starch hydrolysing enzymes Nitrate reduction test (NRT) Peptic digest of animal tissue– 0.5% Meet extract‒ 0.3% KNO3 - 0.1% Nacl - 3.0 % pH – 7.0 Sulfanilic acid (Reagent A) α-naphthylamine (Reagent B) Produced a red precipitate Molecular characterisation by 16S rDNA sequence analysis Procedure  DNA was isolated from the stab-culture. Its quality was evaluated on 1.2 % agarose gel, a single band of high-molecular weight DNA has been observed.  Fragment of 16S rDNA gene was amplified by PCR from the above isolated DNA. A single discrete PCR amplicon of 1500 bp was observed when resolved on agarose gel.  The PCR amplicon was purified.  Forward and reverse DNA sequencing reactions of PCR amplicon was carried out with 8F and 1492R primers using BDT v3.1 Cycle sequencing kit on ABI 3730xl Genetic analyser.  Consensus sequence of 16S rDNA sequence was generated from forward and reverse sequence data using aligner software.  The 16S rDNA gene sequence was used to carry out BLAST with the nucleotide database of NCBI Genbankdatabase. Based on maximum identity score first ten sequences were selected and aligned using multiple alignment software program Clustal W (Priji et al., 2013). Distance matrix was generated using Ribosomal Datatabase Project (RDP database) and the phylogenetic tree was constructed using MEGA 4 (Tamura et al., 2007) Results Isolation and screening of microbes Isolate Five bacterial cultures were isolated from the rumen content of Malabari goat and grown under Benjamin flask (Figure 8). The bacterial cultures were represented as C1, C2, C3, C4 and C5. All the isolates were checked for lipase (Figure 9) and siderophore production (Figure 10). Among the isolates, a bacteria (C3) exhibit positive results for the production of all the two bio-molecules. Characterisation of microbes Microscopic characters of the pure isolate C3, showed a rod-shaped, asporogenous, Gram-negative cells measured 0.4 to 0.8µm (width) by 1 to 2 µm (length) (Figure 11). The biochemical characterisation as such carbohydrate fermentation, nitrate reduction, starch hydrolysis and IMViC tests, of which methyl red and starch hydrolysis tests were positive, whereas others were negative (Table 6). Depends upon the results the pure isolate of C3 showed that it belongs to the genus, Pseudomonas. Based on nucleotide homology and phylogenetic analysis,the isolate was further confirmed as a strain of Pseudomonas aeruginosa, designated as P. aeruginosa strain BUP2. The phylogenetic tree in comparison with related 11 strains of Pseudomonas aeruginosa was constructed (Figure 6). Figure 8. Microbial strains were grown under Benjamin flask containing BUP medium. Figure 9. Screening of P.aeruginosa strain BUP2 for the production of lipase. (A) Tributyrin agar method. Production of lipase was indicated by opaque halo around the well. (B) chromogenic agar plate method. Production of lipase was indicated by the change in color from red to yellow around the well. Figure 10. Screening of the siderphore by CAS asay. (A) Liquid CAS medium in blue color before inoculation; (B) liquid CAS medium changed its color to orange after 48 h incubation indicating siderophore production. (C) CAS agar medium shows siderophore production. Figure 11. Morphology of P.aeruginosa strain BUP2.(A) streak plate on nutrient agar plate; (B) digital image of the single colony appeared slimy and white-to-cream in colour; (C) Gram’s stained colonies. Table 6. Summary of biochemical tests performed on P. aeruginosa strain BUP2 Biochemical tests Results Citrate utilisation test -ve Carbohydrate fermentation test Glucose fermrntation -ve Maltose fermentation -ve Sucrose fermentation -ve Lactose fermentation -ve Nitrate reduction test -ve Indole production test -ve Voges - proskauer test -ve Starch hydrolysis test +ve Methyl red test +ve Figure 12. Evolutionary relationships of Pseudomonas aeruginosa strain BUP2 with other 11 related strains. Discussion The prime objective of the study was to isolate and characterise bacteria capable of producing industrially significant biomolecules; lipase and siderphore from the rumen content of Malabari goat. Among the 5 isolates, one bacterium exhibitsthe potential producer of both the molecules. According to the morphological, biochemical and molecular characterisation result of this bacterium confirmed that it belongs to Pseudomonas aeruginosa BUP2. Pseudomonas aeruginosa (Pa)is a Gram-negative, rod-shaped and asporogenous bacterium isolated from various clinical samples (Oyeleke and Okusanmi, 2008). Painhabits in soil, water, skin flora, and most of the man- made environments throughout the world. Due to its ubiquitous inhabitance in the environment, many animals transiently harbor this bacterium. Apart from humans, various reports