Growth and Lipid Production of L. Starkeyi Mutants

Growth and Lipid Production of L. Starkeyi Mutants CHAPTER 1 INTRODUCTION Diesel is one of the components in fossil fuel. However, the over-use of diesel is producing greenhouse gases such as carbon dioxide gases which are the major elements leading to global warming. Hence, due to increase in demand and source limitation, biodiesel is introduced as a substitute for diesel fuel (Wild et al., 2010). Biodiesel is a diesel fuel substitute that is extracted from renewable biomass. Biodiesel can be produced from plant oils, animal fats and microorganisms. Traditionally, biodiesel is produced from plant oils which were transesterify with methanol (Dai et al., 2007). However, production of biodiesel from plant oils is not suitable due to the quality of tillable land (Li et al., 2008) and competition with food production (Wahlen et al., 2012). Furthermore, the increase in animal fats prices due to the increase in animal feed makes it not suitable as biodiesel feedstock (Li et al., 2008). Hence, oleaginous microorganisms have been introduced as good candidates for biodiesel feedstock. Oleaginous microorganisms can accumulate lipid up to 20% of its cell dry weight (Ageitos et al., 2011). Oleaginous microorganisms have the ability to utilize different carbon source (Ageitos et al., 2011). In this study, Lipomyces starkeyi will be used. This type of yeast has the ability to produce lipid up to 70 % of its cell dry weight (Wild et al., 2010). L. starkeyi can utilize different types of carbon as its sole carbon and it is flexible in terms of culture conditions (Ageitos et al.,2011). However, L. starkeyi is still not economically practical because of the limitations in the wild-type strains (Ageitos et al., 2011). Therefore, in our research, we will be using L. starkeyi mutants in an attempt to produce more lipid more lipid in the fungal cells. The L. starkeyi mutants will be cultured in modified media consists of glucose, (NH4) SO4, yeast extract, Na2HPO4.7H20, KH2PO4, MgSO4. 7H20, CaCl2. 2H20, FeSO4, ZnSO4.H20 and CuSO4 supplied with 2.5% (w/v) and 5.0% (w/v) of glucose and sago effluents in separated schott bottles. pH 5 and pH 6 will also be used in order to optimize the production of lipid. The temperature that will be used is room temperature ( ± 27 °C). In this experiment, sago effluent and glucose would serve as carbon source for L. starkeyi. The total carbohydrate that would be consumed by L. starkeyi will be tested using phenol-sulphuric test. Our objectives in this research are: To optimize growth and lipid production of L. starkeyi mutants To measure the amount of lipid produced by L. starkeyi mutants cultured in 2.5 % and 5 % of glucose medium To measure the amount of lipid produced by L. starkeyi mutants cultured in sago effluent CHAPTER 2:  LITERATURE REVIEW 2.1 Biodiesel Biodiesel consists of alkly ester of fatty acids or triglycerides. Conventionally, triglyceride is produced from soybeans oil with the addition of alcohol and acid or base catalyst. This process is known as transesterifications which will produce Fatty Acid Methyl Ester (FAME) (Wahlen et al., 2012). Basically, biodiesel can be derived from 3 sources which are plants oil, animal fat and microorganisms (Meng et al., 2008). Plant oils that involve in the production of biodiesel are rapeseed, palm oil, soybeans, cottonseed, sunflower and many possible crops (Perritano, 2010). However, the practical used of plant oils raises critical issues on the decreasing in quality of land that is needed to plant the crops could affect the quality of the crops produced (Li et al., 2008). In addition, it also competes with the food production (Wahlen et al., 2012). Animal fat is also not a good biodiesel feedstock due to economical reasons (Meng et al., 2008). Hence, oleaginous microorganisms stand out as a potential feedstock provider. 