Project Code : (for RCMO use only)
RU GRANT
UNIVERSITI SAINS MALAYSIA FINAL REPORT FORM
Please email a softcopy of this report to [email protected]
A PROJECT DETAILS
Title of Research:
PRODUCTION OF BIOSURFACTANT VIA LOCALLY ISOLATED ACTINOMYCETE FERMENTATION
ii Account Number: 1001/PBIOLOGI/815103
iii Name of Research Leader:
ASSOCIATE PROFESSOR AHMAD RAMLI MOHO YAHYA
iv Name of Co-Researcher:
1 .. ASSOCIATE PROFESSOR LATIFFAH ZAKARIA
v Duration of this research:
a) Start Date 15/12/2012 ~
b) Completion Date 14/06/2015
c) Duration 30 MONTHS
d) Revised Date (if any)
8 ABSTRACT OF RESEARCH
(An abstract of between 100 and 200 words must be prepared in Bahasa Malaysia and in English. This abstract will be included in the Report of the Research and Innovation Section at a later date as a means of presenting the project findings of the researcherls to the University and the community at large)
Kajian ini memberi tumpuan kepada penyaringan dan penilaian penghasilan biosurfaktan ekstrasel daripada 33 pencilan actinomycetes. Daripada 33 pencilan, pencilan R1, dicirikan sebagai genus Streptomyces berdasarkan analisis fenotip and genotip, telah dipilih sebagai model organisma berfilamen dalam fermentasi aerobic untuk penghasilan biosurfaktan. Penghasilan biosurfaktan telah disiasat dalam mod kelompok menggunakan kelalang goncang dan 3 L bioreactor tangki teraduk. Penghasilan biosurfaktan maksimum dalam kelalang goncang didapati pada jam 144 dalam medium yang mengandungi 6% (v/v) minyak sawit, 0.6% (w/v) ekstrak yis, 2% (v/v) Tween® 80, 0. 7% (w/v) NaCI dan pH awal 6 dengan 4% (v/v) kepekatan inokulum. Penambahbaikan penghasilan biosurfaktan telah dica ai den an men unakan reaktor tan ki teraduk an dikawal den an baik. Penin katan
RU Grant Final Report I RCMO July 2015 1 sebanyak 3 kali ganda aktiviti pengemulsian dan ukuran ketegangan permukaan terendah (40.5±0.05 dynes/em) telah dicapai apabila kultur diaduk pada 600 rpm. Dalam kultur kelompok, penghasilan biosurfaktan tidak begitu bergantung kepada biojisim tetapi dipengaruhi oleh morfologi kultur semas.a fermentasi tenggelam. Kaedah pengekstrakan pelarut dengan menggunakan methanol, chloroform and 1-butanol (1: 1:1) memberi hasil ekstrak biosurfaktan mentah tertinggi (58.04 g/L) yang dicirikan sebagai biosurfaktan glikolipid. ,.,
The present study focused on screening and evaluating extracellular biosurfactant production from 33 actinomycete isolates. Out of 33 isolates, isolate R1, characterized as the genus Streptomyces based on phenotypic and genotypic analyses, was selected as a model filamentous organism in an aerobic fermentation for biosurfactant production. The production of biosurfactant was investigated in batch mode using shake flasks and 3 L stirred tank bioreactor. The maximum biosurfactant production in shake flasks was obtained at 144 hours in a medium containing 6% (v/v) palm oil, 0.6% (w/v) yeast extract, 2% (v/v) Tween 80, 0.7% (w/v) NaCI and initial pH 6 with 4% (v/v) inoculum concentration. Further improvement of biosurfactant production was achieved using a well-controlled stirred tank reactor. An increase of 3-fold of emulsification activity and lowest surface tension measurement (40.5±0.05 dynes/em) was attained when the culture was agitated at 600 rpm. In batch cultures, the production of biosurfactant was not dependent on biomass as much but rather was influenced by culture morphology during submerged fermentation. Solvent extraction method using methanol, chloroform and 1-butanol (1: 1:1) gave the highest yield (58.04 g/L) of crude biosurfactant extract that was characterized as glycolipid biosurfactant.
C BUDGET & EXPENDITURE
Total Approved Budget : RM RM 243,934.00
Yearly Budget Distributed Year 1 : RM 117,967.00 Year 2 : RM 125,967.00 Year 3 : RM -
Total Expenditure : RM 243,073.25
Balance : RM 890.75
Percentage of Amount Spent(%) : 99.64%
# Please attach final account statement (eStatement) to indicate the project expenditure
ii Equipment Purchased Under Vot 35000
No. Name of Equipment Amount (RM) Location Status
# Please attach the Asset/Inventory Return Form (Borang Penyerahan Asetllnventori)- Appendix 1
RU Grant Final Report I RCMO July 2015 2 D RESEARCH ACHIEVEMENTS
Project Objectives (as stated/approved in the project proposal) i
No. Project Objectives Achievement
To screen potential biosurfactant-producing actinomycetes 1 Achieved from the local environment Identification and characterization of the potential 2 Achieved biosurfactant-producer . To establish good growth conditions of biosurfactant- 3 Achieved producing actinomycete , To enhance the production of biosurfactant via batch 4 Achieved I fermentation 5 6
ii Research Output
a) Publications in lSI Web of Science/Scopus
Status of Publication Publication No. (published/accepted/ (authors,title,journal,year,volume,pages,etc.) under review) Nor Syafirah Zambry, Adilah Ayoib, Nur Asshifa Md Noh, and Ahmad Ramli Mohd Yahya. Production and partial , characterization of biosurfactant produced by Streptomyces Published sp. R 1. Bioprocess and Biosystems Engineering (20 17) (ISSN 1615-7591)
b) Publications in Other Journals
Status of Publication Publication No. (published/accepted/ (authors, title,journal, year, volume, pages,etc.) under review) 1 Nurfarah Aina Mohamed Razalli, Norazurin Syuhada Rusly, Siti Zulaikha Mohd Yusof, Mohd Syafiq Awang, Nor Syafirah Zamry, Shahkillahwati Mohd Ridhwan, Nur Asshifa Md Noh Published , and Ahmad R.M. Yahya, Microbial Biosurfactants,Australian Biotechnology, The Journal of AusBiotech (2016), 26(3), 40-41
c) Other Publications (book,chapters in book,monograph,magazine,etc.)
Status of Publication Publication No. (published/accepted/ (authors, title,journal, year, volume, pages,etc.) under review)
RU Grant Final Report I RCMO July 2015 3 d) Conference Proceeding
Conference Level No. Title of Abstract/Article " (conference name,date, place) (International/National)
1 1st International Conference Screening of Biosurfactant ~ On Sustainable Agriculture Producers from Locally Isolated International and Environment Actinobacterial 2 The Inaugural Asian Effect of Impeller Tip Speed on Conference on the Life Biosurfactant Production by International Sciences & Sustainabilitv Streptomyces sp. R1 3 The Inaugural Asian Isolation and Identification of Conference on the Life Actinobacteriai-Biosurfactant International Sciences & Sustainability Producers
# Please attach a full copy of the publication/proceeding listed above
iii Other Research Ouput/lmpact From This Project (patent, products, awards, copyright, external grant, networking, etc.)
Product : Collection of actinobacterial isolates producing biosurfactants
E HUMAN CAPITAL DEVELOPMENT
a) Graduated Human Capital
Nationality (No.) Student Name National International 1. PhD 2. 1. Nor Syafirah Zambry MSc 2 2. Adilah Ayoib 1. Norazurin Syuhada Rusly 2. lza Syazwani Mardzuan Undergraduate 5 3. Mok Kar Mun 4. Nur Asna Azhar 5. Jonathan Tan Choon Siang
b) On-going Human Capital
Nationality (No.) Student Name National International 1 1 .. PhD 2. 1. MSc 2. 1. Undergraduate 2.
c) Others Human Capital
Nationality (No.) Student Name National International 1. Post Doctoral Fellow 2.
RU Grant Final Report I RCMO July 2015 4 1. Nor Syafirah Zambry * Research Officer 3 2. Nur Asshifa Md Noh 3. Shahkillawati Mohd Ridhwan 1. Nik Farhana Nadiah Nik Mustapha 2. Adilah Ayoib* Research Assistant 5 3. lntan Sakinah Mohd Anuar 4. Mohd Syafiq Awang 5. Siti Zulaikha Mohd Yusof 1. Others(...... ) 2.
F COMPREHENSIVE TECHNICAL REPORT
Applicants are required to prepare a comprehensive technical report explaining the project. The following format should be used (this report must be attached separately): • Introduction • Objectives • Methods • Results • Discussion • Conclusion and Suggestion • Acknowledgements • References
G PROBLEMS/CONSTRAINTS/CHALLENGES IF ANY
(Please provide issues arising from the project and how they were resolved)
H RECOMMENDATION
(Please provide recommendations that can be used to improve the delivery of information, grant management, guidelines and policy, etc.)
Project Leader's Signature:
Name : ASSOC. PROF. AHMAD RAMLI MOHO. YAHYA SCHOOL OF BIOLOGICAL SCIENCES 0 t . , J:NIV~RS I SAINS MALAYSIA a e . ~I c::?r1 Of~PENANG
RU Grant Final Report I RC 0 July 2015 5 COMMENTS, IF ANY/ENDORSEMENT BY PTJ'S RESEARCH ~ONIMITrji~ ,
...... ~.~-~······.•···································································································
Signature and Stamp of Chairperson of PT J's Evaluation Committee
Name: PROFESOR MADYA DR. YAHVA 81N MAT ARIP Tlmbalan Oekan Date : Penyelldlkan. Slswa~ah & Jarlngan Pusat Pengajlan Salns Kajihayat Universiti Sains Malaysia
..._. ... ~ .... :...... PRO::s,:: ::n::?.~~i~:::::~~H S1gn ure and mp o lfttl Director of PT J Uniw~rsiti Sains Malaysia
Name:
Date :
RU Grant Final Report I RCMO July 2015 6 ' •.
FINAL TECHNICAL REPORT
PRODUCTION OF BIOSURFACTANTS VIA LOCALLY ISOLATED ACTINOMYCETE FERMENTATION
RESEARCH UNIVERSITY GRANT (RUI)
1001/PBIOLOGI/8151 03
15/12/2012- 14/06/2015
RM243,934.00
AHMAD RAMLI MOHD. YAHYA, PhD.
School of Biological Sciences f Final Report: Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation ' ' RUJ 1001/PBJOLOGJ/815103 Contents
1.0 INTRODUCTION ...... 1 2.0 OBJECTIVES ...... 1 3.0 RESEARCH METHODOLOGY ...... 2 3.1 Microorganisms ...... 2 3.2 Screening ofBiosurfactant-Producers ...... 2 3.3 Analytical Techniques ...... 2 3.3.1 Surface Tension Measurement...... 2 3.3.2 Emulsification Index (E24) ...... 2 3.3.3 Oil Spreading Technique (OST) ...... 3 3.3.4 Determination of Biomass ...... 3 3.4 Identification of Potential Biosurfactant-Producing Actinomycete ...... 3 3.4.1 Phenotypic Characterization ...... 3 3.4.2 16S rRNA Gene Amplification, Sequencing and Phylogenetic Analysis ...... 3 3.5 Inoculum Preparation ...... 4 3.6 Shake flask cultures ...... 4 3.6.1 Scouting of Suitable Growth Condition for Streptomyces sp. Rl ...... 4 3.6.1.1 Selection of Carbon Source ...... 5 3 .6.1.2 Palm Oil Concentration ...... 5 3.6.1.3 Selection of Nitrogen Source ...... 5 3 .6.1.4 Yeast Extract Concentration ...... 5 3 .6.1.5 Synthetic Surfactant (Tween® 80) Addition ...... 5 3. 6.1. 6 Inoculum Concentration...... 6 3 .6.1. 7 Salinity ...... 6 3.6.1.8 Initial pH ...... 6 3. 7 Bioreactor Studies ...... 6 3. 7.1 Oxygen Requirement ...... 6 3.7.1.1 Determination of Volumetric Oxygen Transfer Coefficient (kLa) ...... 6 3.7.1.2 Determination of Oxygen Uptake Rate (OUR) ...... 6 3.7.2 Batch Cultivation ...... 7 3. 7 .2.1 Influence of Agitation Speed on Growth and Biosurfactant Production ...... 7 3.7.2.2 Influence of Agitation Speed on Morphological of Streptomyces sp. Rl ...... 7 3.8 Biosurfactant Extraction ...... 7 .. Final Report: Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation
RUJ JOOJ/PBIOLOGJ/815103 3.8.1 Solvent Extraction ...... 8 3.8.2 Ammonium Sulphate Precipitation ...... 8 3.8.3 Acid Precipitation ...... 8 3.9 Determination ofBiosurfactant Dry Weight...... 8 3.10 Biosurfactant Characterization ...... 8 3.10.1 Benedict Test ...... 8 3.10.2 Saponification test ...... 9 3.1 0.3 Biuret test ...... 9 3.10.4 Iodine Test ...... 9 3.1 0.5 Fatty Acid Analysis ...... 9 4.0 RESULTS AND DISCUSSION ...... 10 4.1 Screening of Biosurfactant-Producing Actinomycetes ...... 10 4.2 Identification and Characterization ofthe Potential Biosurfactant Producer ...... 10 4.2.1 Phenotypic Characterization ...... 10 4.2.2 16S rRNA Gene Sequence Analysis ...... l3 4.3 Shake flask cultures ...... 15 4.3.1 Scouting for Suitable Growth Condition for Streptomyces sp. Rl...... 15 4.3.1.1 Selection of Carbon Source ...... 15 4.3.1.2 Palm Oil Concentration ...... 15 4.3.1.3 Selection of Nitrogen Source ...... 17 4.3.1.4 Yeast Extract Concentration ...... 18 4.3 .1.5 Synthetic Surfactant (Tween® 80) Addition ...... 19 4.3 .1.6 Inoculum Concentration ...... 20 4.3.1.7 Salinity ...... 21 4.3.1.8 Initial pH ...... 22 4.4 Bioreactor Studies ...... 24 4.4.1 Oxygen Requirement ...... 24 4.4.1.1 Oxygen Transfer Rate (OTR) ...... 24 4.4.1.2 Oxygen Uptake Rate (OUR) ...... 25 4.4.2 Batch Cultivation ...... 26 4.4.2.1 Agitation Speed ...... 26 4.4.2.2 Influence of Agitation Speed on Morphological Streptomyces sp. R1 ...... 27 4.5 Biosurfactant Extraction ...... 29 4.6 Biosurfactant Characterization ...... 31
11 Final Report : Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RUI 1001/PBJOLOGJ/815103 4.6.1 Biochemical Characterization ...... 31 4.6.2 Fatty Acid Analysis ...... 34 5.0 CONCLUSION ...... 34 6.0 RECOMMENDATIONS ...... 35 7.0 ACKNOWLEDGEMENTS ...... 35 8.0 REFERENCES ...... 35
111 Final Report: Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RU/1001/PBIOLOGJ/815103
1.0 INTRODUCTION Biosurfactants are a diverse group of secondary metabolites with surface active properties of microbial origin. The existence of both polar and non-polar domains help biosurfactants to form partition at the water-oil or water-air interfaces, thus effectively reducing the interfacial or surface tension in aqueous and hydrocarbon mixture (Banat et al., 2000). Such characteristics cause more researchers to show interests in the production of biosurfactants, particularly since the biosurfactants can be widely used in a diverse industrial application including microbial-enhanced oil recovery (MEOR), bioremediation, biomedical, cosmetics and food processing (Ron & Rosenberg, 200 I). Current research in our laboratory has established rhamnolipid production by Pseudomonas aeruginosa AR-2, a Gram negative, non-filamentous bacterium. However, this microorganism is an opportunistic pathogen to humans. Moreover, microbial production of biosurfactants in the market currently suffers high cost of production, which is a direct consequence of the cultivation requirements of these microbes and the daunting challenge in product purification and recovery. Alternatively, actinobacteria may be a viable production microbe. The filamentous growth nature of the actinomycetes offers a convenient intrinsic immobilization ability as mycelial pellets can greatly assist downstream processing in the production of biosurfactants. In addition, almost all filamentous bacteria are known as non pathogenic to humans and can be safely utilised in the industry. Therefore, the present study was undertaken to investigate the production of biosurfactant from actinomycetes species. In this project, a wild-type actinomycete isolate was used as a model organism in filamentous fermentation for biosurfactant production.
2.0 OBJECTIVES 1. To screen biosurfactant-producing actinomycetes from the local environment.
2. To identify and characterize of the potential biosurfactant-producers.
3. To establish good growth conditions ofbiosurfactant-producing actinomycete.
4. To enhance the production ofbiosurfactant via batch fermentation. Final Report : Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RUI 1001/PBIOLOGI/815103 3.0 RESEARCH METHODOLOGY
3.1 Microorganisms Previously-isolated actinomycetes from oil contaminated soil samples were used in this study. Pure isolates were maintained in starch casein agar (SCA). For long term preservation, suspension of mycelium and spores were kept in 40% (v/v) glycerol stock at -20°C.
3.2 Screening of Biosurfactant-Producers A total of 33 isolates of actinomycetes were cultivated into starch casein broth with 3% olive oil as the biosurfactant inducer to screen for the production of biosurfactant (Thampayak et al., 2008). The culture was incubated for 7 days in an orbital shaker (B. Braun Certomat® R, Germany) agitated at 200 rpm at 28°C. After 7 days of incubation, the cells were separated by centrifugation (Eppendorf Centrifuge 5424, Germany) at 8000 xg for 15 minutes at 30°C. Cell-free supernatant was analyzed for the presence ofbiosurfactant.
3.3 Analytical Techniques Samples were taken at regular intervals during the biosurfactant production experiments for the determination of surface tension, emulsification activity and oil displacement to monitor the production of biosurfactant. These quantitative methods were carried out to enable the selection of the best strain for the highest biosurfactant production. All tests were done in triplicates.
3.3.1 Surface Tension Measurement The surface tension of the cell-free culture broth was determined using a semi automatic Du-Nouy Tensiometer, model 70535 (CSC Scientific Company, Inc, VA) (de Nevers, 1991), which uses the Du-Nouy ring method. This method measures the surface force between a liquid and air in a liquid medium samples. Prior to measuring the surface tension of samples, the tensiometer was calibrated using distilled water and ethyl alcohol at 28°C.
3.3.2 Emulsification Index (E24) Two mL engine lubricating oil (Carlz, Hi-Tech) was added to 2 mL cell-free culture broth. The solution was mixed thoroughly for 2 minutes on a vortex mixer (KMC-1300V, Korea) and left to stand for 24 hours. The E24 index was calculated as a ratio of the emulsified layer height (em) to the total height of the liquid column (em), expressed as a percentage (Cooper & Goldenberg, 1987).