are available in literature regarding the inhabitance of Pa in the rumen, intestine, milk and fecal matter of cattle and calves including sheep, camel, etc. (Mushin and Ziv, 1973; Duncan et al., 1999; Leitner and Krifucks, 2007). As far as literature says, this is the first report on the inhabitance and characterisation of a Pa strain from the rumen of a goat, especially Malabari goat. In fact, this bacterium was initially a facultative anaerobe, which we tuned to be an aerobe by growing in a specially designed flask, the Benjamin flask (Priji et al., 2013). There are plenty of reports are available for the isolation of Pseudomonas spp. from various sample sources (Dennis and Sokol, 1995). However, no report is available on Malabari goat which is a special breed of domestic goat confined to the Malabar (Northern part) region of Kerala.Thereforethe microflora inhabiting the rumen of this goat were explored. In this study, P. aeruginosa strain BUP2 capable of producing industrially significant lipase and siderphore, were successfully isolated from the rumen of Malabari goat. Conclusion This study demonstrated that the Pseudomonas aeruginosa starin BUP2 is one of the suitable microorganisms which can produce both industrially significant bio-molecules such as lipase and siderophore successfully. Moreover the goat rumen is considered as one of the fine sources for the isolation of microbes which is responsible for the production of industrially significant bio-molecules. But so far the microbial source of goat rumen is still remain an untapped area of the microbiologists. Utility of rubber seed as potent solid substrates for the production of lipase by Pseudomonas aeruginosa strain BUP2 Chapter 4 Utility of rubber seed as potent solid substrates for the production of lipase by Pseudomonas areuginosa strain BUP2 Aim and Rationale The aim of this study is to check the efficiency of flours of rubber seed, coconut and groundnut or deoiled cakes as solid substrate for the production of lipase by P. aeruginosa strain BUP2. India is one of the largest producers of rubber in the world, and every year large quantity of rubber seed emerges as agricultural waste. Studies revealed that higher lipase production could be attained only in the presence of lipidic substrates; since rubber seed oil contains higher levels of fatty acids, which would act as suitable inducer (in addition to providing nutrition) for lipase production. Thus, the flours/cakes of oil seeds utilised as substrate and inducerfor the microbial production of lipase. Introduction Being a versatile biocatalyst, microbial lipases (triacylglycerol acylhydrolase, EC3.1.1.3) offer great potentials in biological as well as industrial applications (Pandey et al., 1999). Though industry mostly prefers submerged fermentation (SmF) strategy for the production of lipase; solid-state fermentation (SSF) received more attention recently due to its cost effectiveness and higher productivity. Numerous studies attempted for the production of lipase on solid substrate, in which different solid agricultural residues such as cakes from deoiled coconut and groundnut kernels; husks of rice, lentil and wheat; residue from banana, melon, soybean and watermelon; and brans from wheat and rice have been used (Benjamin and Panday, 1997; Alkan et al., 2007; Kempka et al., 2008). Oil cake is a cheap byproduct emerged out of oil extraction, which is mainly used in animal and chicken feeds. In this connection, large potential of rubber seed (oil and cake) as substrate for the cultivation of miocroorganisms is seen neglected. India is one of the largest producers of rubber in the world, and every year huge quantity of rubber seed is treated as agricultural waste. Rubber seed oil is rich in fatty acids (Ramadhas et al., 2005): i.e., 39.6% linoleic acid, 24.6% oleic acid, 16.3% linoleic acid, 8.7% stearic acid 2% palmitic acid, apart from carbohydrate (24.21%) and protein (22.17%). Owing to the high nutritional content of the agro-industrial residues, oil cakes are considered as valuable solid substrates for the production of enzymes by growing suitable microorganisms on it under water-restricted environment, the SSF. Moreover, usage of agricultural residues with an industrial perspective is a best strategy for the better agricultural waste management and abatement of environmental pollution problem due to agricultural residues. Though manybacteria, yeast, actinomycetes and fungi shown to have potentials for the production of lipase, species of genera Bacillus, Pseudomonas,Staphylococcus, Candida, Geotrichum,Aspergillus, Mucor, Penicillium, Rhizopus and Rhizomucorare considered as the best producers of lipase (Aravindan et al., 2007). Pseudomonas aeruginosa strain BUP2 (MTCC No. 5924), a