apr.-jun., 2016ssri.pk/psj/psj apr-jun-2016.pdf · trashing of cane in the field or whatever is...

Apr.-Jun., 2016

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Editorial BoardMr. Altaf M. Saleem ChairmanDr. Shahid Afghan Editor-in-ChiefDr. Iftikhar Ahmed MemberDr. Muhammad Zubair MemberDr. Javed Iqbal MemberDr. Aamir Ali MemberMr. Aamir Shahazad Editor

SubscriptionAamir ShahzadShakarganj Sugar Research InstituteToba Road, JHANGPh: +92 47 763 1001-5 Ext. 603, 604Email: [email protected]

Subscription RatePakistan PKR 1,000/-OVERSEAS US$ 50/-

Recognized byHigher Education Commission (HEC) Pakistan

Cited byAsia Net Pakistan (Factiva International)Commonwealth Agriculture & BiologyInternational (CABI-UK)

ISSN 1028-1193

Panel of RefereesDr. P. Jackson: Principal Scientist, CSIRO, AustraliaDr. Raul O. Castillo: Director General, ResearchStation EI Triunfo, EcuadorDr. Benjamin Legendre: Interim Director, AudubonSugar Institute, USADr. Yong-Bao Pan: Research Plant MolecularGeneticist, USDA-ARS, USADr. Jack C. Comstock: Research Leader, ARSUSDA, Canal Point Florida, USADr. Sizuo Matsuoka: Director, Canavialis SA, BrazilDr. Niranjan Baisakh: Asstt. Professor, - SPESS, LSUUSADr. Abdul Rauf: Prof. & Chairman Plant PathologyPMAS Arid Agriculture University, RawalpindiDr. Asif Tanvir: Professor, Dept. of Agronomy, UAFDr. Muhammad Bilal Chattha: Assistant Professor,Agriculture College, Punjab University

CONTENTS

02Reducing cost of productionMuhammad Yousuf Khan, General Manager:Matiari Sugar Mills,

11Isolation and characterization of nitrogen-fixing bacteria from sugarcane rhizosphereK. M. Alam, T. Zhang, Y. Yan, W. Zhang, M. Linand W. Lu

23SUGAR INDUSTRY ABSTRACTS

29INTERNATIONAL EVENTS CALENDAR

30STORY OF SWEETS

i. Gulab Jaman

ii. Sheer Khurma (Vermicelli Pudding)

32GUIDELINES FOR AUTHORS

PAKISTAN SUGAR JOURNAL

April-June 2016 Vol. XXXI, No.02

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Reducing Cost of ProductionMuhammad Yousuf Khan, General Manager: Matiari Sugar Mills, Technical Workshop 2015,

Venue: Hotel Movenpick Karachi, Saturday, June13, 2015

Cost of production is asignificant component of theoverall cost of a product.Keeping the cost of productionat a minimum is a continuousand uphill task. It also meansthat by saving money in thisarea being able to offer a betterprice and services to thecustomer and becoming morecompetitive against other marketplayers. Low cost of productionis synonymous to increasingprofitability of an existingmanufacturing unit. This is theline we always toe to keep theOrganization going, ‘Increasethe Profitability’. The cost ofproduction can be reduced byfocusing on various areas ofoperation, some of which areReduced cost of raw material.Reduced material losses.Reduced rejection and recycling.Reduced process losses.Reduce labor cost. Reduce timeloss. (Reduce down time).Reduce overheads.

Optimize usage of resourcesIncrease productivity. The aboveand many other areas that havea potential for reducing the costof production can be exploredand exploited by making use ofthe following measures. Findingcheaper sources of raw material(cane in our case), includingreduction in transportation costof raw material (cane) fromsource to factory. Locatingimproved quality raw material(Cane) such that it’s conversionto finished product is more

yielding, easier and material lossis also low. Managing rawmaterial inventory such thatmost of the raw material isprocured when prices aresuppressed. For perishableitems optimal stock is held.Obsolescence may beconsidered. Always makingefforts to reduce Milling losses,process losses, steam losses,time losses and electricitylosses. Employing trainedworkers or getting the existingwork team trained, taking care oftheir social aspects such assalaries, health perks etc.Providing security of job andhealth to work team. Providingappropriate incentives to keepthe work team motivated.Applications of Automation andInstrumentation to reducelosses, improve quality of finalproduct, reduce labor cost,reduce time loss, optimizeresources and increaseproductivity. Bring in moreefficient processes andmachines that result in lowerrejection/recycling/ reworkultimately reducing theprocess/material cost. This alsoimproves the quality of finishedproduct. Careful selection ofequipment at the setup stageand better maintenance programin operation results in reductionin down time, increasedproductivity and optimizingusage of resources. OptimizeAdministrative costs, inventory,capitol investment, and reduceaccident/life compensation by

employing better safetymeasures to keep theoverheads under control. Bringabout change within theorganization when ever feltnecessary. This will revealmultifold advantages. Some ofthese measures that areconsidered more important willbe discussed in more detail.