2 Final Report : Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RUJ IOOJ/PBJOLOGJ/815/03 3.3.3 Oil Spreading Technique (OST) The oil spreading technique was performed to detect the presence of biosurfactant. Forty JlL of distilled water was added to a Petri dish. Then, I OJ.tL of crude oil was added to the surface of the water. Ten J.tL of sample was then added onto the centre of the oil film. The diameter of the clear zone on the oil surface was measured with a calliper rule and compared with an uninoculated medium as the control (Youssef eta!., 2004). Any surfactant present in the sample would result in a clear zone with a larger diameter compared to that of the control.
3.3.4 Determination of Biomass A known volume of a culture broth sample was filtered using a dry and pre-weighed filter paper Whatman filter paper No.1). The filtered sample was washed once with acetone and twice by distilled water to remove residual oil from cells. The sample was subsequently dried to a constant weight in an oven at I 05°C.
3.4 Identification of Potential Biosurfactant-Producing Actinomycete
3.4.1 Phenotypic Characterization Phenotypic characterization of the potential biosurfactant producer was based on cell morphology. Generally, microbes are mainly characterized on the basis of their morphological characteristics. The selected biosurfactant-producing actinomycete was grown on SCA and incubated for 7 days at 28°C. After 7 days of incubation, the isolate was identified based on macroscopic and microscopic morphological characteristics such as growth on microbiological media, aerial spore mass colour, substrate mycelium pigmentation and colour of any soluble pigment described by (Waksman, 1961).
3.4.2 16S rRNA Gene Amplification, Sequencing and Phylogenetic Analysis A mycelia pellet was obtained from a five-day old culture of a selected isolate, Rl. The genomic DNA of the isolate was extracted using cetyltrimethylammonium bromide (CTAB) method (Johnson et al., 2012). PCR amplification of the 16S rRNA gene sequence was done in DNA Engine™ Peltier Thermal Cycler Model PTC-100 (USA) using the primer, 27f (5' AGAGTTTGATCMTGGCTCAG-3') and 1525r (5'-AAGGAGGTCWTCCARCC-3'). The PCR amplifications were done using an initial denaturation step at 95 °C for 5 minutes, followed by 30 cycles of 1 minutes at 95°C, annealing at 54°C for 1 minutes and primer extension at 72 °C for 1 minute. The final extension was done at 72 °C for 10 minutes before being cooled to 4°C. The PCR product was purified using a PCR purification kit (Qiagen,
3 Final Report : Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RUI JOOJ/PBJOLOGJ/815103 Germany). The purified PCR product was then sent for sequencing at First Base Laboratories Sdn. Bhd., Selangor using ABI PRISM ®377 DNA Sequencer (Applied Biosystems).
The obtained partial 16S rRNA sequence was corrected manually and aligned using ClusterW Multiple alignment in BioEdit Sequence Alignment Editor version 7 .0.5 (Hall, 1999) to generate a consensus sequence. The consensus sequence of the selected biosurfactant producing actinomycete was then compared to those available in the GenBank database using Basic Local Alignment Search Tool to find the probable identity or the nearest match of the new sequence (http://www.ncbi.nlm.nih.gov/). The consensus sequence of the selected biosurfactant-producing actinomycete and representative sequences from GenBank were used to construct a phylogenetic tree using Molecular Evalutionary Genetic Analysis (MEGA 5) software (Tamura et al., 2011 ).
Determination of genetic distances and evaluation character among the sequences was performed by the neighbor-joining (NJ) and maximum likelihood (ML) method (Saitou & Nei, 1987).
3.5 Inoculum Preparation A fresh, single pure colony isolate of actinomycete was transferred aseptically from a nutrient agar plate into nutrient broth using a sterile wire loop. The inoculated medium was incubated for 48 hours at 28°C on a rotary shaker at 200 rpm (B. Braun Melsungen AG 886344/G, Germany). After 48 hours of incubation, 7 mL sample of the seed culture was homogenized in a 7 mL cell grinder (Safe-Grind, Wheaton) by moving the pestle up and down for 18 times. The optical density of the homogenized mixture was measured using a UV-Vis spectrophotometer (Thermo Spectronic, 40001/4, USA) at the wavelength of 600 nm. The absorbance reading ofthe seed must lie in the range 0.5-2.0. Then, 10% volume aliquot of an overnight preculture grown for 48 hours was transferred to 50 mL of the production medium in 250 mL conical flasks.
3.6 Shake flask cultures
3.6.1 Scouting of Suitable Growth Condition for Streptomyces sp. Rl To find the optimal culture conditions for growth and biosurfactant production of Streptomyces sp. R 1, factors such as carbon and nitrogen sources, effect of Tween® 80, inoculum concentration, salinity and pH, were studied. All experiments were conducted with mineral salt medium containing the following (g/L): casein hydrolysate, 0.3; NaCl, 2.0; KN03,
4 '. Final Report : Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RUJ 1001/PBJOLOGI/815103 2.0; K2HP04, 2.0; MgS04.7H20, 0.05; FeS04. 1H20, 0.01 and CaC03, 0.01. The fermentations were carried out for 7 days at 28°C on an orbital shaker (B. Braun Certormat® R, Germany). After 7 days of cultivation, cells were harvested by filtration. The cells were used to measure the biomass concentration, whereas the supernatant samples were subjected to biosurfactant analysis as described in Section 3.3. Results were analyzed using IBM SPSS Statistics software version 20 (Tukey test) (Universiti Sains Malaysia).
3.6.1.1 Selection of Carbon Source Three types of carbon sources were investigated. These were water-soluble carbon sources (glucose, fructose, sucrose, dextrose, glycerol and starch at a concentration of20 g/L), oils, representing water-insoluble carbon sources (olive oil, palm oil and waste cooking oil at concentration of 1% v/v), and several hydrocarbons (diesel, toluene, kerosene and n-hexane at concentration of 1% v/v). They were screened to find the best carbon source for the growth of Streptomyces sp. R1.
3.6.1.2 Palm Oil Concentration Growth and biosurfactant production by Streptomyces sp. R1 were observed in cultivation medium with different concentrations of palm oil as the sole carbon source at 1 to 7% (v/v).
3.6.1.3 Selection of Nitrogen Source To evaluate the most preferred nitrogen source for the production of biosurfactant by Streptomyces sp. R1, yeast extract, tryptone, urea, peptone, ammonium sulfate, ammonium chloride and KN03 at a concentration of 10 giL were screened with the optimum carbon source (Khopade eta/., 2012).
3.6.1.4 Yeast Extract Concentration Growth and biosurfactant production by Streptomyces sp. R1 were observed in the cultivation medium with different concentrations of yeast extract as the nitrogen source at 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 % (w/v).
3.6.1.5 Synthetic Surfactant (Tween® 80) Addition The growth of Streptomyces sp. R1 was investigated in the culture medium unsupplemented and supplemented with Tween 80 (synthetic surfactant) at a concentration of 1, 2, 3 and 4 % (v/v) in the medium with optimum carbon and nitrogen sources.
5 Final Report : Production of Biosurfactants via Locally-Isolated Actinomycete Fermentation RUI 1001/PBIOLOGI/815103 3.6.1.6 Inoculum Concentration Growth and biosurfactant production by Streptomyces sp. Rl were evaluated in cultivation medium with different concentrations of inoculum ranging from 2, 4, 6, 8 and 10% (v/v).
3.6.1. 7 Salinity The effect of salinity on growth and biosurfactant production by Streptomyces sp. Rl was observed in different concentration ofNaCl at 0.3, 0.5, 0.7 and 1.0% (w/v). Growth and biosurfactant production in culture medium without NaCl was used as the control.
3.6.1.8 Initial pH The effects of initial pH on growth of Streptomyces sp. Rl and biosurfactant production · were investigated by varying the initial pH of the culture medium at pH 5, 6, 7, 8 and 9. The pH was adjusted using 1 M HCL and NaOH. At the end of fermentation, the pH ofthe culture medium was measured.
3. 7 Bioreactor Studies
3.7.1 Oxygen Requirement
3. 7.1.1 Determination of Volumetric Oxygen Transfer Coefficient (kLa) The determination of volumetric oxygen transfer coefficient (kLa) was conducted using the static gassing out method (Garcia-Ochoa & Gomez, 2009). The experiment was evaluated in distilled water with 6% (v/v) of palm oil. The mass balance for dissolved oxygen is as follows;
--dCL- k La (C* - CL ) (1) dt
where CL =dissolved oxygen concentration in the broth
and C* = saturated dissolved oxygen concentration in the broth.
3.7.1.2 Determination of Oxygen Uptake Rate (OUR)
The volumetric oxygen uptake rate,q02 x, was determined during fermentation. During a batch fermentation, the air supply and agitation were turned off. The rate of dissolved oxygen decreasing was monitored and recorded. The descending dissolved oxygen concentration (CL) was plot as a function of time to obtain slope that represents OUR. These conditions can be simplified by following equation: 6 Final Report : Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation '' RUJ 1001/PBJOLOGJ/815103
(2)
3.7.2 Batch Cultivation Batch cultivations were conducted in a 3 L stirred-tank bioreactor with a working volume of 1.5 L, equipped with a Rushton-type impeller (Bioflo 115, New Brunswick, USA). The reactor was equipped with a polarographic dissolved oxygen electrode (InPro6830/12/220, Mettler Toledo, Germany) and steam sterilisable pH probe (405-DPAS-SC-K8S/225, Mettler Toledo, Germany) to monitor the dissolved oxygen concentration and pH in the culture medium, respectively. Operating parameters such as temperature, pH, dissolved oxygen concentration, aeration and agitation were monitored and controlled by computer using a program written in BioCommand OPC (New Brunswick Scientific Co., Inc., New Jersey). All cultivations were carried out at 28°C and aerated at 0.5 vvm. The pH was left uncontrolled during the cultivation. Throughout the cultivation in the reactor, 30 ml of the culture broth were collected at 24-hour intervals for the analysis of biomass growth and biosurfactant production as described in Section 3.3.
3.7.2.1 Influence of Agitation Speed on Growth and Biosurfactant Production The effect of agitation speed on Streptomyces sp. R1 growth and its biosurfactant production was investigated by cultivating at different agitation speeds (200 rpm, 400 rpm, 600 rpm and 800 rpm). All experiments were repeated twice for each agitation speed.
3. 7.2.2 Influence of Agitation Speed on Morphological of Streptomyces sp. Rl The impact of agitation speeds on the morphology of Streptomyces sp. Rl was observed by taking I mL of sample at the end of the fermentation for every agitation speed. The morphology was observed under compound light microscope (Olympus model BX41, Germany) at 4 xg magnification.
3.8 Biosurfactant Extraction To extract the biosurfactant, 200 mL of culture broth was centrifuged at 10,000 xg, 4·c, 10 minutes (Sigma 3-18 K Centrifuge, Germany) to remove the cells. Three methods of biosurfactant extraction were evaluated, namely solvent extraction, ammonium sulphate extraction and acid precipitation. Extracts were confirmed to contain biosurfactant using E24 and ST as described in Section 3.3.
7 Final Report: Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RUJ 1001/PBIOLOGI/815103 3.8.1 Solvent Extraction The cell-free culture broth were lyophilized and extracted with a mixture of solvents (methanol/chloroform/1-butanol) with a ratio of 1:1:1 by volume. The mixtures were then shaken continuously in a rotary shaker (B. Braun Melsungen AG 886344/G, Germany) at 28°C and 200 rpm for 5 hours to allow phase separation. After 5 hours, the solvent layer was removed and the aqueous layer was poured into a clean glass Petri dish. The Petri dish containing a crude extract was left to evaporate in the fume hood until dry. The dried crude extract was obtained and was dissolved in distilled water for further analyses.
3.8.2 Ammonium Sulphate Precipitation The supernatant was precipitated with ammonium sulphate and left overnight at 4 °C. The precipitate was collected by centrifugation (10,000 xg, 4°C, and 10 minutes). After centrifugation, the precipitate was then extracted using acetone. The mixture was transferred into a clean glass Petri dish and left to evaporate in the fume hood. After approximately 24 hours of drying, a dry, crude extract was obtained.
3.8.3 Acid Precipitation The supernatant was acidified by a drop wise addition of 1 M HCL to pH 2 and keeping it overnight at 4 °C for the complete precipitation of the biosurfactant. The precipitate formed was collected by centrifugation (1 0,000 xg, 20 minutes, 4°C). The precipitate was then washed with acetone and left to evaporate until dry in the fume hood.
3.9 Determination of Biosurfactant Dry Weight A known volume of culture broth will be extracted with a mixture of solvents (methanol/chloroform/1-butanol) with a ratio of 1:1:1 by volume. Then, the crude biosurfactant extract will be dried to constant weight to determine the concentration in g/L.
3.10 Biosurfactant Characterization Biochemical analyses such as Benedict test, Saponification test, Biuret test and Iodine test were performed onto the crude extract to characterize the biosurfactant.
3.10.1 Benedict Test The sugar content of the extracted biosurfactant was detected using Benedict method (Thenmozhi eta!., 2011 ). To a 5 mL of Benedict's reagent, 8 drops of crude biosurfactant was added and mixed evenly. Then, the solution was heated at 70°C for 15 minutes. After 15
8 '\ Final Report : Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RUI 1001/PBIOLOGJ/815103 minutes incubation, a reddish orange precipitate would form which indicated positive result. The sterile production medium was used as a control.
3.1 0.2 Saponification test The saponification test was done to detect the presence of lipid (Bhavani & Hemashenpagam, 2013). To every 2 mL of sample, 2 mL of 2% NaOH solution was added. The solution was vigorously shaken using vortex mixer (KMC-1300V, Korea) and the formation of soap was observed. The sterile production medium was used as a control.
3.10.3 Biuret test The detection of proteins was done using the Biuret test (Jamal et al., 2012). Two millilitre of crude extract solution was heated at 70°C before mixed with 1 M NaOH solution. Then, 1% CuS04 was added dropwise slowly into the solution and a change in colour was observed. The formation of a violet or a pink ring indicated the positive result due to the reaction of peptide bonds in proteins or short-chain polypeptides. The sterile production medium was used as a control.
3.10.4 Iodine Test The iodine test was performed to detect the presence of carbohydrate (Bhavani & Hemashenpagam, 2013 ). To a 2 mL of crude biosurfactant, 4-5 drops of iodine solution was added and mixed gently. Any carbohydrate present would cause a reddish brown colour formation. The sterile production medium was used as a control.
3.10.5 Fatty Acid Analysis The fatty acid from the crude extract was extracted using two-step transesterification method. Approximately 10 mg of crude biosurfactant was mixed with 2 mL of 0.5 M methanolic NaOH (2g NaOH in 100 ml methanol) in the test tubes. To every test tube containing solution, 2 to 3 glass beads was added and incubated at 75°C for 10 minutes. After that, 3 mL ofBF3 was added to the mixture and incubated again for 2 minutes at 75°C. At the same temperature, a volume of 1.5 mL heptane was pipette into the mixtures and incubated for 1 minutes. The mixture was then left to cool at 28°C. After a few minutes, about 500 J.lL of saturated NaCl were added to the mixture until the top layer was clearly separated. Then, the top layer that comprised a mixture of fatty acid methyl ester (FAME) was transferred to a 1. 5 mL Eppendorf tube containing a pinch of Na2S04 anhydrous. The tube was shaken for few
9 Final Report : Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation I' RUJ JOOJ/PBIOLOGI/815103 seconds to remove residual water. The clear layer was transferred into gas chromatography (GC) vial and kept at -20°C until GC analysis was carried out.
The fatty acids composition of crude biosurfactant was analysed by gas chromatograph combined with mass spectrometer (GC-MS) (QP201 0 Ultra, Shidmadzu, Japan). The machine was equipped with a flame ionization detector and a SP-2380 polar capillary column (Supelco 30m x 0.25Jlm x 0.20 Jlm). The temperature of column was programmed from 100°C to 250°C at 1.5°C/minutes and detector temperature at 250°C. Helium was used as the carrier gas at flow rate 1.0 mL!minutes (Shubhrasekhar et al., 2013). The resultant peaks from gas chromatography were subjected to mass-spectral analysis. Then, the mass-spectra analysis was done by matching their recorded mass spectra with those obtained from the NIST08s and FAME library spectrum.
4.0 RESULTS AND DISCUSSION
4.1 Screening of Biosurfactant-Producing Actinomycetes Table 1 summarizes the results for the screening experiment using two methods; OST and E24. Out of 33 isolates, 32 isolates showed positive results from OST, showing the higher sensitivity of this method in detecting surfactants. The results from the E24 test demonstrated that all the isolates showed positive results, with good E24 ranging from 84.11-95.80%. Based on the OST and E24 values aforementioned (Table 1), isolate Rl was selected as the model biosurfactant-producer for this study.
4.2 Identification and Characterization of the Potential Biosurfactant Producer
4.2.1 Phenotypic Characterization Morphological characteristics have been used as a method for preliminary determination of the genus for the actinomycetes taxonomy (Khanna & Solanki, 2011 ). The examination of the macroscopic characteristics for isolate Rl grown on SCA at 28°C for 7 days showed that the outer surface of the colonies was perfectly round initially, but later developed aerial mycelium that appeared velvety white colour (Figure 1a) whereas the substrate mycelium was pale brown (Figure 1b). Isolate R 1 showed concentric rings of colonies on the SCA plate as incubation time progressed. This is one of the important diagnostic criteria for genus Streptomyces (Waksman, 1961 ). Observation under the light microscope demonstrated that isolate Rl showed oval shape of conidia (Figure lc). On the basis of morphological characteristics, isolate Rl was presumptively assigned to the genus Streptomyces.
10
------~-- Final Report : Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RUI 1001/PBIOLOGJ/815103 Table 1.0: Screening of biosurfactant-producing actinomycetes using oil spreading technique and emulsification activity.