new bacterial strain reported from this laboratory (Unni et al., 2014) was used in this study. Thus, the present study investigates the efficiency of the ground kernels of rubber seed, coconut and groundnut or deoiled cakes as solid substrate for the production of lipase by P. aeruginosa strain BUP2. Materials and Methods Bacterial culture Pseudomonas aeruginosa strain BUP2 (MTCC No.5924), a new bacterial strain already reported from this laboratory was used throughout the study, which was isolated from the rumen of Malabari goat (Unni et al., 2014). Chemicals Analytical grade chemicals from Hi Media Laboratories (Mumbai, India) were used for the study. The p-nitro phenyl palmitate (pNPP), substrate for lipase assay was purchased from Sigma Chemical Co., USA. Medium and Inoculum P. aeruginosa BUP2 was maintained at 4 oC on mineral salt -oil-agar medium. Seed culture (12 h old) was prepared in the BUP medium by inoculating a loopful of stock culture of bacterium into it (Unni et al., 2014), which was incubated at 37 oC and 130 rpm. Medium for SSF For the cultivation of P. aeruginosa strain BUP2, kernels of 3 types of nuts/seeds (coconut, groundnut and rubber seed) were used in the ground form or as their deoiled cake for this study. Before grinding, the kernel was chopped into small pieces and dried in an oven for 24 h at 60 oC. The coconuts (coconut powder and coconut cake) and groundnuts (groundnuts powder and groundnuts cake) were purchased from local market, while rubber seeds were collected from a local plantation. The deoiled cakes were prepared after extracting oil using an expeller manually. Solid-state fermentation (SSF) strategy Two grams of ground kernel or their cakes was moistened with 10 ml of BUP medium. All preparations in the flask were autoclaved at 121oC for 15 min, and inoculated with 0.1 ml of inoculum (seed culture) under aseptic condition; and the inoculated media were incubated at respective conditions. In order to check lipase production, fermented samples were assayed at regular intervals of 24 h for 4 days. Effect of pH The solid substrate (ground seeds/cake) was moisturised with BUP medium having varying pH concentration (4, 5, 6, 7 and 8) was inoculated with P.aeruginosa strain BUP2 and incubated at 25 oC. At regular intervals of 24h, the fermented matter was analysed for estimating the production of lipase. Effect of temperature To estimate the role of different temperature (25, 28, 30, 32oC) on lipase production, the ground kernel or cake was inoculated with P. aeruginosastrain BUP2 after moisturising it with BUP medium having optimum pH, and assayed for lipase activity at regular intervals of 24 h. Effect of substrate concentration Effect of substrate concentration [(10, 20, 30, 40 and 50% (w/v)] on lipase production was checked under the optimised conditions of pH and temperature. The fermented samples were regularly withdrawn at every 24 h interval, and assayed for lipase activity. Lipase extraction Lipase was extracted from the fermented solid substrate by the method of (Ramachandran et al., 2004). Crude lipase was extracted by mixing 1g of fermented substrate with 5 ml of 0.1M Tris-HCl buffer (pH 8.0), and mixed well on a vertex mixer. After centrifugation (8000 × g for 10 min, 5 oC), the supernatant was collected for lipase (crude) assay. Lipase assay Lipase activity in the cell and debris free supernatant was determined as described by Kilcawley et al., (2002). The reaction mixture (1.8 ml) contained 0.15 M NaCl and 0.5% triton X-100 in 0.1 M Tris-HCl buffer (pH 8.0) and 200 µl supernatant (or ddH2O in cotrol), which was pre-incubated(10 min) at 37 oC in a water bath. Subsequently, 20 µl pNPP (p-nitrophenyl palmitate) in 50 mM acetonitrile was added, and incubated further at 37 oC for 30 min. The quantity of liberated p-nitrophenolwas recorded at λ405. One unit of lipase activity is defined as the quantity of enzyme required for liberating 1 µmol of p-nitrophenol under the standard assay conditions. Lipase activity was calculated using the following formula: Lipase activity (U/gds) = C once ntra tion (µ M) Where, ΔE is the absorbance at 405 nm; Vf is the final volume of reaction mixture; VS is the volume of crude supernatant (lipase) used; Df is the dilution factor (i.e., total extracted volume from 1g fermented matter), Δt is the time of incubation in min; ε is the extinction coefficient (0.017); gds is the dry weight in grams (i.e., dry matter of the 1 g fermented matter used for extracting lipase. Statistics All studies were repeated at least thrice, and an average of 3 values is presented with standard deviation. Microsoft Excel was used to draw the figures. Results Effect of pH The effect of pH on lipase production was investigated at different pH using different ground kernals or their cakes. It showed that slightly acidic or neutral pH supported the maximum production of lipase. Among the different substrates used, rubber seed flour supported the maximum production of lipase (340 U/gds) at pH 6, followed by groundnut flour and its cake (181 and 168 U/gds, respectively) at pH 7; whereas coconut oil flour and its cake supported lipase production at pH 6, but in lesser quantities (130 and 103 U/gds, respectively) (Figure 13). 