Cost and Quality of RawMaterialThis is especially important inthe case of sugar factories. Attimes when the crop is relativelysmall there is an intense caneavailability pressures, panicbuying erupts a price war andthen sky is the limit for caneprice. It is also observed thatfactories at these times evenbuy cane from far off places andalso from each other’s operatingareas in this manner anunhealthy price competitionresults in a cut throat situation.In addition extra transportationcost is incurred. This additionalcane and transportation costthus results in higher cost ofproduction. Care should betaken at the proper forum indeciding these matters. Propercontrol is also necessary onprocured cane movement.Superior verities such asUS633, SPF234, CPF237,HS12, Thutta10, SPSG26, offresh cane, free of trash,sometimes reveals 1 % highersugar recovery as compared topoor quality cane. These factorsshould be critically checked at


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the time of procurement andbefore milling. Extensive de-trashing of cane in the field orwhatever is possible when canearrives at the factory should bedone. This will enable bettercolor removal. It will also reducebagasse and molasses losses.The trashes removed can beused as fuel at the boilers.These factors results in betterquality sugar and reduced costof production. The impact ofsuitable material for processingis elaborated by studyingMatiari’s crushing example.Above 170 tons of cane per houris crushed, having sugar loss %cane 0.75 and below, with fiberup to 14.70%; it is observed thatwith the same mill openings andhigher milling speeds thiscapacity reduces to about 166tons cane per hour or evenlower having sugar loss % cane0.75 and above when the fiberrises beyond 14.8%. Sampledays are shown in Table shownin ‘Annexure I’. This clearlyshows that with higher fiber themilling capacity reduces andsugar loss in milling increases.Cane with higher fiber is thusnot a preferred material formilling. Similarly when canejuice produces massecuits ofhigher viscosity or has highernon sugars then molasseslosses increase, Pan boilingtime increases centrifugingbecomes difficult. Higherquantities of pectin gum starchash and other coloring materialin cane juice make colorremoval difficult. It can be seenfrom Matiari’s example fromTable in ‘annexure 2’ that boththe sugar loss in molasses andthe boiling time increase with

higher NS Pol ratio. Thisconsequently increases the costof production. Thus again canethat produces higher viscosity injuice or has a higher non sugarcontent, is again not a preferredmaterial for processing. In thislight it can be said that it is veryimportant to have material whichis convenient to process. Thiswill again make it possible toimprove the quality of sugar andreduce the Cost of Production.Continuous un-interruptedmilling and other operations arealso important because thismakes it possible to crush freshcane by keeping a low cut tocrush time (24hours or lower).For this plant maintenanceshould be continuouslyimproved. 100% plantavailability should be the target.Continuous cane developmentwith the aim to make availableimproved varieties of healthycane in one’s own operatingarea is essential. Tissue cultureactivity is recommended (this isbeing done at Matiari).Procurement planning to acquirecane according to it’s maturity,should also be considered as animportant component ofreducing production cost.

Reducing Losses, Rejectionand Re-cyclingIt is wisely said ‘a penny savedis a penny earned’. Let us againstudy the example of MatiariSugar Mills to first study theeffect of material losses andlater energy losses on cost ofproduction. Let us consider acrushing of 4,000 tons for 140day with a recovery of 10% thetotal sugar produced in such aseason will be 56,000 tons. If

the total losses at this crushingreduce by 0.05% than the sugargain will be 280 tons. Assuminga sugar price of Rs 53,000 perton without taxes, with this gainin sugar the cost of productionwill reduce by Rs 252 per ton.This is a significant saving in justone area. It is thus imperative tokeep the material losses at aminimum. These losses can becontrolled by getting good caneas already discussed above.Equally important is adaptinggood milling and processingpractices. Some of these goodpractices may be: Continuousoperation; This demands topclass maintenance. The desiredoutcome should be zerostoppage of any equipment.sugar will thus be bagged at thefirst instance. Resulting inminimum loss Cane preparation;More are the cell walls of caneruptured; more juice will beextracted during milling. Andfinally more will be sugarextracted.91 to 92% open cellsis a reasonably excepted figure.Cane feeding into the mills; Auniform blanket of bagasse intothe mill, fed with a pressure ofabout 2 meter bagasse in thefeeding chute of four mills willresult in 94+ extraction.Mill settings; this is an areawhich is mostly dependent uponthe experience of mill engineer.Although many estimationmethods are available, these stillneed practical correctionaccording to mill type, no. ofmills in the tandem, cane type,allowed water addition, expectedthroughput.Mill hydraulic pressure; In a 4mill tandem, a specific hydraulicpressure of about 16 t/dm2 at