No Isolate Diameter of clear zone E24 (em) (%) 1.10±0.17 Control 0.00±0.00 B1 4.40±0.17 1 90.40±5.71 B2 1.35±0.21 2 87.90±0.14 1.27±0.12 3 B3 86.84±3.72 3.17±0.29 4 B4 84.11±0.15 B5 1.17±0.15 5 90.00±4.71 1.53±0.35 6 I1 92.16±0.22
7 12 3.70±0.14 87.25±0.35 2.67±0.29 8 I3 90.84±1.18 14 1.90±0.14 9 90.63±4.42
10 IS 2.40±0.28 90.42±4.12
11 K1 1.25±0.07 89.18±0.41 4.30±0.26 12 K2 92.47±3.20 2.47±0.15 13 K3 88.05±0.07
14 K4 6.03±0.15 92.23±2.16
15 K5 2.53±0.06 89.24±0.33
16 K6 3.70±0.14 90.63±4.42
17 N1 4.10±0.14 85.17±0.77
18 P1 7.1 0±0.14 90.24±0.34
19 P2 6.46±0.24 93.46±3.60
20 P3 7.03±0.06 89.24±0.33
21 P4 5.65±0.07 91.95±4.32
11 Final Report : Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RUJ 1001/PBJOLOGJ/815103 22 P5 3.03±0.06 94.22±0.31 23 P6 6.15±0.21 88.74±1.04 24 P7 3.30±0.17 91.50±0.71 25 P8 3.07±0.12 89.69±1.12 26 P9 2.60±0.14 92.74±3.20 27 P12 0.97±0.06 88.94±2.79 28 Pl3 4.20±0.26 84.61±0.56 29 R1 7.45±0.07 95.80±0.28 30 R2 3.60±0.14 93.00±4.24 31 R3 5.95±0.21 85.36±0.50 32 R5 4.05±0.07 93.08±3.06 33 81 6.35±0.07 84.26±1.32
12 Final Report : Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RUI 1001/PBIOLOGI/815103
Figure 1 (a-c): The morphology of isolate Rl after 7-day incubation on SCA at 28°C. Upper surface of isolate Rl was white in colour, (b) lower surface was pale brown in colour (c) oval-shaped conidia under light microscope with 4Qx magnification.
4.2.2 16S rRNA Gene Sequence Analysis The 16S rRNA sequence data are used in the modem Streptomyces identification system since they provide valuable information about Streptomycetes systematic (Forar et al., 2006a). Thus, further taxonomic characterization of selected biosurfactant-producing actinomycetes was done by 16S rRNA gene sequence analysis. It is evident from the 16S rRNA gene phylogenetic tree that isolate Rl represents a member of the genus Streptomyces (Figure 2). These analyses were in line with the results obtained from morphological characteristics. Isolate Rl formed a phyletic line that was closely related to Streptomyces sp. sharing 16S rRNA gene similarities with the latter at 92%. This taxonomic relationship is supported by 65% bootstrap value. Based on the results from morphological and molecular characterization, isolate Rl is likely a member of the genus of Streptomyces.
13 Final Report : Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RUJ 1001/PBJOLOGI/815103
Streptomyces sp. R9-549 (JQ660003) Streptomyces sp. R9-545 (JQ660000) Streptomyces sp. DA 10202 (HM371156) 97 Actinobacterium 220362 (FJ429804) Streptomyces sp. DA08602 (FJ797603) 65 Streptomyces sp. ACT-0096 (GQ924536) Streptomyces sp. CNR924 PL04 (00448732) 99 1.....------R1 Streptomyces chartreusis strain DSM 41255(F J932482) 89 Streptomyces chartreusis strain HA 10304 (JF728875) Streptomyces bungoensis strain 15721 (JN 180215) Streptomyces gal bus strain NG4 (JF827353) Streptomycescyaneus(AJ399471) 61 Streptomyces curacoi (EF626595) 100 Streptomycescoeruleorubidusstrain NBRC 12761 (NR041217) Streptomyces sp. 5 (EU360169) Streptomyces longisporus strain ISP 5166 (N R025492) 100 Streptomyces flavidovirensstrain NBRC 13039 (NR041 099) 88 Streptomyces longisporus gene (AB184219) Streptomyces coacervatus gene (AB500703) Streptomyces sp. L-2-2 (EF524054) Streptomyces sp. MSC702 (JF325872) 1oo Streptomyces sp. A72 (EF100783) Streptomyces sp. S72 (EF208617)
100 Streptomycessp.A71 (EF100782) Streptomyces sp. A177 (EF1 00784) 100 Streptomycessp. S177 (EF197893) ,------Nocardiopsis sp. MSA 10 (EU563352) L.....-.--1 1oo ~.....-______Microbacterium esteraromaticum strain PA4 (EU647562)
f-----1 0.01
Figure 2: Neighbour-joining tree based on almost complete 16S rRNA gene sequences showing relationships among the isolates R 1, representatives of the genus Streptomyces and biosurfactant-producing actinomycetes using Jukes-Cantor method. The percentage of bootstrap values (1000 replicates) that are higher than 50% are shown next to the branches.
14 -, Final Report : Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RUI 1001/PBJOLOGJ/815103 4.3 Shake flask cultures
4.3.1 Scouting for Suitable Growth Condition for Streptomyces sp. Rl.
4.3.1.1 Selection of Carbon Source The influence of different types carbon sources on growth and production based on
surface tension reduction and E24 by Streptomyces sp. R1 is presented in Figure 3. Based on these observations, it is tempting to speculate that Streptomyces sp. R1 may be able to utilize all types of carbon sources tested for growth although the yield was limited with glycerol. This finding was in agreement with the report ofK.hopade et al. (2012) in which glycerol gave a low biosurfactant production in Streptomyces sp. B3. Out of the various carbon sources that were tested, starch and palm oil were found to be favourable carbon sources for Streptomyces sp. R1 growth and biosurfactant production. Streptomyces sp. Rl showed good growth on starch as a sole carbon substrate but gave a small reduction of ST of distilled water.
The highest biomass of 4.51 g/L was accompanied by biosurfactant production, causing
a reduction of distilled water ST of 11.6%. Moreover, an E24 value of 40.7% was achieved when isolate Rl was grown in palm oil as the sole carbon source. Palm oil was chosen as the most preferred carbon source for Streptomyces sp. Rl because it is abundantly available in Malaysia, as one of the world's largest palm oil-producing countries. Thus, palm oil was selected as the sole carbon source for further experimentation to enhance growth and production biosurfactant by Streptomyces sp. Rl.
4.3.1.2 Palm Oil Concentration The adverse effect of different palm oil concentration on growth and biosurfactant production by Streptomyces sp.R1 is shown in Figure 4. There is a linear increase in biomass
and E24 with increasing initial palm oil concentration up to 6% (v/v). Beyond this value,
biomass and biosurfactant concentration (based on reduction of distilled water ST and E24) were significantly decreased. As shown in Figure 4, the maximum biomass (14.02 g/L) and biosurfactant production were observed when 6% (v/v) palm oil was used in the cultivation of
Streptomyces sp. Rl, indicated by the highest reduction of ST (28.1%) and E24 (85.2%). Therefore, a mineral salt medium supplemented with 6% (v/v) palm oil was chosen as the appropriate culture medium for the next biosurfactant production experiments.
15 Final Report : Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RUJ 1001/PBJOLOGI/815103
50.0 (a) 5.0 -=~ 4.5 40.0 -0 4.0- M 3.5 ~ =-=- 30.0 3.0- """~=~ 2.5 ; E-c ~ 20.0 2.0 e 00~ 1.5 ...= ...... ==- 10.0 1.0 ~ CJ 0.5 "C= ~ 0.0 0.0 ~ Toluene n-Hexane Kerosene Diesel Hydrocarbons
~ 50.0 5.0 = 4.5 0- 40.0 4.0 ;3:' M 3.5 bll ="C '? 30.0 3.0- """~ 2.5 ; E-c=- ~ 20.0 2.0 e 00~ 1.5 .s §- 10.0 ...... 1.0 ~ CJ 0.5 "C= 0.0 0.0 ~ Olive oil Palm oil Waste cooking oil Oils
50.0 (c) 5.0 o_ 4.5
"C-=~ 40.0 4.0- """ .., 3.5 = M ~ E-c ~ 30.0 3.0- 00- 2.5 ; =~ e 20. o 2.0 e ...= ~ .... ~ 1.5 .s CJ = -6 -6' 10.0 1.0 ~ ~- 0.5 0.0 0.0
Soluble Carbon • Reduction ST of dH20 • E24 % • Biomass
Figure 3: Effect of carbon sources on biosurfactant production by Streptomyces sp. R1; (a) hydrocarbon; (b) oils; (c) soluble carbon sources.
16 Final Report: Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RUI 1001/PBIOLOGJ/815103
100.0 16.0 -~ =..., 90.0 -N 14.0 ~ ...... 80.0 12.0 -=~ 70.0 -0 10.0 - N 60.0 ~ I'll ="'0 50.0 8.0 -I'll c.. Q e= E-- 40.0 - 6.0 Q 00. .... 30.0 = ...... =Q 4.0 Col 20.0 "'0= 2.0 ~ 10.0 ~ 0.0 0.0 1 2 3 4 5 6 7 Concentration of Palm Oil (% v/v)
• Reduction ST of dH20 • E24 % +Biomass
Figure 4: Effect of different concentration of palm oil (% v/v) on biosurfactant production by Streptomyces sp. R1
4.3.1.3 Selection of Nitrogen Source The results in Figure 5 showed that the nitrogen source exhibited a significant effect on biosurfactant production. Streptomyces sp. R1 was able to use all nitrogen sources that were tested for growth and biosurfactant production as indicated by the reduction of ST and E24. Among the nitrogen sources tested, yeast extract, tryptone and KN03 are the most preferable, promoting higher biomass, reduction of distilled water ST and E24. The highest percentage of ST reduction of distilled water (29.86%) and E24 (42.86%) were achieved in the culture with yeast extract. Yeast extract is the most widely used nitrogen source for biosurfactant production (Saharan et al., 2011). Consequently, yeast extract was chosen as the best nitrogen sources for Streptomyces sp. R1 growth and biosurfactant synthesis.
17 Final Report: Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RUI IOOI/PBIOLOGI/815103
- 50.0 10.0 ~ t... 45.0 9.0 N""' ::: 40.0 8.0 '!-.- 35.0 7.0 - -0 ~ N 30.0 6.0 ~ -~ =~ 25.0 5.0 e= Q ....Q ~ 20.0 4.0 = 15.0 3.0 Q ...... = CJ 10.0 2.0 "'0= ~ ~ 5.0 1.0 0.0 0.0 Yeast Urea Peptone Tryptone Beef KN03 extract extract Nitrogen sources
• Reduction ST of dH20 (%) • E24% • Biomass (giL)
Figure 5: Effect of nitrogen sources on growth and synthesis ofbiosurfactant by Streptomyces sp. R1.
4.3.1.4 Yeast Extract Concentration Figure 6 shows the results of effect of different yeast extract concentration on growth and biosurfactant production by Streptomcyes sp. R1. The results seem to suggest that a low yeast extract concentration in the medium favoured biosurfactant production. According to Casas and Garcia-Ochoa (1999), when nitrogen was in excess in yeast fermentation, biosurfactant production decreased because the carbon source was used mainly for growth. Thus, restrained condition of these macro-nutrients was preferable in order to obtain high concentration of biosurfactant (Khopade et a/., 20 12). Based on the reduction ST of distilled water (23.6%) and E24 (45.4%), 0.6% (w/v) yeast extract was deemed as the optimum concentration of yeast extract for biosurfactant production by Streptomyces sp. R1.
18 Final Report : Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RUJ 1001/PBIOLOGJ/815103
60.0 12.0 =~ "'"N -~ 50.0 10.0 -=~ 40.0 8.0 - -0 ~ N VJ 30.0 6.0 -VJ ="'CC a~ """= 20.0 4.0 = ~ ·- 00 = 10.0 2.0 ....= ·-CJ "'CC= 0.0 0.0 ~ 0.2 0.4 0.6 0.8 1.0 1.2 Concentration ofYeast extract (% w/v)
• Reduction ST of dH20 (%) • E24% • Biomass (g/L)
Figure 6: Effect of different concentration of yeast extract(% w/v) on growth and synthesis ofbiosurfactant by Streptomyces sp. R1.
4.3.1.5 Synthetic Surfactant (Tween® 80) Addition As aforementioned, palm oil was selected among other carbon sources that were evaluated for growth and biosurfactant production by Streptomyces sp. R1. According to Marsudi et al. (2008), 1 g of palm oil contains 0.94 g of total free fatty acids and 0.09 g of glycerol. The major portion in palm oil, which is the fatty acid, makes it partially soluble in water. Thus, it was hypothesized that an added surfactant might enhance the solubilization of this substrate in water for its uptake by Streptomyces sp. R1, in the culture medium.
Polyethylene glycol sorbitan monooleate (Tween® 80) is a widely used, food grade synthetic surfactant. The addition of Tween® 80 has been reported to enhance the degradation of oil in the culture medium, possibly due to the increased contact between oil and water as the surfactant reduced the interfacial tension between oil and cell surface (Feng et al., 2006). In this study, the effects of different concentration of Tween® 80 on the growth of Streptomyces sp. R1 were observed. As shown in Figure 7, there is a positive correlation between biomass with the increasing concentration of Tween® 80 from 1 to 3% (v/v) compared with the unsupplemented with Tween® 80 (control). It seems to suggest that Tween® 80 is necessary for the utilization of palm oil in the medium, thereby affecting the biomass of Streptomyces sp. 19 Final Report : Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RUI IOOJ/PBJOLOGJ/815103 Rl. This could be due to the ability of this synthetic surfactant to act as an oil-in-water emulsifier and hence increase the availability of insoluble substrates to the cell.
5.0 -4.0 ~_3.0 rll rll ~ § 2.0 ·-= 1.0
0.0 1 2 3 4 Control Tween 80 (% v/v)
Figure 7: The effect of different concentration of Tween® 80 on growth of Streptomyces sp. R1
The present study showed that the highest biomass densities, at 4.1±0.3 g/L and 4.0±0.7 g/L, were attained when the culture medium was supplemented with 2 and 3% (v/v) Tween® 80, respectively. However, when the concentration of Tween® 80 was increased to 4% (v/v), the resulting biomass was rather low, at 2.2±0.4 giL, which was equivalent to that of the culture unsupplemented with Tween® 80 (2.1±0.1 g/L) that served as a control. This is because the presence of a surfactant above its critical micelle concentration (CMC) value could suppress adhesion of bacteria to the surface of liquid hydrocarbon droplets (Rodrigues et al., 2006). Hence, the present investigations conclude that the supplementation of Tween® 80 at concentrations within the range of 1 to 3% (v/v) is necessary to increase the growth of Streptomyces sp. Rl when using palm oil as a substrate.
4.3.1.6 Inoculum Concentration Inoculum concentration is another bioprocessing parameter that influences the growth and production of biosurfactant for every microorganism. Typically, in most microbial fermentation, the density of inoculum or seed culture will determine the duration of the lag phase, the specific growth rates, the biomass yield, and the quantity of the final product. The
20 ' \ Final Report : Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RUJ 1001/PBIOLOGJ/815103 effects of different inoculum concentrations (2 to 10% (v/v)) on the production ofbiosurfactant by Streptomyces sp. R1 is shown in Figure 8.
As seen in Figure 8, the highest percentage of surface tension reduction (29.1± 1.1 %) and the highest emulsification activity (60.0± 0.0 %) was achieved at 4% (v/v) inoculum of Streptomyces sp. Rl. Indeed, the maximum biomass density (13.2± 2.0 g/L) was also attained at this inoculum concentration. This could be due to the higher inoculum concentration (not exceeding the optimum level) which provided a higher cell density at the beginning of the fermentation. Increasing the inoculum concentration any further resulted in lowering the biomass yield and amount of surface tension reduction. The results can be explained in that the 4% (v/v) inoculum gave the highest cell density, which provided the microbial cell factories for the biosurfactant, leading to the highest uptake rate of insoluble substrate in the fermentation broth.
..,. M 70 16 ~ ...... 60 14 0 M 50 12- ="0 10 ~ '+-; 40 rll ~ 8 rll E--~ 30 eo!$ ~~ 6 5 .....~ ~ - 20 ~ ...... = 4 CJ 10 2 "0= ~ 0 0 2 4 6 8 10 ~:::!e Inoculum (%)
•% Reduction ST of dH20 •E24 (%) +Biomass (giL)
Figure 8: The effect of the inoculum concentration on biomass and biosurfactant production by Streptomyces sp. R1.
4.3.1. 7 Salinity Environmental factors such as salinity have been reported to affect biosurfactant production through their influence on cellular growth or activity in the culture medium. An experiment was carried out to investigate the effect of different NaCl concentrations within the
21 J' Final Report: Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RUI IOOIIPBIOLOGI/815103 range of0.3 to 1.0 (w/v) on growth and yield ofbiosurfactant by Streptomyces sp. Rl. Growth and biosurfactant production in a culture medium without NaCl was used as a control.
As seen in Figure 9, the maximum growth occurred at a salinity of 0.5 % w/v (1.38± 0.05 g/L) and 0.7% w/v (1.54± 0.23 giL). However, biomass growth decreased to 0.93± 0.11 g/L when NaCl concentration was increased further to 1.0% (w/v). These observations suggest that the growth of Streptomyces sp. R1 is sensitive to high salts concentration.
Although cell dry weight seemed to increase with an increase in NaCl concentration, the excreted biosurfactant did not show significant surface tension reduction and emulsification activity. It is tempting to speculate that the production ofbiosurfactant by Streptomyces sp. Rl is less tolerant to salinity due the low biomass densities obtained in this experiment. From the results of the present study, it was concluded that the growth of Streptomyces sp. R1 was optimum at 0.7 % (w/v) of NaCl, giving a surface tension reduction of 22.82± 0.50% and emulsification activity of 61.53±4.90 %.
-so 2.0 ~ '-;: 70 1.8 N 1.6 ::: 60 1.4 ~ ~50 1.2- =;;: 40 1.0 ; 0 E-o 30 0.8 § 00. 0.6 § 20 = ...... 0.4 :; 10 0.2 "0 ~ 0 0.0 ~ 0.3 0.5 0.7 1.0 Control NaCI (% w/v)
•% Reduction ST of dH20 • E24 (%) +Biomass (g/L)
Figure 9: The effect of salinity on the biomass density and biosurfactant production by Streptomyces sp. R1
4.3.1.8 Initial pH In general, biosurfactant production is dependent by the initial pH of the culture broth. In the present study, as anticipated, the biomass density and biosurfactant production by Streptomyces sp. R1 was indeed affected by the initial pH of culture broth (Figure 10). The cell growth of Streptomyces sp. R1 was the highest at 19.07±0.03 g/L when grown in a medium
22 Final Report : Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RUI 1001/PBJOLOGI/815103 with the initial pH of6. Nonetheless, cell growth decreased as the initial pH of the fermentation broth was increased from pH 6 to 9. This confirms the inability of Streptomyces sp. Rl to grow in an alkaline environment. Generally, the optimum pH range for Streptomyces growth lay between pH 6.5 and 8.0 (Waksman, 1961).
""'N 80 25 ~ ...... 70 0 20 N 60 ='t:l • - ""- 50 ~ Q • 15 1ll -1ll rJ'jQE--~ 40 10 a= =Q - 30 Q ...... CJ 20 ~ 't:l= 5 Cl.l 10 ~ . ·r--· "'""1 .T .. Q~ 0 0 5 6 7 8 9 Initial pH
• Reduction ST of dH20 (%) •E24 (%) • Biomass (g/L)
Figure 10: The effect of initial pH of culture broth on growth of Streptomyces sp. Rl and biosurfactant production in shake flask.