4 5 6 7 8 0 50 100 150 200 250 300 350 400 COP COC GOP GOC RSP pH L ip as e ac ti vi ty (U /g ds ) Figure 13. Effect of pH on lipase production by P. aeruginosa strain BUP2 on various flours and cakes moisturised with BUP medium. Effect of culture temperature The effect of temperature on the production of lipase by P. aeruginosa strain BUP2 was determined at various temperature (25 to 32 oC); of which, all the substrates supported the maximum production of lipase at temperature range between 28 and 30 oC. Rubber seed flour, coconut flour and groundnut cake supported the maximum lipase production (871, 174 and 203 U/gds, respectively) at 28 oC; while coconut cake (344 U/gds) and groundnut four (397 U/gds) supported the maximum production of lipase at slightly higher temperature (30 oC) (Figure 14). Object 8 Figure 14. Effect of temperature on lipase production by P. aeruginosa strain BUP2 on various flours and cakes moisturised with BUP medium. Effect of substrate concentration on lipase production Five solid substrates (coconut flour, coconut cake, groundnut flour, groundnut cake and rubber seed flour) enriched with BUP medium were tested for their effect on lipase production by P. aeruginosa strain BUP2. Of them, the maximum lipase production (871 U/gds) was supported by 20 % (w/v) of rubber seed flour under optimised condition (pH 6, 28oC and 48h incubation) (Figure 15); and 20 % (w/v) of groundnut flour (pH7, 30 oC and 48h incubation) supported the production of 398 U/gds lipase (Figure 16). Coconut cake (20 %, w/v) and coconut flour (40 %, w/v) supported 327 and 311 U/gds lipase production, respectively (Figures 17 and 18). Groundnut cake (30%, w/v) supported comparatively lesser lipase production (244 U/gds), which was at 72 h of incubation (Figure 19). Object 9 Figure 15. Lipase production profile of P. aeruginosa strain BUP2 on 10, 20, 30, 40 or 50 % (w/v) of rubber seed flour enriched with BUP medium. Object 10 Figure 16. Lipase production profile of P. aeruginosa strain BUP2 on 10, 20, 30, 40 or 50% (w/v) of groundnut flour enriched with BUP medium. Object 11 Figure 17. Lipase production profile of P. aeruginosa strain BUP2 on 10, 20, 30, 40 or 50 % (w/v) of coconut cake enriched with BUP medium. Object 12 Figure 18. Lipase production profile of P. aeruginosa strain BUP2 on 10, 20, 30, 40 or 50 % (w/v) of coconut flour enriched with BUP medium. Object 13 Figure 19. Lipase production profile of P. aeruginosa strain BUP2 on 10, 20, 30, 40 or 50% (w/v) of groundnut cake enriched with BUP medium. Discussion The primary objective of this work was to investigate the suitability of agricultural product-based fermentation medium for lipase production by P. aeruginosa strain BUP2 (MTCC No. 5924). Large quantities of deoiled kernels of seeds/nuts are generated as by-product during the extraction vegetable oil, which would fetch low price to the farmers. Rubber seed (oil or kernel) is not considered as a healthy source of cooking oil or its industrial uses are limited. Thus, a few of them were explored in this study for their utility as substrate for the production of lipase, thereby increasing their market value. SSF is mainly employed with fungi for the production of extracellular enzymes such as lipase, cellulase, amylase, alkaline protease and xylanase. In general, bacterial cultures are not considered suitable for performing SSF due to the higher water activity requirement for their growth. However, numerous studies showed that bacterial cultures can well be adapted or it can be manipulated for SSF processes (Chakraborty and Srinivasan, 1993; Benjamin and Pandey, 1998; Kaur et al., 2001). Bacteria can efficiently produce α- amylase, alkaline protease and inulinase by species of Bacillus, Pseudomonas and Staphylococcus (Chakraborty and Srinivasan, 1993; Selvakumar and Pandey, 1999; Prakasham et al., 2006; Smitha et al., 2013; Jisha et al., 2014). In the light of several advantages of SSF, lipase production from the strain of P. aeruginosa strain BUP2 was attempted on agricultural products. The rubber seed flour supported the maximum production of lipase. To the best of our knowledge, this is the first report showing rubber seed flour as a potent substrate for the production of lipase via SSF. A few studies reported oil cakes as the