2.2 Oleaginous microorganisms Oleginous yeasts (OY) are known producers of single cell oil (SCO). SCO produced from this organism are triacylglycerides (TAG) that have long-chain of fatty acids and have similar properties with plant oils. TAG acts as source of energy and it assist in phospholipid membrane formation. OY also utilizes various its carbon sources from waste substrate thus the cost to culture this microorganism is low (El-Fadaly et al., 2009). There are four groups of oleaginous microorganisms that capable of producing biodiesel which are bacteria, algae, filamentous fungi and yeast (Kitcha and Cheirsilp, 2011). The genera of oleaginous yeast are Yarrowia, Candida, Rhodotorula, Rhodosporium, Crytococcus, Trichosporon and Lipomyces (Ageitos et al., 2011). The specific name for the most preferable candidates for production of lipid are Cryptococcus albidus, Rhodosporidium toruloides, Rhodotorula glutinis, Lipomyces starkeyi and Yarrowia lipolytica. These microorganisms are capable of producing intracellular lipid more than 20% of its cell dry weight (Tapia et al., 2012). The duplication rate of yeast is lower than 1 hour and it is easy to culture compared to other microalgae. Other than that, certain oily yeast also has the ability to produce lipid up to 80% of their dry weight, while utilizing different carbon source including the lipid present in media (Ageitos et al., 2011). 2.3 Factors affecting lipid accumulations in Oleginous yeast Lipid accumulations occur when yeast is cultured under high amount of carbon source but in limited source of nitrogen. This is due to the nutrient imbalance that helps in triggering the accumulation of lipid because the remaining substrate would be assimilated by the yeast’s cells hence convert it into fat for storage (Ageitos et al., 2011). The fat that accumulated could be extracted to produce biodiesel. In addition, the accumulations of lipid also affected by other factors such as the present of microelements and inorganic salts in media. These elements help in ATP (AdenosineTriPhosphate) citrate lyse which important in lipid production (Ageitos et al., 2011). 2.4 Lipomyces starkeyi L. starkeyi is one of the members of Saccharomycetales and considered as true inhabitant of soil which have a worldwide distribution (Ansschau et al., 2014). L. starkeyi have the ability to accumulate lipid up to 70% of its dry weight (Wild et al., 2010). It also has a high flexibility in utilization of carbon source and culture environment. Other than that, fatty acid produced by L. starkeyi is almost similar to the vegetable oil (Tapia et al., 2012). According to Wild et al. (2010), L. starkeyi need a high ratio of carbon to nitrogen in order to optimize the production of lipid. The lipid bodies (LB) of L. starkeyi will receive the excess carbon source in the form of triglycerides (TAGs) (Ageitos et al., 2011) 2.5 Sago effluent Sago effluent is a form of sago liquid waste. In normal processes, this effluent would be channeled into the river, thus polluting the river and environment (Awang-Adeni et al., 2010). The releasing of sago effluent into the river can cause decreasing in water pH and increase in biochemical oxygen demand (BOD) and chemical oxygen demand (COD) (Ayyasamy et al., 2008) Sago effluent contains a high amount of organic materials and non-starch polysaccharide (NSP) (Awang-Adeni et al., 2010). NSP are made of cellulose, hemicellulose and lignin. In cellulose, the sub-components are 89% glucose and small amount of xylose, rhamnose, arabinose, mannose, fructose and galactose. In contrast to cellulose, hemicellulose main components are glucose and xylose accompanied with arabinose, galactose, rhmnose, fucose and uranic acid. Lignin functions in rigidity and stability of the wood. To sum up, sago effluent contains up to 66% of starch, 14 % fiber and 25 % lignin (Awang-Adeni et al., 2010). Sago effluents which flow from the sago mill usually have the ratio of carbon to nitrogen high which is 105: 0.12 (Awang-Adeni et al., 2010). As stated by Ageitos et al. (2011), L. starkeyi have the ability to utilize starch as its sole carbon. Hence, sago effluent is an excellent choice because it has a high amount of starch which can helps in optimizing the lipid production. 