The data presented in Figure 10 also demonstrated that the highest reduction in surface tension (39.37±1.73%) was attained when the initial pH of the culture broth was adjusted to pH 6, which coincided with the pH for the highest biomass density. This observation suggests that the biosurfactant excreted in the fermentation broth has superior surface tension activity which helps in breaking the oil for consumption by Streptomyces sp. R1, thereby resulting in the high biomass density at this initial pH. Alternatively, this could also mean that the amount of biosurfactant produced at this initial pH was the highest compared to those at the other pH values. The present study also revealed that the surface tension reduction was decreased when the initial pH of fermentation broth was increased to more than 6. Based on biomass density and surface tension reduction, the optimum initial pH for growth and biosurfactant production by Streptomyces sp. R1 is pH 6.
23 Final Report : Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RUI IOOI/PBIOLOGI/815103 4.4 Bioreactor Studies
4.4.1 Oxygen Requirement
4.4.1.1 Oxygen Transfer Rate (OTR) The dissolved oxygen concentration in an aerobic submerged fermentation is strongly influenced by the rate of oxygen transfer from the gas phase to the fermentation broth. The oxygen transfer rate (OTR) must be determined to achieve the optimum design and operation of a bioreactor. The volumetric oxygen transfer coefficient (kLa) is often used to gauge the OTR in the bioreactor system. Therefore, the investigation on the influence of agitation speed on the kLa value using static gassing out method was performed in this study (Figure 11 ).
60 50.94 -50 '1 40 -30 ~ 20 ~ 10 0
Agitation
Figure 11: Volumetric oxygen transfer coefficient (kLa) as a function of agitation speed.
The data depicted in Figure 11 show that the kLa value increased from 11.82 to 50.94 h- 1 with increasing agitation speeds from 200 to 600 rpm. Increasing impeller speed will cause rapid breakage of gas bubbles into smaller sized bubbles and hence enhance gas-liquid interfacial area for mass transfer in the aqueous solution. Increasing the agitation speeds from 600 to 1000 rpm in the present study resulted in a decreasing kLa value. This is generally in contrast with other findings that revealed increasing kLa values with increasing impeller speeds. It is hypothesized that at high impeller speeds (800 to 1000 rpm), the vigorous dispersion of oil causes the oil droplet in the aqueous solution to become more soluble, effectively creating an emulsion, since palm oil is partially soluble in water. This behaviour leads to a viscous
24 Final Report: Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RUJ 1001/PBIOLOGJ/815103 broth, which will negatively affect kLa value since it provides resistance to the oxygen transfer from the gaseous to the liquid phase.
1 In the present study, the highest kLa value (50.94 h" ) was determined at the agitation speed of 600 rpm. The knowledge of the ha can be used to gauge the efficiency of agitation speed in this bioreactor system. More importantly, it can be used to ensure ample oxygen transfer during the aerobic bioprocess in this study.
4.4.1.2 Oxygen Uptake Rate (OUR) The rate at which oxygen is being consumed in a bioreactor provides the oxygen demand, appropriately termed as the oxygen uptake rate (OUR). The oxygen transfer rate (OTR) must be equal to or more than the oxygen uptake rate (OUR) to satisfy the oxygen requirement of the aerobic organisms. In batch culture, the rate of oxygen uptake varies with time due to the increasing cell density throughout the cultivation. The present study was conducted to evaluate the volumetric oxygen uptake rate of Streptomyces sp. R1 as a function of agitation speed.
As tabulated in Table 2, the OUR of Streptomyces sp. R1 increased from 6.60 to 19.36 mmol Oz L" 1 h" 1 when the agitation speed was increased from 200 to 800 rpm. This can be explained by the increase in biomass density (4.35±0.01 to 8.47±0.02 g/L) as a function of agitation speed.
Table 2: Oxygen Transfer Rate (OTR) and Oxygen Uptake Rate (OUR) as a function of agitation speed in stirred tank bioreactor. Aeration Agitation Impeller OTR OUR Biomass (vvm) (rpm) Tip Speed 0.95*ku C* qOz•X (giL) 1 1 1 (m.s- ) (mmol 02 L- h" ) (mmol 02 L-1 h-1)
0.5 200 0.52 14.15 6.60 4.35 ±0.01 400 1.05 42.52 15.65 6.33±0.00 600 1.57 60.98 8.70 7.70±0.03 800 2.09 47.76 19.36 8.47±0.02
Here, the data also show the increment of OTR from 14.15 to 60.98 mmol Oz L" 1 h"1 with increasing agitation speeds from 200 to 600 rpm. By comparing the data of OTR and OUR, it can be concluded that the oxygen supply in the present study at the aeration rate of 0.5 vvm was ample for Streptomyces sp. Rl culture.
25 Final Report : Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RUI1001/PBJOLOGI/815103 4.4.2 Batch Cultivation
4.4.2.1 Agitation Speed As often highlighted, agitation speed is one of the important bioprocess parameter that provide effective oxygen transfer rate during aerobic fermentation, thereby affecting the cell growth and its productivity. Therefore, the present study was initiated by manipulating the agitation speed from 200 to 800 rpm at 0.5 vvm to enhance the production ofbiosurfactant by Streptomyces sp. Rl in a batch cultivation.
Table 3 shows the positive influence of agitation speed on biosurfactant production by Streptomyces sp. Rl. The results from the batch fermentation showed that as agitation speed increased from 200 to 800 rpm, the biomass density of Streptomyces sp. Rl also increased from 4.35±0.01 to 8.47±0.02 g/L. It is noted that the increasing agitation speed facilitate better heat and mass transfer of oxygen and nutrient in the fermentation broth. Thus, the factors led to better Streptomyces sp. Rl growth in the present study.
Table 3: Growth and biosurfactant production by Streptomyces sp. R1 at different agitation speed Agitation Biomass Biosurfactant production (rpm) (giL) E24 ST (%) (dynes/em) 200 4.35 ±0.01 48.85±1.0 47.17±0.29 400 6.33±0.00 49.40±1.0 56.80±0.80 600 7.70±0.03 67.80±2.0 40.50±0.50 800 8.47±0.02 48.28±0.0 50.20±0.03
Albeit biomass density increased proportionally with agitation speed, the production of biosurfactant indicated by surface tension measurement and emulsification activity did not follow a similar trend in this study. At first, the production of biosurfactant increased when the cultures were agitated from 200 to 600 rpm. However, further increasing the agitation speed to 800 rpm caused a reduction to emulsification activity (48.28±0.0%) and led to the highest value of surface tension measurement (50.20±0.03 dynes/em). This could be due to the mechanical damage of mycelia or pellet structure caused by shear forces at this agitation speed, which in tum affected the production of biosurfactant.
26 Final Report: Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RUI 1001/PBIOLOGI/815103 The maximum emulsification activity (67.80±2.0%) and surface tension reduction (40.50±0.5 dynes/em) were attained at 600 rpm. Interestingly, based on the previous study, the 1 highest kLa value (50.94 h- ) was also found at this agitation speed (Figure 11). It seems to suggest that the higher kLa value promoted better biosurfactant production by Streptomyces sp. Rl regardless ofthe agitation speed.
As a conclusion, the best biomass growth was attained when the culture was agitated at 800 rpm (8.47±0.02 g/L). Nonetheless, 600 rpm was found to be the optimal agitation speed for biosurfactant production. The results suggest that the production of biosurfactant by Streptomyces sp. Rl did not depend solely based on biomass concentration.
4.4.2.2 Influence of Agitation Speed on Morphological Streptomyces sp. Rl Unlike microbial fermentation, the cultivation of filamentous organisms is far more complex because of bioprocessing parameters such as agitation speed that influences the morphology of the cell, thereby affecting the production of secondary metabolite. Nevertheless, data on the effects of agitation speed on morphology of Streptomyces sp. and its biosurfactant production is still limited. Hence, the influence of different agitation speeds (200 to 800 rpm) towards the cell morphology of Streptomyces sp. R1 and its correlation to the biosurfactant production were investigated in the present study.
As illustrated in Table 4, the agitation speed gave a significant effect on the pellet morphology of Streptomyces sp. Rl during batch cultivation. In general, increasing agitation speed from 200 to 800 rpm led to a more freely dispersed filamentous growth morphology (Figure 12) with a mean pellet diameter range of within 0.175±0.04 to 0.094±0.01 mm.
Table 4: Effects of agitation speed on the morphology of Streptomyces sp. Rl. Agitation speed Fungal morphology Mean pellet diameter (rpm) (mm) 200 Aggregate of 3-4 pellets 0.175±0.04 of various size
400 Big rounded pellets and 0.366±0.14 filamentous
600 Freely dispersed mycelia 0.106±0.02
800 Freely dispersed mycelia 0.094±0.01
27 Final Report: Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RUJ JOOIIPBIOLOGJ/815103 It was noted that at 200 rpm, the pellets were uniform in shape in the form of 3-4 aggregates. This is probably because the relatively mild agitation enables the pellet to agglomerate. In addition, the mean pellet diameter at this agitation speed was smaller (0.175±0.04 mm) than those seen at 400 rpm (0.366±0.14 mm) which are consistent with the results of biomass obtained in Table 3 (4.35 ±0.01 g/L at 200 rpm and 6.33±0.00 g/L at 400 rpm), possibly due to the low amount of dissolved oxygen in the cultivation medium. The pellet morphology at 400 rpm contained a mixture of big rounded pellet and filamentous form. It seems to suggest that a good mixing of oxygen, temperature and nutrients at this agitation speed promoted the pelleted growth. However, increasing agitation speed from 600 to 800 rpm resulted in smaller mean pellet diameter (0.1 06±0.02 mm and 0.0904 ±0.01 mm) with dispersed filamentous growth form.
Upon closer inspection of Table 3 and Figure 12, it appears that the maximum biosurfactant production indicated by lowest surface tension measurement (40.50±0.5 dynes/em) and maximum emulsification activity (67.80±2.0%) was obtained when the morphology of Streptomyces sp. R1 was in dispersed form of growth (Figure 12c) at 600 rpm of agitation speed. Consequently, it is tempting to speculate that the production of biosurfactant is influenced by culture morphology.
The results clearly demonstrate that there is a profound correlation between agitation speed, morphology of Streptomyces sp. Rl and biosurfactant production. It is thought that the production of biosurfactant in the fermentation broth is responsible for controlling broth surface tension, which in turn influences the morphological of microbial filamentous culture. Therefore, the production of biosurfactant by Streptomyces sp. R1 may be a morphological auto-regulation mechanism.
28 Final Report : Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RUI 1001/PBJOLOGJ/815103
Figure 12: Pellet morphology of Streptomyces sp. Rl as a function of agitation speed under light microscope (a) 200 rpm; (b) 400 rpm; (c) 600 rpm; (d) 800 rpm.
4.5 Biosurfactant Extraction Figure 13 shows that all three methods namely ammonium sulphate precipitation, acid precipitation, and solvent extraction are successful in extracting biosurfactant from Streptomyces sp. R1 fermentation broth. As depicted in Figure 14, the brown oily paste, containing 39.54 g/L ofbiosurfactant was successfully precipitated using ammonium sulphate precipitation. The crude extract showed a surface tension reduction to 40 dynes/em and an emulsification activity of 37.04±1.81 %. As extensively discussed in the literature, the precipitation method has been widely used due to the amphipathic nature of biosurfactant. Ammonium sulphate precipitation used the salting-out effect to isolate biosurfactant from the culture broth (Kim et al., 2000).
29 Final Report : Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RUI 1001/PBJOLOGJ/815103
70 70 -~ 60 -.., 60- ~50 ...... 50~..... 4o 40 ~ 9 't ~ 30 ~ 30 ct ~20 20 ~ "C ~ -~ 10 10 ~ 00 0 0 Control Ammonium Acid Solvent medium sulphate precipitation extraction Recovery method
• ST (dynes/em) • E24 (%) • Biosurfactant (g/L)
Figure 13: Different recovery method for biosurfactant produced by Streptomyces sp. R1.
Figure 14: The brown oily paste of crude extract biosurfactant produced by Streptomyces sp. Rl.
Like ammonium sulphate precipitation, the acid precipitation also gave sticky brown oily paste. However, the concentration of crude extract was slightly lower at 27.15 giL which showed a surface tension reduction to 40.0±0.29 dynes/em and emulsification activity of 53.33±2.31 %. In the acid precipitation method, the amphipathic biosurfactant is converted into a hydrophobic molecule to extract biosurfactant from culture broth. The addition of hydrochloric acid causes the low pH of the broth.
30 Final Report: Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RUI 1001/PBJOLOGI/815103 This causes the biosurfactant to be positively charged, reducing the effectiveness of the hydrophilic region, thereby causing aggregation. Consequently, this results in the insoluble molecule and the compound precipitates as a solid (Baker & Chen, 201 0). This technique is often chosen to recover biosurfactant from most fermentation broth due to its simplicity and low process cost.
From, the three recovery techniques that have been evaluated, the solvent extraction method, using a mixture of methanol, chloroform and !-butanol, gave the highest yield of biosurfactant at 58.04 g/L in the form of a brown oily paste. It also showed the lowest surface tension reduction to 38 dynes/em and the highest emulsification activity (60%) compared to those observed in the other recovery methods. This could be due to the presence of a hydrophobic molecule which enables the entire compound to completely dissolve in the solvent mixture and hence easily separated by evaporation method. Hence, it was concluded that solvent extraction with a mixture of methanol/chloroform/1-butanol ( 1: 1: 1) is the best recovery method for biosurfactant produced by Streptomyces sp. R1.
4.6 Biosurfactant Characterization
4.6.1 Biochemical Characterization Four biochemical tests, namely Benedict test, saponification test, biuret test, and iodine test, were carried out to determine the type of biosurfactant produced in this study. Table 5 summarizes the results for biochemical analysis of the extracted biosurfactant from Streptomyces sp. Rl.
The Benedict test is used to detect the presence of sugar in the crude extract biosurfactant (Thenmozhi et a/., 2011 ). The saponification test is employed for the characterization of lipid. The presence of NaOH serves to saponify lipid in the solution containing biosurfactant (Bhavani & Hemashenpagam, 2013 ). Thus, the formation of soap in the solution confirms the presence of lipid in the extracted biosurfactant.
The biuret test was performed to detect the presence of lipopeptide. In the presence of lipopeptide, the biuret reagent will change to violet or pink due to the reaction with a peptide bond proteins or short-chain polypeptide with the copper salt in the biuret reagent (Jamal et al., 2012). As shown in Table 5, the biuret reagent did not change colour to the reddish brown precipitation which indicated the absence of lipopeptide in the crude biosurfactant extract.
31 Final Report : Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RUI 100//PBIOLOGJ/815103 The iodine test was performed to detect the presence of carbohydrate in the isolated biosurfactant (Bhavani & Hemashenpagam, 2013). The presence of reddish brown complex in the solution containing crude extract biosurfactant indicates a positive result for carbohydrate. The presences of lipid and carbohydrate compound in the crude biosurfactant suggest that the biosurfactant produced by Streptomyces sp. Rl is a glycolipid type biosurfactant. This finding is consistent with the results described by Khopade et al. (20 12) and Manivasagan et al. (2013) who reported a glycolipid-type biosurfactant produced by Streptomyces sp. B3 and Streptomyces MAB36.
32 Final Report: Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RUJ 1001/PBJOLOGJ/815103
Biochemical analysis Result Observation
Control Crude extract
No reddish brown Benedict test precipitation
Formation of soap Saponification test
No colour change to violet Biuret test
Reddish brown complex
Iodine test
Table 5: Biochemical analysis of extracted biosurfactant from Streptomyces sp. Rl
33 Final Report: Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RUJ 1001/PBIOLOGI/815103 4.6.2 Fatty Acid Analysis The presence of fatty acids in the hydrophobic tails of the crude extract biosurfactant was further analysed using GC-MS. As demonstrated in Figure 15, the chromatograms obtained from the brown oily paste sample (Section 4.5) exhibited two major peaks at the relative retention time of 39.901 min and 50.843 min which were identified as hexadecanoic acid (34.9%) and 9-octadecenoic acid (48.3%).
lnlensity
' 2soooo I
i I 200000-l
150000
100000
50000-'
.;: • 5 ~ i JJ .... __ L 0 t ~~~- j l"" 0 min
Figure 15: GC-MS chromatogram of fatty acid methyl ester from crude extract glycolipid biosurfactant.
5.0 CONCLUSION All objectives in this project have been successfully achieved. A model of filamentous biosurfactant producer was developed, identified as Streptomyces sp. Rl, based on morphological and molecular characterization. Streptomyces sp. Rl produced a glycolipid type biosurfactant. The medium formulation for biosurfactant production is as follow; 6% (v/v) palm oil, 0.6% (w/v) yeast extract, 2% (v/v) Tween 80, 0.7% (w/v) NaCl and initial pH 6 with 4% (v/v) inoculum concentration. The production of biosurfactant is not solely dependent on
34 Final Report : Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RUI JOOIIPBIOLOGI/815103 the biomass density but rather on the culture morphology during batch cultivation in the stirred tank reactor. Among the recovery techniques that were evaluated, solvent extraction method using methanol, chloroform and !-butanol ( 1: 1: 1) was found as the most efficient recovery process, giving the highest concentration of biosurfactant.
6.0 RECOMMENDATIONS To further enhance the growth and production ofbiosurfactant by Streptomyces sp. Rl, fed batch cultivation is proposed for the next project since palm oil was the limiting substrate in this study. Product formation in submerged fermentation of filamentous microorganisms is often influenced by culture morphology. To control the morphology of filamentous microbes during cultivation, several parameters such as viscosity, shear forces, and surfactant inclusion should be further studied to improve the production of biosurfactant. The secretion of biosurfactant into the culture broth and the biomass morphological development of the culture will affect the gas-liquid mass transfer in fermentation broth. Thus, this interaction should be investigated to improve the productivity of biomass and biosurfactant production of Streptomyces sp. Rl.
7.0 ACKNOWLEDGEMENTS This research was financially supported by Grant Research University (1001/PBIOLOGI/815103), Universiti Sains Malaysia, Malaysia.