solid substrate-cum-inducer for the production of lipase by SSF (Benjamin and Pandey, 1996; Ramachandran et al., 2007; Singhania et al., 2008). Benjamin and Pandey (1998) employing Candida rugosa, demonstrated the utility of mixed-solid substrate containing wheat bran and coconut oil cake for lipase production. Recently, efficacy of Pseudomonas sp. strain BUP6 for the production of lipase (107 U/gds) on groundnut cake was illustrated by Faisal et al., (2014). Deoiled cake from Jatropha seed was used as a support for the production of lipase (1084 U/gds) from P. aeruginosa PseA through SSF (Mahanta et al., 2008). Conclusion This study showed the utility of agricultural products, especially cheaply available rubber seed as a solid medium for the production of valuable lipase. This would enable the rubber farmers to get additional income from their agriculture. Moreover, the ability of P.aeruginosa strain BUP2 to grow on rubber seed medium and secrete extracellular lipase is highly promising, as rubber seed is known to embody limarin, a toxin. In this context, efficiency of P.aeruginosa strain BUP2 to grow on other cakes and secrete lipase is yet another promising sign of utility of this novel bacterium. Production, optimisation, purifcation and Chracterisation of lipase produced by Pesudomonas aeruginosa strain BUP2 Chapter 5 Production, optimisation, purification andcharacterisation of lipase produced byPseudomonas aeruginosa strain BUP2 Aim and Rationale The aim of this study was to optimise a suitable fermentation medium for lipase production by Pseudomonas aeruginosa BUP2, followed by its purification and characterisation. Strains ofP. aeruginosa are reported as potent producers of lipase on lipidic substrates. Nowa-days, lipase has increasing demand in food, textile and detergents industries; and thus, the relevance of this study. Introduction Lipases (E.C. 3.1.1.3), a subclass of the esterases catalyse the hydrolysis of long chain triglycerides (fat) into fatty acid and glycerol. At limited water activity, it can reverse the reaction towards ester synthesis; and this pliable nature of lipases is exploited in a host of bioconversion and catalytic reactions such as hydrolysis of lipids, alcoholysis, acidolysis, aminolysis, esterification, interesterification, transesterification, racemic solution, stereoselective and chiral syntheses (Benjamin and Pandey, 1998; Reis et al., 2009). Lipases show cosmopolitan distribution in animals, plants, and microorganisms; of this, microbial lipases (of bacteria, yeast, and fungi) gained much attention, and are widely used for commercial purpose; especially in biotechnological applications (Jaeger and Eggert, 2002). Bacterial lipases are used in dairy, food, detergents, pharmaceuticals, textile, cosmetic and biodiesel industries, apart from the synthesis of fine chemicals, agrochemicals and new polymeric materials. Stability, selectivity, and broad substrate specificity make microbial lipases more attractive to the bio-industry. Microbial enzymes may either be secretory (extracellular) or cell-bound (intracellular); the former is preferred over the latter due to its ease in downstream processing (Treichel et al., 2010). Commercially, species of Bacillus (e.g., alcalophilus, coagulans, licheniformis, pumilus, stearothermophilus, subtilis, etc.) and Pseudomonas (especially aeruginosa and putida)claim the major share of bacterial lipases; and Burkholderia cepacia, Burkholderia multivorans, Staphylococcus caseolyticus also contribute significantly to the microbial lipases (Lang et al., 1998; Treicher, et al., 2010). The Gram-negative Pseudomonas, a prolific producer of lipase is demonstrated to have a great deal of metabolic diversity with ability to colonise a wide range of niches (Lang et al., 1998; Unni et al., 2014). Bacterial lipases as mostly produced by submerged fermentation strategy. Several physical factors such as pH, temperature, agitation, substrate concentration and inoculum size play crucial role in lipase production; normally, optimisation of single variable is adopted as the production strategy. The major drawback of this one at a time strategy is that it does not address the interaction effects among the variables, i.e., the coexistence of variables. Moreover, it does not describe the net effect of the different medium components on the enzyme production; In addition, is also tedious, and a number of trails required for determining the optimum levels. Unlike this classical method, statistical tool like response surface methodology is applied for optimising the fermentation conditions (Kaushik et al., 2010; Liu et al., 2006; He and Tan, 2006). Thus, the objectives of the present study are: statistical optimisation (using RSM) of the production conditions