2.6 Phenol-sulphuric test Phenol-sulphuric test is the quantitative assays which often used in estimation of carbohydrate. This test could detect the presence of neutral sugar in oligosaccharides, proteoglycan, glycoproteins and glycolipids (Albalasmeh et al., 2013). When phenol-sulphuric is added, the glucose that presence in samples would dehydrate thus forms hydroxymethyl furfurax. It would yield a yellow-brown product and the OD could be checked at 490 nm (Albalasmeh et al., 2013). CHAPTER 3:  MATERIALS AND METHOD 3.1 Materials Modified media as suggested by Wild et al. (2010). Lipomyces Starkeyi mutants (LS R1 and LS R2) 2.5 % (w/v) and 5.0 % (w/v) of glucose (Ee Syn, Malaysia) 2.5 % (w/v) and 5.0 % (w/v) of sago effluent (Pusa, Malaysia) 80 % (w/v) of Glycerol stock (HmbG, Germany) 5 % Phenol (Nacalai Tesque, Japan) Hexane (Reagents, USA) Isopropanol (Amresco, USA) Microcentrifuge (Hettich EBA 21, England) Schotts bottles (Duran, Germany) 3.2 Glycerol stock A single colony of L. starkeyi mutants R3 will be inoculated into 100 ml of modified media. 800 ÃŽ ¼l of L. starkeyi mutants R3 that have grown will be transferred into vial that contained 1200 ÃŽ ¼l of glycerol stock. The glycerol stock steps of L. starkeyi will be repeated for L. starkeyi mutants R4. The solution will be stored in freezer at -20  °C. 3.3 Propagation of cell 1.5 L of modified media with pH 5 will be prepared into two Liter schott bottles and L. starkeyi mutants R3 and R4 will be inoculated in respective bottles (Wild et al., 2010). This step will be repeated for pH 6. For day 1 until day 6, three (3) falcon tubes will be autoclave and weight. After that, 50 ml of the cultured from first bottle will be transferred into each three (3) falcon tubes and it will be weighted again. The sample will be sent for centrifuge for 5 minutes at 5000 rpm. The supernatant will be discarded and the pellet with falcon tube will be weight again for its wet weight. The sample will be dry in the oven for 1 or 2 days. After that, the sample will be weight again for its dry weight. All experiments will be performed in duplications. 3.4 Standard curve for L. starkeyi 1 ml of culture which will be incubated for 3 days earlier will be added into 9 ml of modified media in test tube. Serial dilution will take place with the factors of 10-1 until 10-7. For factors of 10-1 until 10-7, their OD will be checked for 600 nm. For factors 10-5 until 10-7, 300 ÃŽ ¼l from each sample will be taken and poured onto plate count agar. The plate will be incubated overnight before colony counting will be performed. 3.5 Lipid accumulation stage for L. starkeyi mutants The L. starkeyi mutants culture will be incubated for 3 days (optimum growth) at room temperature. After 3 days, 750 ml of 10.0% (w/v) of glucose will be added into 750 ml modified media to achieve final concentration of 5% (w/v) in the schott bottle and it will be incubated further for 6 days. From day 1 to day 6, 150 ml of cultured will be harvested into each three (3) falcon tubes. This step will be repeated for pH 5 with 5.0% (w/v) of glucose and pH 6 with 10.0% (w/v) and 5.0% (w/v) of sago effluent. 