8.0 REFERENCES Baker, S. C., & Chen, C. Y. (2010). Enrichment and Purification of Lipopeptide Biosurfactants. Banat, I M, Markkar, R S, & Cameotra, S S. (2000). Potential commercial applications of microbial surfactants. Applied Microbiology Biotechnology, 53, 495-508. Bhavani, T. M. B., & Hemashenpagam, N. (2013). Production of Biosurfactant and Characterization by 16S rRNA Sequencing Technique of Bacteria Degrading Hydrocarbon Isolated from Petroleum Contaminated Sites.International Journal of Advanced Research, 1(5), 300-305. Casas, J A, & Garcia-Ochoa, E. ( 1999). Sophorolipid production by Candida bombicola: medium composition and culture methods. Journal ofBioscience and Bioengineering, 88(5), 488-494. Cooper, D.G., & Goldenberg, B.G. (1987). Surface-active agents from two Bacillus species. Applied Environmental Microbiology, 53, 224-229. de Nevers, N. (1991). Fluid mechanics for chemical engineers. Feng, J, Zeng, Y, Ma, C, Cai, X, Zhang, Q, & Tong, M. (2006). The surfactant tween 80 enhances biodesulphurization. Appl Environ Microbial, 72(11 ), 7390-7393. 35 Final Report: Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RUIJOOJ/PBIOLOGI/815103 Forar, L R, Amany, K, Ali, E, & Bengraa, Ch. (2006). Taxonomy, identification and biological activities of a novel isolate of Streptomyces tendae. Arab J Biotech., 9, 427-436. Garcia-Ochoa, Felix, & Gomez, Emilio. (2009). Bioreactor scale-up and oxygen transfer rate in microbial processes: An overview. Biotechnology Advances, 27, 153-176. Hall, T A. (1999). BioEdit: A User-Friendly Biological Sequence Alignment Editor and Analysis Program for Windows 95/98/NT. Nucl. Acids. Symp. Ser, 41, 95-98. Jamal, P., Wan Nawawi, W. M. F., & Alam, M. Z. (2012). Optimum Medium Components for Biosurfactant Production by Klebsiella pneumoniae WMF02 Utilizing Sludge Palm Oil as a Substrate.Australian Journal ofBasic and Applied Sciences, 6(1 ), 100-108. Johnson, J A, Citarasu, T, & Helen, P. A. Mary. (2012). Screening of antibiotic producing actinomycetes from streams. Journal of Chemical, Biological and Physical Sciences, 2(3), 1363-1370. Khanna, Monisha, & Solanki, Renu. (20 11 ). Selective isolation of rare actinomycetes producing novel antimicrobial compounds. International Journal of Advanced Biotechnology and Research, 2(3), 357-375. Khopade, A., Ren, B., Liu, X. Y., Mahadik, K., Zhang, L., & Kokare, C. (2012). Production and characterization of biosurfactant from marine Streptomyces species B3. J Colloid Interface Sci, 367(1), 311-318. Kim, S. H., Lim, E. J., Lee, S. 0., Lee, J. D., & Lee, T. H. (2000). Purification and Characterization of Biosurfactants from Nocardia sp. L-417. Biotechnol Appl Biochem, 31,249-253. Manivasagan, P., Sivasankar, P., Venkatesan, J., Sivakumar, K., & Kim, S.-K. (2013). Optimization, Production and Characterization of Glycolipid Biosurfactant from the Marine Actinobacterium, Streptomyces sp. MAB36. Bioprocess Biosyst Eng. Marsudi, S., Unno, H., & Hori, K. (2008). Palm Oil Utilization for the Simultaneous Production of Polyhydroxyalkanoates and Rhamnolipids by Pseudomonas aeruginosa. Appl Microbial Biotechnol, 78, 955-961. Rodrigues, L., Banat, I. M., Teixeira, J., & Oliveira, R. (2006). Biosurfactants: Potential Applications in Medicine. J Antimicrob Chemother, 57( 4 ), 609-618. Ron, E. Z., & Rosenberg, E. (2001). Natural role of biosurfactants. Journal Environmental Microbiology, 3, 229-236. Saharan, B.S., Sahu, R. K., & Sharma, D. (2011). A review on biosurfactants: Fermentation, Current Developments and Perspectives. Genetic Engineering and Biotechnology Journal, 1-14. Saitou, N, & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4(4), 406-425. Shubhrasekhar, C., Supriya, M., Karthik, , L., Gaurav, K., & Bhaskara Rao, K. V. (2013). Isolation, Characterization and Application of Biosurfactant produced by Marine Actinobacteria isolated from Saltpan Soil from costal area of Andhra Pradesh, India. Research Journal ofBiotechnology, 8(1 ), 18-25. Tamura, K, Peterson, D, Peterson, N, Stecher, G, Nei, M, & S, Kumar. (2011). MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary
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Final Report: Production ofBiosurfactants via Locally-Isolated Actinomycete Fermentation RUJ 1001/PBIOLOGJ/815103 Distance, and Maximum Parsimony Methods. Molecular Biology and Evolution (submitted). Thampayak, I, Cheeptham, N., Pathom-Aree, W., Leelapompisid, P., & Lumyong, S. (2008). Isolation and identification of biosurfactant producing actinomycete from soil. Research Journal ofMicrobiology, 3(7), 499-507. Thenmozhi, R., A.Somalaksmi, D.Praveenkumar, & A.Nagasathya. (2011). Characterization of Biosurfactant Produced by Bacterial Isolates from Engine Oil Contaminated Soil. . Advances in Environmental Biology, 5(8), 2402-2408. Waksman, Selman A. ( 1961 ). The Actinomycetes Volume II: Classification, Identification and Descriptions ofGenera and Species. Youssef, Noha, H., Duncan, Kathleen, E., Nagle, David, P., Savage, Kristen, N., Knapp, Roy, M., & Mcinerney, Michael, J. (2004). Comparison of methods to detect biosurfactant production by diverse microorganism. Journal of Microbiological Methods, 56, 339- 347.
37 flllla-MI , Tarikh C~takan: 16/12/2015 UNIVERSITJ SAINS MALAYSIA JABATAN BENDAHARI PENYATA PERBELANJAAN SEHINGGA 16 DISEMBER 2015
Projek PRODUCTION OF BIOSURFACTANTS VIA LOCALLY ISOLATED ACTINOMYCETE FERMENTATION KETUA PENYELIDIK: PROFESOR MADYAAHMAD RAMLI MOHO YAHYA PENYELIDIK BERSAMA: PROFESOR MADYA LATIFFAH ZAKARIA TEMPOH: 15 DISEMBER 2012 HINGGA 14 DISEMBER 2014 (LANJUTAN SEHINGGA 14 JUN 2015) PUSAT PENGAJIAN SAINS KAJIHAYAT No. Akaun: 1001.PBIOLOGI.815103.
111 I GAJI I 86,400.00 120,747.91 -34,347.91 0.00 -34,347.91 0.00 17,025.04 17,025.04 -51,372.95
221 I PERJALANAN DAN SARA HIDUP I 24,000.00 22,041.39 1,958.61 0.00 1,958.61 0.00 558.00 558.00 1,400.61
222 I PENGANGKUTAN BARANG-BARANG I 1,000.00 0.00 1,000.00 0.00 1,000.00 0.00 0.00 0.00 1,000.00
224 I SEWAAN I 2,000.00 0.00 2,000.00 0.00 2,000.00 0.00 0.00 0.00 2,000.00
226 I BEKALAN BAHAN MENTAH I 6,000.00 0.00 6,000.00 0.00 6,000.00 0.00 0.00 0.00 6,000.00
227 I BEKALAN DAN BAHAN LAIN I 110,534.00 46,158.41 64,375.59 0.00 64,375.59 0.00 3,984.60 3,984.60 60,390.99
228 I PENYELENGGARAN & PEMBAIKAN KECIL I 10,000.00 8,480.00 1,520.00 0.00 1,520.00 0.00 300.00 300.00 1,220.00
229 I PERKHIDMATAN IKTISAS & HOSPITALITI I 4,000.00 18,948.90 -14,948.90 0.00 -14,948.90 0.00 0.00 0.00 -14,948.90
441 I BIASISWA DAN GERAN PELAJARAN I 0.00 4,829.00 -4,829.00 0.00 -4,829.00 0.00 0.00 0.00 -4,829.00
Penyata ini adalah cetakan komputer tiada tandatangan diperlukan Penyata ini adalah dianggap tepa! jika tiada maklumbalas dalam tempoh masa 14 hari dari tarikh penyata ' ,i .•· I , 'f i).' ' I .-'! i' '; ~ .~
Bioprocess Biosyst Eng (I) CrossMark DOl 10.1007/s00449-017-1764-4
Production and partial characterization of biosurfactant produced by Streptomyces sp. Rl
1 1 1 Nor Syafirah Zambry • Adilah Ayoib • Nur Asshifa Md Noh • Ahmad Ramli Mohd Yahya 1
Received: 2 December 2016/ Accepted: 29 March 2017 ©Springer-Verlag Berlin Heidelberg 2017
Abstract The present study focused on developing a Introduction wild-type actinomycete isolate as a model for a non-patho genic filamentous producer of biosurfactants. A total of 33 Biosurfactants are a diverse group of secondary metabo actinomycetes isolates were screened and their extracellular lites with surface active properties of microbial origin. biosurfactants production was evaluated using olive oil as They have amphiphilic structures, having both hydrophobic the main substrate. Out of 33 isolates, 32 showed positive and hydrophilic domains. The presence of these domains results in the oil spreading technique (OST). All isolates causes the molecules to line themselves at the water-oil showed good emulsification activity (~ 4 ) ranging from or water-air interfaces, consequently reducing the inter 84.1 to 95.8%. Based on OST and E24 values, isolate Rl facial or surface tensions in aqueous and oil mixtures [I]. was selected for further investigation in biosurfactant pro Such characteristics make them good candidates in diverse duction in an agitated submerged fermentation. Phenotypic industrial applications spanning petroleum, pharmaceuti and genotypic analyses tentatively identified isolate Rl as a cal, biomedical and food industrial processes [2]. In recent member of the Streptomyces genus. A submerged cultiva decades, the increasing awareness of eco-friendly pro tion of Streptomyces sp. Rl was carried out in a 3-L stirred cesses has resulted in an increased interest in the produc tank bioreactor. The influence of impeller tip speed on tion of biologically synthesized surfactants. Microbes have volumetric oxygen transfer coefficient (kLa), growth, cell the ability to utilize various organic compounds. These morphology and biosurfactant production was observed. microbes self-replicate under certain conditions, collec It was found that the maximum biosurfactant produc tively producing copious amounts of biosurfactants in their tion, indicated by the lowest surface tension measurement vicinity. Microbial biosurfactants offer several advantages (40.5 ±0.05 dynes/em) was obtained at highest kLa value over synthetic surfactants, namely lower toxicity, inher 1 (50.94 h- ) regardless of agitation speed. The partially ent good biodegradability and ecological acceptability [3]. purified biosurfactant was obtained at a concentration of Hence, in many instances, biosurfactants are more pre 7.19 g L -I, characterized as a Iipopeptide biosurfactant ferred to synthetic surfactants. and was found to be stable over a wide range of tempera Although biosurfactant production is more environmen ture (20-121 °C), pH (2-12) and salinity [5-20% (w/v) of tal friendly compared to that for its chemical alternatives, NaCl]. industrial production via fermentation is challenging due to the high production cost. As is the case with many fer Keywords Actinomycetes · Streptomyces · Lipopeptide mentation products, downstream processing accounts for a biosurfactant · Bioreactor · Fermentation · Morphology major portion of the total biosurfactant production costs [4, 5]. Hence, reducing product recovery costs remains para mount in the effort to reduce overall production cost. The 123:1 Ahmad Ramli Mohd Yahya filamentous growth nature of actinomycetes can offer sig armyahya @usm.my nificant savings in product recovery, especially for extra School of Biological Sciences, Universiti Sains Malaysia, cellular microbial products since the separation of biomass 11800 Penang, Malaysia from product typically become much easier.
Published online: 07 April 2017 ~Springer )< , I
Bioprocess Biosyst Eng the cultivation. Throughout the cultivation in the reactor, [27] using a semi-automatic Du-Nouy Tensiometer, model 30 mL samples of the culture were collected at 24-h inter 70535 (CSC Scientific Company, Inc, VA). This method vals for analyses of biomass growth and biosurfactant pro measures the surface force between a liquid and air in liq duction through measurement of surface tension. uid medium samples. Prior to measuring the surface ten The effect of impeller tip speeds on Streptomyces sp. R 1 sion of samples, the tensiometer was calibrated using dis growth and its biosurfactant production were investigated tilled water and ethyl alcohol at 28 °C. by agitating the culture at different stirrer speeds (0.52, 1 1.05, 1.57 and 2.09 m s- ) corresponding to 200, 400, 600 Cell growth determination and 800 impeller rpm. In the interest of better measurement accuracy, especially Determination of volumetric oxygen transfer coefficient for filamentous biomass grown in a hydrophobic substrate, (kLa) the direct method of cell dry weight was chosen instead of an indirect method such as optical density. A known vol The determination of volumetric oxygen transfer coef ume of a culture broth sample was filtered using a dry and ficient (kLa) was conducted using the static gassing-out pre-weighed filter paper (Whatman filter paper No. 1). The method [26]. The kLa was evaluated in distilled water con filtered sample was washed once with acetone and twice by taining 6% (v/v) of palm oil with a DO probe. After cali distilled water to remove residual oil from cells. The sam brating the DO probe, the oxygen was stripped from the ple was subsequently dried to a constant weight in an oven system by sparging with nitrogen gas. Then, air was intro (UFE 600 Memmert, Germany) at 105 oc. duced to the system at different stirrer speeds (0.52, 1.05, 1 1.57 and 2.09 m s- ) while aeration rate was held constant Biosurfactant recovery at 0.5 vvm. The increased DO tension was recorded every 30 s till reaching its saturation. The mass balance for dis The biosurfactant was recovered from the cell-free broth by solved oxygen is as follows: acid precipitation, followed by solvent extraction method [ 19]. The cell-free broth was acidified to pH 2 using 2 M (1) HCl and incubated overnight at 4 oc for complete precipita tion of the biosurfactant. The precipitate was then collected CL represents DO tension in the broth at a specific time by centrifugation at 10,000xg, 4 °C for 30 min. The pre point, C* means the saturated DO tension in the broth while cipitate obtained was further purified three times using a ~ represents the DO change with time. The kLa value mixture of chloroform:methanol (2: 1 v/v) in a separatory 1 (h- ) was calculated as the slope of ln(c* -C) versus time. funnel at 28 °C. The partially purified biosurfactant was concentrated using a rotary evaporator (EYELA Oil Bath Influence of impeller tip speed on morphology OSB-2000, Japan), weighed and stored at -20°C for fur of Streptomyces sp. Rl ther use.
The impact of impeller tip speeds on the morphology Biochemical composition of biosurfactant of Streptomyces sp. R1 was observed by taking 1 mL of sample at the end of the fermentation for every impeller The chemical composition of partially purified biosur tip speed. Two slides were prepared for every impeller tip factant was determined using standard methods [9]. Carbo speed. The slides were observed under compound light hydrate content was determined by the phenol-sulfuric acid microscope (Olympus model BX41, Germany) at 4x mag method Dubois et al. [28] using o-glucose as a standard. nification. At this magnification, ten images were captured Protein content was determined according to the methods for the morphological analysis. The diameter of pellet was described by Lowry et a!. [29], using bovine serum albu measured using image analysis software Cel(B (Olympus, min as a standard and lipid content was determined using Germany). method adopted by Folch et a!. [30].
Effect of temperature, pH, and salinity on biosurfactant Analytical techniques stability
Surface tension measurement The thermal stability of the biosurfactant at different tem peratures was observed by heating the sample in a boiling The surface tension of the cell-free culture broth was deter water bath (20-100 °C) (WNB 22 Memmert, German) for mined by the DuNouy ring method De Nevers and Grahn 60 min. To study the stability of biosurfactant at 121 °C,
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Bioprocess Biosyst Eng the extracted biosurfactant was autoclaved. All the samples This is one of the important diagnostic criteria for the were cooled at 28 oc prior to measurement of the surface genus Streptomyces [23]. Observation under the light tension. microscope demonstrated that isolate Rl gave non-motile The effect of the biosurfactant at different pH values was oval shape spores (Fig. lc) and showed the smooth surface investigated by adjusting the pH of the extracted biosur mycelia under scanning electron micrograph observation. factant to pH 2, 5, 7, 10 and 12 using 1 M NaOH and 1 M On the basis of morphological characteristics, isolate Rl HCI. The pH adjusted extracts were then subjected to sur was presumptively assigned to the genus Streptomyces. face tension measurements. The 16S rRNA sequence data have been used in the The effect of salinity on the biosurfactant stability was modern Streptomyces identification system since they pro determined by manipulating NaCI concentration. The vide valuable information about streptomycetes system extracted biosurfactant was mixed evenly with the specific atic [32, 33]. Thus, further taxonomic characterization of concentrations of NaCI [5, 10, 15 and 20% (w/v)] while selected biosurfactant-producing actinomycete was done keeping the biosurfactant concentration constant. The sur by 16S rRNA gene sequence analysis. From the BLAST face tension for each mixture was measured. search, isolate Rl was identified as Streptomyces sp. Rl (KJ669362) with 92% similarity. Based on the consist ency of results from morphological and molecular char Results and discussion acterization, isolate Rl is likely a member of the genus of Streptomyces. Screening for biosurfactant-producing actinomycetes Production of biosurfactant by Streptomyces sp. rl A total of 33 isolates of actinomycetes have been screened in a stirred-tank bioreactor for biosurfactant production in starch casein broth contain ing olive oil as the biosurfactant inducer. Olive oil was cho Influence of impeller tip speed on kLa, growth sen as a sole carbon source in the screening process since it and biosurfactant production was proven in enhancing the production of biosurfactant by actinomycetes [3 I]. Table 2 summarizes the results of the As often highlighted, agitation or impeller tip speed is one screening experiment using two measurements; OST and of the important bioprocess parameters that provides effec E24· tive oxygen transfer rate (OTR) during aerobic fermenta Out of the 33 isolates, 32 showed positive results in OST tion, thereby affecting the cell growth and its productiv when compared to the control of uninoculated medium. ity. The volumetric oxygen transfer coefficient (kLa) is These results highlight the reliability and sensitivity of often used to gauge OTR the in the bioreactor system. It this technique in detecting very low concentrations of is important to have a good estimate of kLa value to ensure biosurfactant. In a similar screening experiment, Youssef adequate transfer of oxygen in the bioreactor and for scal et al. [21] demonstrated that 16 strains that showed nega ing up [26]. Therefore, the investigation on the influence of tive results in the drop collapse method, gave positive oil agitation speed on the kLa value, growth and biosurfactant spreading result (0.5-0.9 em), demonstrating the higher production by Streptomyces sp. Rl in a batch cultivation sensitivity of the oil spreading technique in detecting sur was performed in this study. factants. The E24 test gave positive results for all isolates, Table 3 shows that as the speed of the impeller 1 with good E24 values ranging from 84.11 to 95.80%. Based increased from 0.52 to 2.09 m s- , the biomass density on the OST and E24 values aforementioned (Table 2), iso of Streptomyces sp. R 1 also increased from 4.35 ± 0.01 to late Rl was selected as the model biosurfactant producer 8.4 7 ± 0.02 g L -I. This may be due to the fact that higher for further studies. impeller tip speed facilitates oxygen transfer, consequently leading to better Streptomyces sp. Rl growth. However, the Identification and characterization result presented here was contradictory to other reports. of biosurfactant-producing actinomycete The increased agitation speed in cultivation of Streptomy ces clavuligerus, Streptomyces hygroscopicus, and Strep The examination of the macroscopic characteristics for iso tomyces flocculus would result in a depletion in biomass late Rl grown on SCA at 28 oc for 7 days showed that the density due to the shear stress that is introduced by the agi outer surface of the colonies was perfectly round initially, tation intensity [34-36]. but later developed aerial mycelium that appeared velvety Although biomass density increased proportionally white colour (Fig. la) whereas the substrate mycelium was with stirrer speed, the highest biosurfactant production pale brown (Fig. lb). Isolate Rl showed concentric rings of indicated by the lowest surface tension value for samples colonies on the SCA plate as incubation time progressed. from each agitation did not follow a similar trend. It was
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1 Table 2 Screening of biosurfactant-producing actinomycetes using biosurfactin production (0.0485 g- L-t h-1) by Bacil OST and E24 lus subtilis ATCC 6633 was found at maximum OTR, 1 1 1 No. Isolate Diameter of clear E24 (%) 0.01 mol- L- h- . zone (em) Influence of impeller tip speed on morphological Control 1.10±0.17 0.00±0.00 of Streptomyces sp. Rl I BI 4.40±0.17 90.40±5.7I 2 B2 1.35±0.2I 87.90±0.I4 Unlike microbial fermentation, the cultivation of filamen 3 B3 1.27±0.I2 86.84±3.72 tous organisms is more complex due to the tendency of the 4 B4 3.I7±0.29 84.II±O.I5 culture to be non-homogeneous and greatly influenced by 5 B5 1.17±0.15 90.00±4.71 bioprocessing parameters such as impeller tip speed. These 6 II 1.53±0.35 92.16±0.22 parameters affect the morphology of the cell, which con 7 12 3.70±0.14 87.25±0.35 sequently dictates the production of secondary metabolites. 8 13 2.67±0.29 90.84± 1.18 For instance, increasing impeller tip speed led to a lower 9 14 1.90±0.I4 90.63±4.42 production of avermectin B in S. avermitilis but a higher IO 15 2.40±0.28 90.42±4.I2 18 production of clavulanic acid in S. clavuligerus [36, 39]. II KI 1.25±0.07 89.18±0.41 Nevertheless, data on the effects of impeller tip speeds on I2 K2 4.30±0.26 92.47±3.20 the morphology of Streptomyces sp. and its biosurfactant 13 K3 2.47±0.15 88.05±0.07 production are yet to be reported. Hence, the influence of 14 K4 6.03±0.I5 92.23±2.16 1 different impeller tip speed (0.52-2.09 m s- ) towards the I5 K5 2.53±0.06 89.24±0.33 cell morphology of Streptomyces sp. Rl and its correlation I6 K6 3.70±0.I4 90.63±4.42 to the biosurfactant production were investigated in the pre I7 N1 4.10±0.14 85.17±0.77 sent study (Table 4). I8 PI 7.10±0.14 90.24±0.34 As is illustrated in Table 4 and Fig. 2, the impeller tip 19 P2 6.46±0.24 93.46±3.60 speeds gave a significant effect on the growth morphol 20 P3 7.03±0.06 89.24±0.33 ogy of Streptomyces sp. Rl during batch cultivation. It was 2I P4 5.65±0.07 91.95±4.32 noted that at 0.52 m s-1 impeller tip speed, the pellets were 22 P5 3.03±0.06 94.22±0.3I uniform in shape in the form of 3-4 aggregates (Fig. 2a). 23 P6 6.15±0.21 88.74± 1.04 This is probably because the mild agitation enabled the pel 24 P7 3.30±0.I7 91.50±0.7I let to agglomerate [40]. In addition, the mean pellet diam 25 P8 3.07±0.12 89.69± 1.12 eter at this agitation speed was smaller (0.175 mm) than 26 P9 2.60±0.I4 92.74±3.20 those seen at 1.05 m s-1 tip speed (0.366 mm), which are 27 P12 0.97±0.06 88.94±2.79 consistent with the results of biomass obtained in Table 3 28 PI3 4.20±0.26 84.6I ±0.56 (4.35 ±0.01 g L -I at 0.52 m s-1 and 6.33 ±0.00 g L-1 at 29 RI 7.45±0.07 95.80±0.28 1 30 R2 3.60±0.14 93.00±4.24 1.05 m s- ), possibly due to the low amount of dissolved oxygen, 14.15 mmol 0 L- 1 h-1 at 0.52 m s-1 compared to 31 R3 5.95±0.21 2 85.36±0.50 1 1 1 42.52 mmol 0 L- h- at 1.05 m s- , respectively, in the 32 R5 4.05±0.D7 93.08±3.06 2 cultivation medium. The growth morphology of the culture 33 S1 6.35±0.D7 84.26± 1.32 at 1.05 m s-1 tip speed contained a mixture of big round pellet and filamentous form (Fig. 2b). However, increasing the speed of impeller from 1.05 to 2.09 m s-1 resulted in found that the surface tension measurement correlated smaller mean pellet diameters (0.366-0.094 mm, respec with kLa value. In this study, the lowest surface tension tively) with dispersed filamentous growth form (Fig. 2c, d). 1 measurement (40.50±0.50 dynes cm- ) was obtained at A similar finding was reported by Xia et al. [35], namely 1 1 highest kLa value (50.94 h- ) at 1.57 m s- stirrer speed that the pellet diameter of Streptomyces flocculus decreases (600 rpm). It seems that the higher kLa value promoted as the agitation speed increased from 400 to 800 rpm. This better biosurfactant production by Streptomyces sp. R1 was probably due to the intense stirrer speed creating a regardless of the agitation speed. This preliminary finding higher shear stress, preventing filamentous organisms to was supported by Ray [37], who published that the maxi grow in larger sized and dense pellets. mum biosurfactant production indicated by lowest surface Upon closer inspection of Table 3 and Fig. 2, it appears 1 tension reduction (22 dynes cm- ) was attained at a high that the maximum biosurfactant production, assumed 1 kLa value (129.76 h- ) in Bacillus sp. (m28) fermentation. as indicated by the lowest surface tension measurement In addition, Jokari et al. [38] also claimed that the high (40.50±0.5 dynes/em) was obtained when the morphology
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Fig. 1 The morphology of isolate R I after 7 -day incuba tion on SCA at 28 oc. a Upper surface of isolate Rl showed white colour and b lower sur face showed pale brown colour. Microscopic features of isolate Rl (c) oval-shaped spores (X40) (d) scanning electron micro graph showing smooth surface mycelium (6.27 xK)
Table 3 The kLa value, growth and biosurfactant production by relied heavily on the culture morphology during submerged Streptomyces sp. Rl at different impeller tip speed fermentation. The results clearly demonstrate that in the Agitation Impeller tip kLa (h-1) Maximum Surface ten- present study, the production of biosurfactant by Strep rate (rpm) speed (m s-1) biomass sion (dynes/ tomyces sp. R I may be a morphological auto-regulation (gL-1) em) mechanism. 200 0.52 11.82 4.35±0.01 47.17±0.29 400 1.05 35.52 6.33±0.00 56.80±0.80 Biochemical composition of partially purified 600 1.57 50.94 7.70±0.03 40.50±0.50 biosurfactant 800 2.09 39.90 8.47±0.02 50.20±0.03 The recovery techniques, namely acid precipitation, fol lowed by the solvent extraction method used in this study, Table 4 Effects of impeller tip speed on the morphology of Strepto were successful in extracting biosurfactant from Streptomy myces sp. RI ces sp. R1 fermentation broth, giving a viscous light brown Impeller tip Morphology Mean pellet matter. These are the most common and effective recov speed (m s-1) diameter ery techniques that have been used by many researchers to (mm) extract biosurfactant from culture broth [42-44]. A similar 0.52 Aggregate of 3-4 pellets of various size 0.175 result was obtained by Allada [ 12] where the yellowish, oily 1.05 Big rounded pellets and filamentous 0.366 crude biosurfactant was successfully recovered from Strep 1.57 Freely dispersed mycelia 0.106 tomyces Coelicoflavus NBRC (15399T) fermentation broth 2.09 Freely dispersed mycelia 0.094 using the same recovery techniques applied in this study. The concentration of the partially purified biosurfactant 1 was 7.19 g L- • This value is high compared to the con of Streptomyces sp. Rl was in dispersed form of growth centration of biosurfactant reported by Bhuyan-Pawar et al. 1 (Fig. 2c) at 1.57 m s- tip speed. Consequently, it is tempt [19], 56.7 mg L -I from Streptomyces sp. V2. The chemi ing to speculate that the production of biosurfactant is also cal characterization revealed that the partially purified bio influenced by culture morphology. A similar finding was surfactant produced was of a lipopeptide nature, primarily also reported by 6 Cleirigh [41] stating that the produc consisting of lipid with the relative percent of 80.5% (w/w) tion of biosurfactant by Streptomyces hygroscopicus var. and 11.11% (w/w) protein. Most studies reported that the geldanus was not dependent on the biomass density but biosurfactant produced by the genus Streptomyces sp. is
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Fig. 2 Pellet morphology of Streptomyces sp. Rl as a func tion of impeller tip speed under 1 light microscope. a 0.52 m s- ; 1 1 b 1.05 m s- ; c 1.57 m s- ; d 2.09m - 1
glycolipid [6, 9-12]. This is the first report of a lipopeptide caused no significant effect on the biosurfactant perfor biosurfactant produced by genus Streptomyces. mance indicated by the surface tension measurements (38.67 ± 0.29 dynes/em). A similar finding was reported Effect of temperature, pH, and salinity on biosurfactant by Khopade et al. [I 0] which claimed that the surface stability tension of glycolipid biosurfactant produced by Strep tomyces sp. B3 was stable after heating at 100 °C. This Figure 3 shows the effect of temperature, pH, and salin meant that the biosurfactant produced can potentially be ity on the stability of glycolipid biosurfactant produced applied in food, pharmaceutical and cosmetic industries by Streptomyces sp. Rl. Figure 3a shows that the biosur where heating to attain sterility is a common practice factant produced by Streptomyces sp. Rl was thermosta [45], pending safety considerations. ble. Heating glycolipid biosurfactant from 20 to 121 oc
50 (a) (b) (c)
40+---~--~---.--_.----+ i ~ ~ 30 ~ .§ a 20 ~ ..u ~ ~.. 10
0~--~--~--~--~--~~ 20 40 60 80 100 120 2 4 6 8 10 12 5 10 15 20
Temperature ("C) pH NaCl o/o (w/v)
Fig. 3 Ell'ect of a temperature, b pH, and c salinity on the stability of glycolipid biosurfactant produced by Streptomyces sp. Rl
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Figure 3b shows the effect of different pHs within the 4. Santos DKF, Rufino RD, Luna JM, Santos VA, Sarubbo LA range of 2-12 on the surface tension measurement of the (2016) Biosurfactants: multifunctional biomolecules of the 21st century. Int J Mol Sci 17(3):401 glycolipid biosurfactant. The surface tension measurements 5. Desai JD, Banat IM (1997) Microbial production of surfactants (38.83 ± 0.28 dynes/em) of the glycolipid biosurfactant and their commercial potential. Microbiol Mol Bioi Rev remained relatively stable over a wide range of pH tested. 61(1):47-64 The ability of the glycolipid biosurfactant to withstand a 6. Yan X, Sims J, Wang B, Hamann MT (2014) Marine actino mycete Streptomyces sp. ISP2-49E, a new source of Rham wide range of pH promotes its application in extreme con nolipid. Biochem Syst Ecol 55:292-295 ditions such as in acidophilic and alkalophilic environments 7. Lakshmipathy TD, Prasad AA, Kannabiran K (2010) Produc [46]. Similar to temperature and pH, the surface tension of tion of biosurfactant and heavy metal resistance activity of the glycolipid biosurfactant was unaffected with increased Streptomyces sp. VITDDK3-a novel halo tolerant actinomy cetes isolated from saltpan soil. Adv Bioi Res 4(2):108-115 salinity from 5 to 20% (w/v) (Fig. 3c). This characteris 8. Richter M, Willey JM, Submuth R, Jung G, Fiedler HP (1998) tic gives the biosurfactant an advantage to be used in the Streptofactin, a novel biosurfactant with aerial mycelium extreme salinity condition that is common in many oil res inducing activity from Streptomyces tendae Tii 901/8c. FEMS ervoirs for MEOR applications and bioremediation of spills Microbiol Lett 163(2):165-171 9. Manivasagan P, Sivasankar P, Venkatesan J, Sivakumar K, in the marine environment [45]. Kim S-K (2014) Optimization, production and characterization of glycolipid biosurfactant from the marine actinobacterium, Streptomyces sp. MAB36. Bioprocess Biosyst Eng 37(5):783- 797. doi: I 0.1007 /s00449-0 13-1048-6 10. Khopade A, Ren B, Liu XY, Mahadik K, Zhang L, Kokare Conclusion C (2012) Production and characterization of biosurfactant from marine Streptomyces species 83. J Colloid Interface Sci The present study showed that, out of the 33 confirmed 367(1):311-318 actinomycete isolates, 32 were able to produce extracel 11. Kalyani ALT, Naga SG, Girija SG, Prabhakar T (2014) Isola tion, identification and antimicrobial activity of bio-surfactant lular biosurfactant when cultured in starch casein broth in from Streptomyces matensis (PLS-1). lnt J Pharm Sci Rev Res the presence of olive oil as the biosurfactant inducer. From 25(1): 165-170 these isolates, isolate Rl, characterized and identified as the 12. Allada LTK (2016) Characterization of bioactive compound genus Streptomyces based on morphological and molecu obtained from Streptomyces coelicojlavus NBRC (15399T) and its anticancer activity. Int J Chern Pharm Anal 2 (4 lar characterization was selected as a model biosurfactant 13. Thampayak I, Cheeptham N, Pathom-Aree W, Leelapornpisid producer for studies in a stirred-tank bioreactor. The results P, Lumyong S (2008) Isolation and identification of biosur of batch cultivation in the stirred-tank bioreactor demon factant producing actinomycetes from soil. Res J Microbiol strated that the production of biosurfactant was dependent 3(7):499-507. doi: I 0.3923/jm.2008.499.507 14. Karthik L, Kumar G, Rao KVB (2010) Comparison of meth on kLa value and cell morphology rather than biomass den ods and screening of biosurfactant producing marine act sity alone. The partial characterization studies presented inobacteria isolated from Nicobar marine sediment. IIOAB J here suggests that the biosurfactant produced by Streptomy 1(2):221-227 15. Chaudhary P, Sharma R, Singh SB, Nain L (2011) Bioremedia ces sp. Rl belongs to lipopeptide type biosurfactants. This tion of PAH by Streptomyces sp. Bull Environ Contam Toxicol is the first report on lipopeptide biosurfactant produced by 86(3):268-271. doi:IO.I007/s00128-0II-0211-5 genus Streptomyces. Interestingly, this compound was sta 16. Shubhrasekhar C, Supriya M, Karthik L, Gaurav K, Bhaskara ble across a wide range of pH, temperature, and salinity. Rao K (20 13) Isolation, characterization and application of bio Thus, Streptomyces sp. Rl presents an alternative to the surfactant produced by marine actinobacteria isolated from salt pan soil from costal area of Andhra Pradesh, India. Res J Bio current biosurfactant producers to produce viable industrial technol 8: 18-25 biosurfactant production. 17. Panjiar N, Gabrani R, Sarethy IP (2013) Diversity of biosur factant-producing Streptomyces isolates from hydrocarbon-con taminated soil. lnt J Pharma Bio Sci 4(1):524-535 18. Korayem A, Abdelhafez A, Zaki M, Saleh E (2015) Optimiza tion of biosurfactant production by Streptomyces isolated from References Egyptian arid soil using Plackett-Burman design. Ann Agric Sci 60(2):209-217 I. Oh KT, Kang CM, Kubo M, Chung SY (2006) Culture condi 19. Bhuyan-Pawar S, Yeole RP, Sanam VM, Bashetti SP, Mujum tion of Pseudomonas aeruginosa F722 for biosurfactant produc dar SS (2015) Biosurfactant mediated plant growth promotion in tion. Biotechnol Bioprocess Eng 11(6):471-476. doi:IO.I007/ soils amended with polyaromatic hydrocarbons bf02932069 20. Doshi DV, Maniyar JP, Bhuyan SS, Mujumdar SS (2010) Studies 2. Fontes GC, Fonseca Amaral PF, Nele M, Zarur Coelho MA on bioemulsifier production by Actinopolyspora sp. A 18 isolated (2010) Factorial design to optimize biosurfactant produc from garden soil. Indian J Biotech no! 9(4 ):391-396 tion by Yarrowia lipolytica. J Biomed Biotechnol 2010:8. 21. Youssef NH, Duncan KE, Nagle DP, Savage KN, Knapp RM, doi: 10.1155/2010/821306 Mcinerney MJ (2004) Comparison of methods to detect biosur 3. Banat IM (2000) Les biosurfactants, plus que jamais sollicites. factant production by diverse microorganisms. J Microbiol Meth Biofutur 2000(198):44--47 ods 56(3):339-347. doi: 10. 1016/j.mimet.2003.Il.OOI