3.6 Sampling biomass The samples will be weighted in wet condition before dry in the oven. After that, the samples will be dried in the oven for 3 days. The dried mass will be taken and weighted again for dry weight. 3.7 Lipid extraction Hexane: propanol in the ratio of 3:2 will be added into the falcon tubes consists of the dry mass. The mixture will be homogenized for 2 minutes. The homogenized sample will be incubated for 1 hour before centrifuge for 5 minutes. The supernatant will be taken and placed in an empty beaker and weight. The supernatant will be heated until the hexane and propanol solution have evaporated completely. The remaining oil will be weighted again. This step will be repeated for 5.0% (w/v) of glucose, 2.5% (w/v) of sago effluent and 5.0% (w/v) of sago effluent. 3.8 Phenol-sulphuric carbohydrate test Phenol test is used to detect the amount of carbohydrate that is not consumed by L. starkeyi. For each sample, phenol-sulphuric carbohydrate test will be performed by adding 0.2 ml of 5% (w/v) of phenol and 1 ml of 96% (w/v) of sulphuric acid. After that, 1 ml from each mixture will be placed into a clean cuvette and read at 490 nm in a spectrophotometer. EXPECTED OUTCOME By the end of this experiment, we expect to measure the amount of lipid produced by Lipomyces starkeyi mutants in 2.5% (w/v) and 5.0% (w/v) concentration of glucose and sago effluent at different pH. WORK SCHEDULE Project Activities 2014 2015 Aug Sept Oct Nov Dec Jan Feb March Apr May Data collection à ¢-  Proposal writing and presentation à ¢- º à ¢- º à ¢-  Bench work and sample processing à ¢- º à ¢- º à ¢- º à ¢-  Progress report à ¢- º à ¢- º à ¢- º à ¢-  Data analysis à ¢- º à ¢- º à ¢-  Data validation: Statistical analysis à ¢- º à ¢- º à ¢- º à ¢-  Report writing and presentation à ¢- º à ¢- º à ¢- º à ¢- º à ¢-  Legends à ¢- º: In progress à ¢-  : End of progress REFERENCES Ageitos, J.M., Vallejo, J.A., Veiga-Crespo, P., Villa, T.G. (2011). Oily yeast as oleaginouscell factories. Applied Microbiology and Biotechnoogy, 90(12), 1219-1227. Albalasmeh, A.A., Berhe, A.A., Ghezzehei, T.A. (2013). Method for rapid determination of carbohydrate and total carbon concentrations using UV spectrophotometetry. Carbohydrate Polymers, 97(2), 253-261. Anschau, A., Xavier, M.C.A., Hernalsteens, S., Franco, T.T. (2014). Effect of feeding  strategies on lipid production by Lipomyces starkeyi. Bioresource Technology, 157, 214-222. Ayyasamy, P.M., Banuregha, R., Vivekanandhan, G., Rajakumar, S., Yasodha, R., Lee, S., Lakshmanaperumalsamy, P. (2008). Bioremediation of sago industry effluent and its impact on seed germination (green gram and maize). Journal of Microbiology and Biotechnology, 24(11). 2677-2684 Awang-Adeni, D.S., Abd-Aziz, S., Hassan, M.A. (2010). Bioconversion of sago residue into value added. African Journal of Biotechnology, 9(14), 2016-2021. El-Fadalay, H.A., El-Naggar, N.E., Marwan, E.M. (2009). Single Cell Oil Production by an Oleginous Yeast Strain in a Low Cost Cultivation Medium. Research Journal of Microbiology, 4(8), 301-313. Kitcha, S., Cheirsilp, B. (2011). Screening of Oleaginous Yeasts and Optimization for Lipid. Energy Procedia, 9, 274-282. Li, Q., Du, W., Liu, D. (2008). Perspectives of microbial oils for biodiesel production. Applied Microbiology and Biotechnology, 80(5), 749-756. Meng, X., Yang, J., Xu, X., Zhang, L., Nie, Q., Xian, Mo. (2008). Biodiesel production from oleaginous microorganisms. Renewable Energy, 34(2009), 1-5. Perritano, J. (13, December 2010). 10 top biofuel crops. Retrieved from HowStuffWorks:http://auto.howstuffworks.com/fuel-efficiency/biofuels/10biofuelcrops. htm#page=2 Tapia, E. V., Anschou, A., Coradini, A. L., Franco, T. T., Deckmann, C. (2012). Optimization of lipid production by the oleaginous yeast Lipomyces starkeyi by random mutagenesis coupled to cerulenin screening. AMB express, 2(64), 1-8.