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22. Cooper DG, Goldenberg BG (1987) Surface-active agents from 36. Rosa J, Neto AB, Hokka C, Badino A (2005) Influence of dis two Bacillus species. Appl Environ Microbial 53(2):224-229 solved oxygen and shear conditions on clavulanic acid produc 23. Waksman SA (1961) The actinomycetes. Vol. II, classification, tion by Streptomyces clavuligerus. Bioprocess Biosyst Eng identification and descriptions of genera and species. Williams 27(2):99-1 04 and Wilkins, Baltimore 37. Ray S (2012) Optimization of process conditions for biosur 24. Johnson J, Citarasu T, Helen PM (2012) Screening of antibiotic factant production from mutant strain of Bacillus sp. (m28) producing actinomycetes from streams. J Chern Bioi Phys Sci in a 5 L laboratory fermenter. J Microbial Biotechnol Res 2(3): 1363-1370 2(3 ):431-439 25. Hall TA (1999) BioEdit: a user-friendly biological sequence 38. Jokari S, Rashedi H, Amoabediny G, Yazdian F, Rezvani M, alignment editor and analysis program for Windows 95/98/NT. Hatarnian Zarmi A (2012) Effect of aeration rate on biosurfac In: Nucleic acids symposium series, pp 95-98 tin production in a miniaturized bioreactor. Int J Environ Res 26. Garcia-Ochoa F, Gomez E (2009) Bioreactor scale-up and oxy 6(3):627-634 gen transfer rate in microbial processes: an overview. Biotechnol 39. Ki SS, Jeong YS, Kim PH, Chun GT (2006) Effects of dissolved Adv 27(2): 153-176. doi: 10.10 16/j.biotechadv.2008.1 0.006 oxygen level on avermectin B 1a production by Streptomyces 27. De Nevers N, Grahn R (1991) Fluid mechanics for chemical avermitilis in computer-controlled bioreactor cultures. J Micro engineers. McGraw-Hill, New York bioi Biotechnol 16(11): 1690-1698 28. Dubois M, Gilles KA, Hamilton JK, Rebers P, Smith F (1956) 40. Gomaa MO, Bialy A (2009) Pellet morphology, broth rheology Colorimetric method for determination of sugars and related sub and stalin production in submerged fermentation of P. citrinum. stances. Anal Chern 28(3):350-356 Glob J Biotechnol Biochem 4(2):75-83 29. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Pro 41. 6 Cleirigh C (2005) Quantification and regulation of pellet mor tein measurement with the Falin phenol reagent. J Bioi Chern phology in Streptomyces hygroscopicus var; geldanus cultures. 193(1):265-275 PhD. Dublin City University, Dublin 30. Folch J, Lees M, Sloane-Stanley G (1957) A simple method for 42. Joshi SJ, Geetha SJ, Desai AJ (2015) Characterization and the isolation and purification of total lipids from animal tissues. J application of biosurfactant produced by Bacillus licheniformis Bioi Chern 226(1):497-509 R2. Appl Biochem Biotechnol 177(2):346-361. doi:I0.1007/ 31. Kiran GS, Thomas TA, Selvin J, Sabarathnam B, Lipton s12010-0J5-1746-4 A (2010) Optimization and characterization of a new lipo 43. Gandhimathi R, Seghal Kiran G, Hema TA, Selvin J, Rajeetha peptide biosurfactant produced by marine Brevibacterium Raviji T, Shanmughapriya S (2009) Production and characteriza aureum MSA13 in solid state culture. Bioresour Techno! tion of lipopeptide biosurfactant by a sponge-associated marine I 0 I (7):2389-2396 actinomycetes Nocardiopsis alba MSAIO. Bioprocess Biosyst 32. Lee JY, Lee JY, Jung HW, Hwang BK (2005) Streptomyces koy Eng 32(6):825-835. doi: 10.1007/s00449-009-0309-x angensis sp. nov., a novel actinomycete that produces 4-phenyl- 44. Dhasayan A, Selvin J, Kiran S (2015) Biosurfactant produc 3-butenoic acid. Int J Syst Evol Microbial 55( I ):257-262 tion from marine bacteria associated with sponge Callyspon 33. Kim HJ, Lee SC, Hwang BK (2006) Streptomyces cheonanensis gia diffusa. 3. Biotechnology 5(4):443-454. doi:l0.1007/ sp. nov., a novel streptomycete with antifungal activity. Int J Syst sl3205-014-0242-9 Evol Microbio156(2):471-475 45. Prieto L, Michelon M, Burkert J, Kalil S, Burkert C (2008) The 34. Zhu X, Zhang W, Chen X, Wu H, Duan Y, Xu Z (2010) Gen production of rhamnolipid by a Pseudomonas aeruginosa strain eration of high rapamycin producing strain via rational meta isolated from a southern coastal zone in Brazil. Chemosphere bolic pathway-based mutagenesis and further titer improvement 71(9): 1781-1785. doi: 10. 1016/j.chemosphere.2008.01.003 with fed-batch bioprocess optimization. Biotechnol Bioeng 46. Kim SH, Lim EJ, Lee SO, Lee JD, Lee TH (2000) Purifica I 07(3):506-515. doi: I 0.1 002/bit.22819 tion and characterization of biosurfactants from Nocardia sp. 35. Xia X, Lin S, Xia X-X, Cong F-S, Zhong J-J (2014) Significance L-417. Biotechnol Appl Biochem 31 (3):249-253. doi:l0.1042/ of agitation-induced shear stress on mycelium morphology and BA19990111 lavendamycin production by engineered Streptomyces flocculus. Appl Microbial Biotechnol 98(10):4399-4407. doi: 10.1007/ s00253-0 14-5555-4
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Several actinobacterial isolates from mangrove areas MCROBIAL BIOSURF ACT ANTS BY NURFARAH AINA MOHAMED RAZALLI, NORAZURIN SYUHADA RUSL Y, SITI ZULAIKHA, MOHO YUSOFF, MOHO SYAFJQAWANG, NOR SYAFIRAH ZAMBRY, SHAHKILLAHWATI MOHO RIDHWAN, NUR ASSHJFA MD NOH AND AHMAD R. M. YAHYA, SCHOOL OF BIOLOGICAL SCIENCES, UNIVERSITJ SAJNS MALAYSIA, PENANG, MALAYSIA
Surfactants have a wide range of applications Biosurfactants are amphiphilic, having both hydrophilic in many industries. Most are of chemical and hydrophobic domains in one molecule. They tend origin, which may make them hazardous to the to accumulate at interfaces by reducing the surface environment, and interest has been increasing (gas-liquid) and interfacial (liquid-liquid or solid- in finding biosurfactants as an alternative to liquid) tension, reducing the repulsive forces between chemical surfactants. two dissimilar phases, and allowing the different phases to mix and interact. The special properties of Microbial-derived surfactants, or 'biosurfactants', are biosurfactants include their higher biodegradability, biological compounds that exhibit high surface-active lower toxicity, better environmental compatibility, properties. They are produced by a wide variety of higher surface activity and ability to be synthesised microorganisms, including bacteria, fungi and yeast, from renewable sources. on microbial cell surfaces, or excreted extracellularly. In addition, they are also preferred over chemical Their production is often considered an integral trait of surfactants due to their specific action, effectiveness microorganisms capable of utilising water-immiscible and stability at extremes of temperatures, as well substrates. as pH, ionic strength, salinity and widespread
40 Australasian BioTechnology •Volume 26! Number 3 @th c:Jt Ausr3iotech's lnternat1onal BioF est
Oil droplet remains beaded with sterile medium
Rod-shaped P. aeruginosa USM-AR2 under scanning electron Oil droplet collapses when added with spent broth of microscope (SEM) P. aeruginosa USM-AR2 containing biosurfactant applicability. These properties make them desirable substrates, the use of other microbial producers in various industrial processes, such as in food may offer simpler bioprocessing. One possible processing, pharmaceutical formulations, health alternative is the filamentous actinobacteria, such care and the beauty industry; and as biocontrol as Actinomycetes nocardiopsis sp. strain A17. The agents and biopesticides in agriculture, enhanced oil filamentous nature of actinobacteria can simplify recovery, wastewater treatment and environmental downstream processing, reducing the cost of bioremediation. biosurfactant production. They are non-pathogenic, Microorganisms utilise a variety of organic thereby extending their biosurfactant applications into the human health industry. compounds as a source of carbon and energy. Many are capable of synthesising biosurfactants Actinobacteria are traditionally known as prolific when grown on different carbonaceous substrates. producers of antibiotics with a broad range of Typically, water-immiscible compounds have been biological activities, making them important the substrate of choice to produce biosurfactants. microorganisms in the pharmaceutical industry. They Water-soluble carbon sources, such as glycerol and belong to the family Actinomycetaceae, and have glucose, can also be used to produce biosurfactants; fungal-like morphology and high-GC content in however, the biosurfactant production is generally their DNA It is thought that many novel secondary much lower since these substrates are soluble metabolites from this group remain undiscovered, since and thus nullify the need for the cells to excrete each strain of actinobacteria has the genetic potential biosurfactants to improve their solubility. for the production of many secondary metabolites. Among the various types of biosurfactants, Unlike antibiotics, studies into actinobacterial rhamnolipid, a glycolipid-type biosurfactant, is one of biosurfactant production is still in its infancy. the most versatile. It is attracting attention in various In recent years, the rapid emergence of new fields as a multifunctional material for the new century, pathogens and antibiotic resistance among and is considered as a 'green' alternative to synthetic microorganisms has become a significant threat to surfactants. It has a strong emulsifying activity and a human health, globally. In 2015, the World Health low critical micelle concentration (CMC). Bacteria of Organization (WHO) created a campaign to increase the genus Pseudomonas are the best producers of the awareness of global antibiotic resistance and to rhamnolipids. The opportunistic pathogenic nature provide public precautions in preventing its spread. of however, presents a Pseudomonas aeruginosa, This problem brought researchers to employ new concern during mass cultivation and necessitates an strategies in the search of novel actinobacteria and adequate product recovery and purification process. secondary metabolites. Exploring new and special Industrial productions are still limited due to the high niches, such as marine and mangrove sediments, hot production cost. springs and untapped caves, may increase the chances The fermentation process is the key to improving the of isolating a new actinobacteria with novel secondary overall process economics in enhancing biosurfactant metabolites. Many actinobacterial biosurfactants are production. Besides using cheaper and renewable yet to be characterised and identified. >11
Australasian BioTechnology· Volume 26: Number 3 41 PRE-PROCEEDINGS
of the 1st International Conference on Sustainable Agriculture and Environment
·./2(0))/Z·~ and environment J)
Solo City, Indonesia 271h-2gth June, 2013 Sebelas Maret University i
BIODIVERSITY OF BACTERIAL COMMUNITIES IN SUPPRESSIVE AND CONDUSIVE LANDS FOR FUSARIUM BASAL ROT ON GARLIC ...... 89 1 1 2 1 1 Vita Ratri Cahyanl , Hadlwlyono , Ratna Setyanlngtyas ,Zainal Djauhari Fatawi , Dedy Prasetyo , And Agus 1 Siswanto ...... 89 SPECIES RICHNESS ESTIMATION OF GROUNDDVER VEGETATION IN SOME PARTS OF 2010 PYROCLASTIC FLOWS AREAS OF MT. MERAPI USINGEST/MATES ...... 92 Sutomo I' and d. Fardila 2 ...... 92 Area of Interest : BIOTECHNOLOGY ...... 103 SCREENING OF BIOSURFACTANT PRODUCERS FROM LOCALLY ISOLATED ACTINOBACTERIAL...... 104 Nor Syafirah Zambry, Adllah Ayoib, Latiffah Zakaria, And Ahmad Ramll Mohd Yahya ...... 104 IMPROVEMENT OF EXTRACELLULAR LIPASE FROM SELECTED FUNGAL STRAIN USING TAGUCHI DOE METHODOLOGY ...... 114 F. Fibriana, A. Upaichit, and T. Hongpattarakere ...... 114 PRELIMINARY DETECTION OF IMMUNOMODULATORY MOLECULES FROM Carica papaya EXTRACT FOR TREATMENT OF DENGUE FEVER ...... 120 Y. Mat·arip, A. A. Amirul, and A. M. N. Zatll ...... 120 THE RESEARCH OF SOME CASSAVA VARIETY AS RAW MATERIALS FOR GREEN ENERGY BIOETHANOL.147 M.C. Tri Atmodjo ...... 147 Sansevieria trifasciata PRAIN PROPERTIES AS PB(II) IONS BIOSORBENT ...... 153 1 2 3 Lela Mukmilah Yuningsih , lrmanida Batubarau, Latifah K Darusman · ...... 153 RAPD CLUSTERING BASED ON TANNIN CONTENTOF VARIOUS SALAK (Sa/acca za/acca Gaertner Voss) ACCESSIONS ...... 163 1 2 3 4 Nandariyah , Hartati,S. , Wartoyo , And Pardono ...... 163 Area of Interest : HORnKULTUR ...... 168 TOMATO YIELD AND NUTRIENT UPTAKE AS AFFECTED BY COW MaNURE COMPOST IN TWO SEASONS .. 169 Darwin H. Pangaribuan•, And Rizka Novl Sesantt•• ...... 169 EFFECT OF PLANT SPACING AND NPK FERTILIZER RATES ON PRODUCTION AND SEED QUALITY OF SWEET CORN (Zea mays saccharate sturt) ...... 178 Sri Hartatik ...... 178
Area of Interest: CUMATE CHANGE ...... 185 IMPACT OF CLIMATE CHANGE ON FOOD SECURITY INDEX AND POVERTY IN INDIA: AN EMPIRICAL ANAL YSIS ...... 186 A)ay Kumar• and Dr. Pritee Sharma ...... 186 ASSESSING THE CURRENT INDONESIA'S ELECTRICITY MARKET ARRANGEMENTS AND THE OPPORTUNITIES TO REFORM ...... 202 Dhanl Setyawan ...... 202 BARRIERS TO ENERGY EFFICIENCY: A COMPARISON ACROSS 3 ASIAN COUNTRIES ...... 212 Dhanl Setyawan ...... 212 ASSESSMENT OF INDONESIAN ELECTRICITY MARKET TO SUPPORT CLIMATE CHANGE ISSUES ...... 224 Dhani Setyawan ...... 224
PRE-PROCEEDINGS of the 1'1/nternatlona/ Conference on Sustainable Agriculture and Environment 5 1si/CSAEIIAM001 1s/ICSAEJBIOTECH001
The Role of Environmental Management System Screening of Blosurfactant Producers Toward Agroindustry Corporate Performance from Locally Isolated Actlnobacterlal
NS. Zemry, A. Ayolb, L. Zekarie, and AR. Mohd Yahya I Gusti Putu Diva Awatara School of Biological Sciences, Un/v&rsUI Sa/ns Malaysia, Malaysia Postgraduate Student of Environmental Sciences of Sebetas Meret Unlvsrslty, Indonesia ABSTRACT Email: [email protected] Biosutfactants are secondary metabolites with sutface active properties that are synthesized by a diverse microbial community. Interest in biosurfactant production remains high as they are widely used In many ABSTRACT diverse commercial applications spanning petroleum, pharmaceutical, biomedical and food Industrial processes. Although blosurfactant This study Aimed to Determine the role of environmental management production Is more environmentally friendly compared to Its chemical systems In terms of aspects of the corporate commitment, corporate counterpart, Industrial production via fermentation Is challenging due to orientation, corporate culture and Implementation of cost toward the relatively low yield and the consequential unattractiVe cost of corporate petformance of Agro Industry in Central Java. This type of production. The present study focused on screening and evaluating survey research conducted in Agro Industry companies In Central Java extracellular biosurtactant production from a tote! of 33 Isolates of using proportional stratified random sampling. Methods of Data collection actinomycete& in starch casein broth with olive oil as the sole carbon using questionnaire and analysis using multiple linear regression source. Actlnomycetes was chosen for their unique filamentous growth techniques. pattern, which can ease downstream processing, Blosutfactant The results of this study show that: 1) the influence of corporate production was confirmed by two screening methods. namely the oil commitment toward corporate petformance of Agro Industry, 2) the spreading technique and emulslflcaUon activity. Both are main methods influence of corporate orientation toward corporate petformance of Agro to detect blosurtactant production. Oil spreading technique Is a reliable Industry, 3) the influence of corporate culture toward corporate and sensitive method In detecting very low concentrations of performance of Agro Industry, 4) the Influence or cost or implementation biosurfactants. The method gives a quantitative assessment by toward corporate performance Agro Industry. measuring the diameter of clear zone formation when a drop of blosutfactant-containing solution Is placed on oil-water sutface. Out of 33 Isolates, 32 isolates showed positive results from oil spreading Keywords: co1porate commitment, co1p0rate orientation, co1porate culture, costlmplementallon, cotporate performance technique, highlighting the high sensiiMty of this method In detecting sutfactants. The emulsifying activity IS evaluated by the emutslftcation Index, e,.. The results from emulsification activity suggest that isolates of actlnomycetes used In the present sbJdy showed good emulsification activity thai ranged from 84·96%. Basad on the oil spreading technique and E2• values, Isolate R1 was selected for blosurfactant production In a larger scale. Following morphological and molecular characterization, Isolate R 1 was tentatively Identified as a member of the Streptomyces genus. The Isolate R 1 was closely related to Streptomyces sp. sharing only 92% 16S rRNA gene similarities suggesting that the Isolate may represent a novel species. Streptomyces sp. R1 was used as a model for a filamentous producer of blosurfllctant. Keywords: Biosurfactant, Actlnomycetes, o/1 spreading technique. emulsification aclivity, mOlpho/oglcal characteristic, 16S rRNA gene.
32 ICSAE- 1" lnlematfonttJ Collfanmco On Sustainable AQf(culture and Envttonmenl ICSAE· 1"' lnternt~lionsl COnference On Suatalltlfble Agrlcunuro and E'ivlronment I 33 SCREENING OF BIOSURFACTANT PRODUCERS FROM LOCALLY ISOLATED ACTINOBACTERIAL Nor 8yafirah Zambry•, Adilah Ayoib, Latiffah Zakaria, and Ahmad Ramli Mohd Yahya School ofBiological Sciences, Universiti Sains Malaysia, Malaysia
ABSTRACT Biosurfactants are secondary metabolites with surface active properties that are synthesized by a diverse microbial community. Interest in biosurfactant production remains high as they are widely used in many diverse commercial applications spanning petroleum, phannaceutical, biomedical and food industrial processes. Although biosurfactant production is more environmentally friendly compared to its chemical counterpart, industrial production via fermentation is challenging due to the relatively low yield and the consequential unattractive cost of production. The present study focused on screening and evaluating extracellular biosurfactant production from a total of 33 isolates of actinomycetes in starch casein broth with olive oil as the sole carbon source. Actinomycetes was chosen for their unique filamentous growth pattern, which can ease downstream processing. Biosurfactant production was confirmed by two screening methods, namely the oil spreading technique and emulsification activity. Both are main methods to detect biosurfactant production. Oil spreading technique is a reliable and sensitive method in detecting very low concentrations of biosurfactants. The method gives a quantitative assessment by measuring the diameter of clear zone formation when a drop of biosurfactant-containing solution is placed on oil-water surface. Out of 33 isolates, 32 isolates showed positive results from oil spreading technique, highlighting the high sensitivity of this method in detecting surfactants. The emulsifying activity is evaluated by the emulsification index, E24· The results from emulsification activity suggest that isolates of actinomycetes used in the present study showed good emulsification activity that ranged from 84-96%. Based on the oil spreading technique and E24 values, isolate Rl was selected for biosurfactant production in a larger scale. Following morphological and molecular characterization, isolate Rl was tentatively identified as a member of the Streptomyces genus. The isolate Rl was closely related to Streptomyces sp. sharing only 92% 168 rRNA gene similarities suggesting that the isolate may represent a novel species. Streptomyces sp. R1 was used as a model for a filamentous producer of biosurfactant. Keywords: Biosurfactant, Actinomycetes, oil spreading technique, emulsification activity, morphological characteristic, 168 rRNA gene
1. INTRODUCTION 8urfactants or surface active agents are amphiphilic molecules that posses both hydrophilic and hydrophobic moieties that are capable of reducing interfacial tensions in both aqueous solution and hydrocarbon mixtures (Desai and Banat, 1997). The majority of them is chemically synthesized from petroleum. These compounds have been reported to be non-biodegradable and toxic to the environment (Ghribi and Chaabouni, 2011). Alternatively, surfactants also can be produced by microorganisms such as bacteria, fungi and yeasts. These compounds are called biosurfactants. In
1 recent years, due to the increasing awareness of eco-friendly processes, more researchers have started to show interest in the production of biologically-synthesized surfactants. Biosurfactant production process can utilize various organic compounds including municipal wastewaters (Thampayak et al., 2008). Compared to chemical surfactants, biosurfactants have unique advantages including lower toxicity, higher biodegradability, better environmental compatibility, higher foaming, higher selectivity and specific activity at extreme temperature, pH and salinity (Ghribi and Chaabouni, 2011 ). It is thus necessary to understand the microbial surfactants since there are many advantages over the chemical counterparts.
Most downstream waste by-products are dumped in landfills, where they accumulate in the soil. Organic waste is degraded biologically in the soil, often with the help of a diverse group of naturally occurring soil microbes. Among the most abundant soil inhabitants are the actinobacteria. They are Gram-positive filamentous bacteria with high G+C content in DNA of 69-78% (Stackebrandt et al., 1997; Deepika et al., 2009). These filamentous bacteria were chosen as the desired microbe to produce biosurfactant in this study due to their abundance and diversity in the soil. In addition, unlike many bacteria, actinobacteria have the ability to secrete copious amounts of many valuable secondary metabolites including enzymes, antibiotics and biosurfactants ·(Ritcher et al., 1998; Oskay et al., 2004; Augustine et al., 2005; Imasda, 2005).
However, only a few reports are available for biosurfactant production in actinobacteria. Therefore, the present study is aimed at screening and evaluating extracellular biosurfactant production from local isolated actinomycetes. The presence of biosurfactant was confirmed by oil spreading technique and emulsification index. The potential biosurfactant-producing actinobacteria was identified using morphological characteristic and 168 rONA technology.
2. MATERIALS AND METHODS
2.1. Microorganisms A total of33 isolates ofactinomycetes were used in this study, taken from soil and water samples from Northern states in Malaysia.
2.2. Screening of Biosurfactant-Producers Seven-day old actinobacteria were cultivated in starch casein broth with 3% olive oil as the biosurfactant inducer to screen for biosurfactant-producers (Thampayak et al., 2008). The culture was incubated for 7 days in an orbital shaker agitated at 200 rpm at 28°C. After 7 days of incubation, cells were separated by centrifugation (Eppendorf Centrifuge 5424, Germany) at 8000 x g for 15 min at 30°C. Cell-free supernatant was analyzed for the presence ofbiosurfactant.
2 2.3. Analytical Techniques
2.3.1. Emulsification Index
Two mL kerosene was added to 2 mL cell-free culture broth. The solution was mixed thoroughly for 2 minutes on a vortex mixer and left to stand for 24 hour. The E24 index is given as a ratio of the emulsified layer height (em) to the total height of the liquid column (em) expressed as a percentage (Cooper and Goldenberg, 1987).
2.3.2. Oil Spreading Technique The oil spreading technique was performed to detect the presence of biosurfactant. Fourty mL of distilled water were added to a Petri dish. Then, 1Of.lL crude oil was added to the surface of the water. Ten JJL of sample was added onto the centre of the oil film. The diameter of the clear zone on the oil surface was measured with a caliper rule and compared with the control with an uninoculated medium (Youssef et al., 2004 ).
2.4. Identification of Potential Biosurfactant-Producing Actinomycete
2.4.1. Morphological Characteristics
Microbes are mainly characterized on the basis of their morphological characteristics. The selected biosurfactant-producing actinomycete was grown on starch casein agar and incubated for 7 days at room temperature. After 7 days incubation, the isolate was identified using macroscopic and microscopic morphological characteristics such as growth on microbiological media, aerial spore mass colour, substrate myceliwn pigmentation and color of any soluble pigment described by Waksman (1961).
2.4.2. 168 rRNA Gene Amplification, Sequencing and Phylogenetic Analysis The mycelia pellet was obtained from five-day-old culture ofRl. The genomic DNA of isolate was extracted using CTAB method ~ohnson et al., 2012). PCR amplification of 16S rRNA gene sequence was done in DNA Engine 1 Peltier Thermal Cycler Model PTC-1 00 (USA) using primer 27f (5'-AGAGITfGATCMTGGCTCAG-3') and 152Sr (S'-AAGGAGGTCWTCCARCC-3'). The PCR amplifications were done using an initial denaturation step at 95 °C for S min, followed by 30 cycles of 1 min at 95°C, annealing at 54°C for 1 min and primer extension at 72 °C for 1 min and a final extension at 72 °C for 10 min and cooled to 4°C. The PCR product was purified using PCR purification kit (Qiagen, Germany). The purified PCR product was then sequenced at First Base Laboratories Sdn. Bhd., Selangor using ABI PRISM®377 DNA Sequencer {Applied Biosystems). The obtained partial 16S rDNA sequence was corrected manually and aligned using ClusterW Multiple alignment in BioEdit Sequence Alignment Editor version 7.0.5 (Hall, 1999) to generate consensus sequence. The consensus sequence of selected biosurfactant-producing actinomycete was then compared with those available in the GenBank database using Basic Local Alignment Search Tool to find the probable identity or nearest match of new sequence (http://www.ncbi.nlm.nih.govD. The consensus sequence of the selected biosurfactant-producing actinomycete and representative sequences from GenBank were used to construct phylogenetic tree using Molecular Evalutionary Genetic 3 Analysis (MEGA 5) software (Tamura eta/., 2011). Determination of genetic distances and evaluation character among the sequences was performed by the neighbor-joining (NJ) and maximwn likelihood (ML) method (Saitou and Nei, 1987) . ..
3. RESULTS AND DISCUSSION
3.1. Screening for Biosurfactant Producing Actinobacteria
A total of 33 isolates of actinobacteria have been screened for biosurfactant production in starch casein broth containing olive oil as the biosurfactant inducer. Kiran eta/., (2010) has shown that olive oil is capable in enhancing the production of biosurfactant in actinobacteria. Consequently, it was chosen as one of the carbon sources in the screening process of the present study. Table 1 swnmarized the results for the screening experiment using two methods; oil spreading technique (OST) and emulsification activity (E24). A similar study has been conducted by Karthik et a/., (2010) that screened biosurfactant-producing marine actinobacteria. Out of the 33 isolates, 32 isolates showed positive results on oil spreading technique when compared with the control of uninoculated mediwn. In a similar screening exercise, Youssef et a/., (2004) demonstrated that 16 strains that showed negative results in the drop collapse method, gave positive oil spreading result (0.5-0.9 em), showing the higher sensitivity of this method in detecting surfactants. The formation of a clear zone after a drop of supernatant containing biosurfactant is shown in Figure I. The results from the emulsification activity test demonstrated that all the isolates showed positive results, with good emulsification activity range of 84-96%. The extent of emulsification activity is shown in Figure 2. According to the OST and E24 value aforementioned (Table 1), isolate Rl was further studied for the biosurfactant production.
4 Table 1: Screening of biosurfactant-producing actinobacteria using oil spreading technique and emulsification activity •
No Isolate Diameter of clear zone E24 {em} {%~ Control 1.10±0.17 0.00±0.00 1 Bl 4.40±0.17 90.40±5.71 2 B2 1.35±0.21 87.90±0.14 3 B3 1.27±0.12 86.84±3.72 4 B4 3.17±0.29 84.11±0.15 5 B5 1.17±0.15 90.00±4.71 6 11 1.53±0.35 92.16±0.22 7 12 3.70±0.14 87.25±0.35 8 13 2.67±0.29 90.84±1.18 9 14 1.90±0.14 90.63±4.42 10 IS 2.40±0.28 90.42±4.12 11 Kl 1.25±0.07 89.18±0.41 12 K2 4.30±0.26 92.47±3.20 13 K3 2.47±0.15 88.05±0.07 14 K4 6.03±0.15 92.23±2.16 15 KS 2.53±0.06 89.24±0.33 16 K6 3.70±0.14 90.63±4.42 17 N1 4.10±0.14 85.17±0.77 18 P1 7.10±0.14 90.24±0.34 19 P2 6.46±0.24 93.46±3.60 20 P3 7.03±0.06 89.24±0.33 21 P4 5.65±0.07 91.95±4.32 22 PS 3.03±0.06 94.22±0.31 23 P6 6.15±0.21 88.74±1.04 24 P7 3.30±0.17 91.50±0.71 25 P8 3.07±0.12 89.69±1.12 26 P9 2.60±0.14 92.74±3.20 27 P12 0.97±0.06 88.94±2.79 28 Pl3 4.20±0.26 84.61±0.56 29 R1 7.45±0.07 95.80±0.28 30 R2 3.60±0.14 93.00±4.24 31 R3 5.95±0.21 85.36±0.50 32 RS 4.05±0.07 93.08±3.06 33 SI 6.35±0.07 84.26±1.32 5 •
(a) (b)
Figure 1 (a-b): Oil spreading test, (a) control with uninoculated medium, (b) displacement of oil by supernatant of isolate Rl.
(a) (b) (c)
Figure 2 (a-d): The emulsion of engine oil after 24 hours of incubation period (a) control, (b)- (d) emulsion forms in the presence biosurfactant from actinobacteria.
6 3.2. Identification and Characterization of Biosurfactant-Producing Actinomycete
3.2.1. Morphological Characterization • Morphological characteristics have been used as a method for preliminary determination of the genus for the actinobacteria taxonomy (Khanna et a/., 2011). The examination of the macroscopic characteristics for isolate Rl grown on starch casein agar at 28°C for 7 days showed that the outer surface of the colonies was perfectly round initially, but later developed aerial mycelium that appeared velvety white colour (Figure 3a) whereas the substrate mycelium was pale brown (Figure 3b). Isolate Rl showed concentric rings of colonies on the starch casein agar plate as incubation time progressed. This is one of the important diagnostic criteria for genus Streptomyces (Waksman, 1961). Observation of isolate Rl under the light microscope showed the presence of oval-shaped conidia (Figure 3c). On the basis of morphological characteristics, isolate R 1 was presumptively assigned to the genus Streptomyces.
(a) (b)
(c)
Figure 3 (a-c): The morphology of the isolate Rl after 7 days incubation on SCA at 28°C. (a) Upper surface of isolate Rl showed white colour, (b) lower surface showed pale brown colour (c) oval-shaped conida under light microscope witb 40x magnification.
7 Streptomyces sp. R9-549 (JQ660003) Streptomycessp. R9-545 (JQ660000) Streptomycessp. DA10202(HM371156) 97 Actinobacterium 220362 (FJ429804) Streptomyces sp. DA08602 (FJ797603) 65 Streptomyces sp. ACT-0096 (GQ924536) Streptomyces sp. CNR924 PL04 (00448732) 99 ~------R1 Streptomyces chartreusls strain DSM 41255(F J932482) 89 Streptomyces chartreusls strain HA 10304 (JF728875) Streptomyces bungoensis strain 15721 (JN180215) Streptomycesgalbus strain NG4 (JF827353) Streptomyces cyaneus (AJ399471) 61 Streptomyces curacoi (EF626595) 100 Streptomycescoeruleorubldusstraln NBRC 12761 (NR041217) Streptomyces sp. 5(EU360169) Streptomyces longlsporusstrain ISP 5166 (NR025492) 100 Streptomyces ftavidovirensstraln NBRC 13039 (NR041099) 88 Streptomyces longisporus gene (AB184219) Streptomyces coacervatusgene (AB500703) Streptomyces sp. L-2·2 (EF524054) Streptomyces sp. MSC702 (JF325872) 100 Streptomyces sp. A72 (EF 100783) Streptomyces sp. 572 (EF208617) 100 Streptomyces sp. A71 (EF100782) Streptomyces sp. A1n (EF100784) 100 Streptomycessp. s1n (EF197893) r------Nocardlopslssp. MSA 10 (EU563352) ----t 100'------Microbacterium esteraromaticum strain PA4(EU647562)
~ 0.01 Figure 4: Neighbour-joining tree based on the almost complete 16S rRNA gene sequences showing relationships among the isolates Rl, representatives of the genus Streptomyces and biosurfactant-producing actinobacteria using Jukes-Cantor method. The percentage of bootstrap values (1000 replicates) that are higher than 50% are shown next to the branches.
8 3.2.2. 16S rRNA Gene Sequence Analysis
The 168 rDNA sequence data have been used in the modem Streptomyces identification system s since they provide invaluable information about Streptomycetes systematic (Lee et al., 2005; Forar et ,. a/., 2006a; Hyo et al., 2006). Thus, further taxonomic characterization of selected biosurfactant producing actinobacteria was done by 168 rRNA gene sequence analysis. It is evident from the 168 rRNA gene phylogenetic trees that isolate Rl is a member of the genus Streptomyces (Figure 4). These analyses were in line with the results obtained from morphological characteristics. Isolate Rl formed a phyletic line that was closely related to Streptomyces sp. sharing 168 rRNA gene identities with the latter at 92%. This taxonomic relationship is supported by 65% bootstrap value. Based on the results from morphological and molecular characterization, isolate Rl was tentatively identified as member of the genus of Streptomyces.
4. CONCLUSIONS There are very limited studies published on the ability of the actinobacteria to produce extracellular biosurfactants. The present study showed that 32 actinobacterial isolates were able produce extracellular biosurfactant when grown in starch casein broth with olive oil as the biosurfactant inducer. From a pool of 32 isolates, isolate Rl that characterized and identified to the genus Streptomyces based on morphological and molecular characterization have been selected as a model filamentous bacterium to be studied further to produce biosurfactant in a bioreactor. The filamentous growth nature of isolate R 1 can simplify downstream processing which consequently reduce the recovery cost of the biosurfactant. Hence, further study will be conducted for strain and process improvement.
5. ACKNOWLEDGMENTS This research was fmancially supported by Research University Grant, Universiti 8ains Malaysia, Malaysia.
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10 The Asian Conferences on the Life Sciences & Sustainability The Asian Symposium on Water, Sanitation and Hygiene August 27-29,2014 at the KKR Hotel Hiroshima, Japan
ORAL SESSION A 1 -. Thursday, August 28, 2014 Oral Session A 13:00 to 14:15
Biotechnology I Takasago Room Presenters: 0138, 0141, 0144
13:00to 13:25 Biotechnology 0138 Effect ofImpeller Tip Speed on Biosurfactant Production by Streptomyces sp. Rl Nor Syafirah Zambry, School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia1 Latiffah Zakaria, School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia2 Ahmad Ramli Mohd Yahya, School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia3
13:25 to 13:50 Biotechnology 0141 Isolation and Identification ofActinobacterial-Biosurfactant Producers Intan Sakinah Mohd Anuar, School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia1 Mor Kar Mun, School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia2 Ahmad Ramli Mohd Yahya, School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia3
13:50 to 14:15 Biotechnology 0144 Rhamnolipid production in Pseudomonas aeruginosa USM-AR2 fed~ batch fermentation Nur Asshifa Md Noh, School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia 1 Ahmad Ramli Mohd Yahya, School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia1
Sponsored by The PRESDA Foundation 9 www .presdafoundation.org "
ACLS 2014 t Global Trends in the Life Sciences ,-
The Asian Conference on the Life Sciences and Sustainability
Official Proceedings ISSN 2188-3971
The 2014 Asian Conference on the Life Sciences and Sustainability KKR Hotel Hiroshima, Japan 27- 29 August 2014 http://www .esdfocus.org/life-sciences-conference/ The 2014 Asian Conference on the Life Sciences and Sustainability Hiroshima, Japan Official Proceedings - Abstracts Section August 27-29, 2014
Conference: ACLS Serial Number 2014_0138 • Presenter Name: Nor Syafirah Zambry Abstract Title: Effect of Impeller Tip Speed on Biosurfactant Production by Streptomyces sp. Rl
Abstract: Biosurfactant are known as secondary metabolite with surface active properties that synthesized by microbial community. Among diverse microbial community, non-filamentous bacteria such as Pseudomonas sp. and Bacillus sp. are well known with the ability to produce biosurfactant. However, these non-filamentous bacteria offer high cost ofbiosurfactant production due to challenging in product recovery and purification process. Unlike these microorganisms, Actinomycete, a Gram positive filamentous bacteria offer the immobilization ability since the mycelia structure greatly assist the downstream processing which consequently reduce the cost ofbiosurfactant production. The present study was focused on a developing Streptomyces sp. Rl, as a model filamentous non pathogenic organism for biosurfactant production. The cultivation of Streptomyces sp. R I was done in a 3 L well-controlled stirred tank bioreactor. The impeller tip speeds was manipulated to improve the biosurfactant productivity. In batch culture, there is a profound correlation between impeller tip speed, morphology of Streptomyces sp. Rl and biosurfactant production. A Rushton-turbine impeller of 17.45 em min-I tip speed was the optimal impeller tip speed for biomass growth. Nevertheless, the maximum biosurfactant production, indicated by the highest surface tension reduction (40.5±0.05 dynes/em) and emulsification activity (67.8±2.0%) was attained when the culture was agitated at 13.09 em min-I tip speed and results the free filamentous dispersed growth form.
ISSN 2188-3971 The 2014 Asian Conference on the Life Sciences and Sustainability Hiroshima, Japan Official Proceedings - Abstracts Section August 27-29, 2014
Conference: ACLS Serial Number 2014_0141 .. Presenter Name: In tan Sakinah Mohd Anuar
Abstract Title: Isolation and Identification of Actinobacterial-Biosurfactant Producers •
Abstract: Biosurfactants are surface-active compounds that are produced extracellularly or as part of the cell membrane that are useful in environmental, food industrial, pharmaceutical and medical applications. This study was conducted to isolate, screen and identify actinobacterial biosurfactant producers from tropical soil. A total of 27 isolates of actinobacteria were isolated and subsequently screened for the ability to produce extracellular biosurfactant by using drop collapse, oil displacement, emulsification index and surface tension measurement. The biosurfactant-producing actinobacterial isolates were identified using morphological characteristic, biochemical test and 16S rRNA gene. Out of 27 isolates, 4 isolates that showed positive results from the drop collapse assay were further screened using oil displacement, emulsification index and surface tension measurement. Isolate SJS 1 showed the highest potential as a biosurfactant producer. Based on morphological and molecular characteristics, isolate SJS 1 was identified as Streptomyces sp.
ISSN 2188-3971