‘Explore the ways that writers present strong feelings to interest the reader or audience’

Various techniques are used by writers to present strong feelings which evoke emotion from the reader or audience. Literary techniques are used in great lengths to both emphasise strong feelings in a literary piece and to also evoke strong feelings from an audience. The techniques embody language, structure and form. The experimentation of structure and poetic techniques used by writers create strong feelings within both the contemporary and present audience, ensuring audiences were and always will be interested in the literary piece.In the prologue of the play ‘Romeo and Juliet’ the audience learn that two dignified households in the city of Verona hold an ‘’ancient grudge’’ towards each other, which remains a source of the violent conflict which is central to the play. It can be suggested that hatred has grown stronger over a long period of time. Similarly, the structure of the poem undermined traditional Elizabethan sonnets which were tradit ionally love poems. Shakespeare’s, however, changed this form to show hatred, violence, conflict and death to foreshadow the ending of ‘Romeo and Juliet’.An Elizabethan audience would have recognised this, creating a feeling of excitement and curiosity within them. Likewise in ‘sonnet 43’ Browning has also undermined the traditional form of a sonnet to create religious imagery to describe her lover. Browning’s sonnet discusses and compares her strong feelings for her lover and as her description develops she illustrates that she loves him with the emotions of an entire life from childhood right through to death. ‘’I love thee with the breath, smiles, tears, of all my life!– And, if God choose, I shall love thee better after death’’. She worships her lover with all her heart and respects him much more than she does her religion. He touches all aspects of her life and gives meaning to her whole existence. The au dience would be shocked as during the Victorian era religion was paramount in the lives of the people. However, it is not just love for one person that is described but the feeling of love itself. Similarly, as Romeo and Juliet become innocent victims of an atrocious ‘‘strife’’ between their families when they ‘’take their lives’’.Considerably, the poet evokes strong feelings towards the emotion of love when Browning is describing the intensity of religion and the link between death and love, as Shakespeare does when he links the idea of love and death in the prologue, allowing both the audience and the reader to openly question the content without profanity. In the play ‘Romeo and Juliet’ segregated from society is a dominant feeling and a reoccurring theme. Romeo and Juliet go to extreme lengths to preserve their love together.They did this as according to Elizabethan society it was nor their ‘Fate’ nor ‘Destiny’ to ever be together. Therefore by choosing to be together consequences to them both mocking society. Juliet was Romeos second love which is ironic and therefore mocks society because Elizabethans believed in fate and destiny and that you could only ever love one person. If you loved again you weren’t really in love or you previous relationship wasn’t love but lust. Romeo’s feelings power his actions contradicting the Elizabethan norm which would undoubtedly evoke strong feelings such as disgust and shock from the audience.Alternatively in the poem ‘My last duchess’ the writer evokes strong feelings from the reader by focusing on the dominance and control of the Duke towards his wife. The audience in this case is this the ambassador acting on behalf of Ferdinand referred to in the poem as ‘The Count, your master’ but in reality it is the reader. This makes the reader feel rebellious as they are ‘eavesdroppin g’ on an interesting conversation. This completely contrasts to Romeos relationship with Juliet. They respect and accept each other as equals whereas the Duke doesn’t respect his wife or even acknowledge her.The duke refers to his wife, not by her name, by ‘she’. A contemporary reader would accept this as men were the dominant spouse. ‘’Half flushed that dies along the throat’’. This is ironic as it is said that the duke killed or had his wife killed, we could interpret that he beheaded or had her beheaded. This would surprise the reader as the duke earlier in the poem he compliments his wife, calling her a ‘wonder’. Although his words and actions are brutal the duke would have been accepted by society as men were believed to be higher than women.Romeo and Juliet’s love was not accepted by an Elizabethan society but they choose to ignore their friends and family, therefore appalling their audiences and mocking their entire beliefs. In the play Romeo and Juliet confusion, doubt and uncertainty are common emotions. During the balcony scene Juliet is speaking her mind unaware that Romeo had been listening. ‘’Be sworn my love, and I shall no longer be a Capulet’’. She would disown her family to be with Romeo, someone whom she had just met. This would have stunned an Elizabethan audience as she came from a rich, well respected family.There is more confusion when Juliet’s feelings change, ‘’ it is too rash, too unadvised, too sudden’’. Shakespeare uses the ‘rule of three’ to emphasise on the word ‘too’. He does this to show how strong Juliet’s doubt towards Romeo is. An Elizabethan audience would be confused as to how she could change her mind as they believed in love at first sight and this was going against that belief. Shakespeare mocks his entire society though his characters by showing them that fate and destiny is not written in the stars but is decided by you.Equally, in the poem ‘The Laboratory’ there is a lot of confusion between the character and the reader. The woman in the poem is searching for the perfect poison to commit a murder. She cannot seem to decide what poison she would like and is distracted by the â€Å"exquisite blue† colours of the poisons. ‘’Yonder soft phial†¦ sure to taste sweetly, – is that poison too? ’’. Her actions are very child-like and due to her frequent change of mind the reader will begin to doubt her motive and seriousness.The reader feels confused at her motives and could assume the poem is comic and not serious. By using a question mark, the poet emphasises the protagonists confused mind. This confusion is further highlighted by the â€Å"-â€Å"which separates the question from the rest of the stanza and draws it to the attention of the reader. Clearly, Shakespeare and Mr and Mrs Browning all convey strong emotion to the audience and readers whether it is through language, structure or form. This is one of the main reasons why their literature has lasted and is greatly treasured.

Facebook Assignment

Nathan Barry – S10001606 Business Context Introduction The following assignment was created by our group [ ] Our chosen organisation for the assignment is the online social networking site Facebook. As a group we feel as though this would give us a good opportunity to analyse the factors of business content in an in-depth way. Business could be defined as â€Å"the transformation of inputs into outputs. This transformation process takes place against a background of external influences which affect the firm and its activities†. (Worthington and Britton 2000:3) Facebook BackgroundFacebook was co-founded in February 2004 by Mark Zuckerburg and roommate Dustin Moskovitz, both American entrepreneurs who attended Harvard University, Massachusetts. Zuckerburg now owns around a quarter of the organisation with Dustin still having a share of around 6-7% as of 2010. Facebook’s mission statement simply reads â€Å"to give people the power to share and make the world more open and connected†. (www. facebook. com) Economics â€Å"The study of how human beings seek to use resources to satisfy the whole range of their needs, wants and desires†. Wetherly and Otter 2008:3) We need resources in order to satisfy our needs, wants and aspirations. The economy provides this in four categories which are land, labour, capital and enterprise. The economy surrounding Facebook is undoubtedly large and still growing rapidly with investors continuingly looking to pump money into the company. The site attracts businesses from all over the world and of all sizes with its expansive advertising space which reaches out to millions of users daily.Recent figures published by Facebook themselves estimate a figure in the region of â€Å"five hundred million users with 50% of chose actively logging into the site daily†. (www. Facebook. com/stats). This therefore is an extremely attractive proposition for any business looking to advertise as most target audi ences are reachable. Supply and demand is an extremely important factor for Facebook. They are the market leader in there sector and therefore control the price as investors and company’s looking to advertise have little choice when it comes to substitutes (compliments).There is a huge demand for what Facebook offer and this demand is only met by small supply which as a result increases the price. Legal/Technology Technology is â€Å"the sum of knowledge of the means and methods of producing goods and services† (Worthington and Britton 2000:138). Technology is massive issue for any online business as falling behind could be very costly in terms of gaining a competitive advantage. Facebook specifically must concentrate on information technology (i. c. t) as they handle huge quantities of data about their users.Businesses must comply with legislation and Facebook are a complex example with issues such as eBullying, â€Å"the term used to refer to bullying and harassment by use of electronic devices such as mobile phones, e-mail, instant messaging, text messages and websites†. (www. ebullying. co. uk). Facebook attempts to reduce this with an extensive rights and responsibility page which must be agreed to when the user signs up to the website such as an age restriction policy although this is easily flawed as users can enter the wrong D. O. B without checks being carried out.

The Middle East Is Clearly Out Of Hand Essay - 1628 Words

Jeffrey A. Beery Professor Carlin Mackie English 102 9 November 2016 Introduction: The Middle East is clearly out of hand. Why is this region prone to so much bloodshed? All we need to do is refer back to our history books. In preparation for the Collapse of the Ottoman Empire and the end of WW1 the Sykes and Picot agreement was signed, which called for British and French influence in the region and the creation of states. This agreement was to benefit French and British interests and had very little concern regarding the ethnic and religious makeup of the region. After analyzing the region it is evident that the current border situation is ineffective and causing much of the problems. The current foreign policies the Western world has towards the Middle East need to be seriously reconsidered. The Vision of T.E. Lawrence: The Sykes and Picot agreement was conceptualized as a great idea among western leaders at the time, however according to Yassamine Mather in his article titled The Fall of the Ottoman Empire and Current Conflict in the Middle East â€Å"even then the agreement had its critics: T.E. Lawrence, who knew a lot more than Sykes about national and religious differences in the Arab world, warned of conflicts within the region† (471). Time has proven that T.E. Lawrence made a rather accurate prediction. Lawrence was a rather popular man regarding this period, and is still very much so today. In fact, Lawrence’s book titled Seven Pillars of Wisdom has become a mustShow MoreRelatedMERS-CoV: Middle East Respiratory Syndrome726 Words   |  3 Pagesthe Middle East, became ill in Saudi Arabia (WHO). He visited the hospital once he started seeing symptoms of an flu-like illness (WHO). 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