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the crusher and about 21t/dm2at the last mill is considered togive a good float of about 35 to15mm to the mill top rollersacross the tandem. Thiscorresponds to a mill hydraulicpressure of about 160 kg/cm2 atthe 1st mill and about 200Kg/cm2 at the last mill, with ajournal size of about450mm*500mm. Mill extractioncan be increased in excess to96% with s.h.p. up to 30t/dm2but this value is limited due to 21t/dm2 due to considerations ofexcess steam consumption andexcess stresses in the millingequipment. Sometimes it is alsoconsidered beneficial to keepthe hydraulic pressure at thenon drive end about 10 Kg/cm2higher than what is kept at thedrive end.Imbibition water quantity; 40%water added on cane isconsidered to be good. Now adays mills are using higherquantities of water and gainingadvantage in mill extraction,reduced bagasse pole andincreased undiluted juice.Matiari and Faran Sugar Millsare adding about 45% water oncane Point of application ofImbibition water; the best pointof application of Imbibition wateris where the bagasse is exitingfrom a mill, here it has notcaptured air and is susceptibleto absorb maximum water andreplace pure juice with it.Imbibitions water quality; (usecondensate if available inexcess), more is the quantity ofnon sugar in juice more is theloss of sugar in final molasses,any impurities if added to mixedjuice will increase sugar loss.Thus water with as little

contamination as possible maybe used.Juice liming; Free CaO is mostcommonly used for effectiveprecipitation of impurities inmixed juice. Lime with 95% CaOis considered to be excellentmaterial. In our country thisquality is seldom available, thuslime with about 78% CaO isfrequently used. Milk of lime isprepared so that lime isuniformly mixed in juice. withproper mixing and retention timeof about 20 minutes can revealgood results. When encounteredwith difficult to treat juices thanliterature refers to employing hotliming. Juice heating; Primaryheating up to 70oC with thirdvapor and secondary heating upto 104 oC with first vapor andexhaust steam-if considerednecessary would reveal themost economical results.Syrup brix; 65 brix is good syrupto boil at Pans. In seasons 1991to1996 in Sakrand Sugar mills acontinuous brix of 68 wasmaintained. This resulted insteam % cane of 56 which inthose days was considered tobe good since sophisticatedequipment of today was notavailable.Skillful pan boiling; This is a vastfield with numerous aspects tolook after. One aspect which isconsidered important is thevacuum in boiling. Too low avacuum is associated with hightemperature boiling this willresult in grain melting andconsequent reduced exhaustion,where as too high a vacuummay cause entrainment ofmaterial at low brix and alsoenergy loss due to excess waterconsumption at condensers.

Excess water consumption onsome Pans may cause waterstarvation at pans placed at theend of the injection header. Avacuum of 610 to 670mm ofmercury column is consideredefficient, depending on whatsugar is being boiled.Some other points to ponderare: pH control, proper molassesand massecuite conditioning,proper massecuite purging andPlant sanitation. Besides allthese it is also important thatsugar extracted should beprocessed at an optimum speedand bagged as soon aspossible, this will minimizeinversion losses. Spillages andleakages should be reduced to aminimum. The target in this areashould be ‘zero losses’.Rejection and consequentrecycling are usually done dueto quality issues, for exampleexcess color formation orimproper color removal,conglomeration, grainirregularities, burning of sugarand collected spillages. It is acommon misperception that allleaked and spilled material isreclaimed and there is no lossafter recycling. This is incorrectsince all inherent losses of allthe processes through which allthe collected material passesduring recycling are added up tothe production cost. More overin cases of spillage andleakages all material cannot beand is never recovered, some ofit essentially goes down thedrain. In addition to the materialcost there is energy cost,chemicals cost, labor cost andequipment’s capacity loss,associated to recycling. All theseadd up to cost of production thus


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rejection and subsequentrecycling should be avoided asmuch as possible. Themanufacturing process mostsuited to a plant’s prevalentconditions should be employed.For example Carbonitation maybe suitable for one plant havingjuice with low starch and highash; but this may be consideredenergy inefficient at a plant withjuice having high starch and lowash where floatation may bemore effective. The juicecharacteristic of the operatingarea should essentially bedetermined before setting up theplant and then after beexamined periodically.Besides recycling andinappropriate manufacturingprocess material and energylosses also occur in other areas,such as having unsuitable,obsolete, inefficient, improperlysized equipment and Incorrectplant operation. Equipment withexcess capacity requires moreenergy to operate, moreover,this equipment results inadditional material loss and it’sidle capacity makes a waste ofresources. All these factorsrelating to excess capacity addto the cost of production.Modern mills, steam turbines,electric motors, drives pans,evaporators centrifugalmachines, pumps and heatersare amazingly efficient,replacing of all obsolete andinefficient equipment withefficient and time savingequipment is cost effective theseequipment usually also have alow maintenance cost. Thereplacement will definitelyreduce the cost of production.

We at Matiari have observedsaving of about:250 Kw electricity by replacingour condensers and sprayInjection pumps with modernautomated equipment.Consequently saving 4830 tonsbagasse per 140 days season.150 Kw electricity by installingVFDs and capacitors on motors.Consequently saving 2890 tonsbagasse per 140 days season.30,000 Kcal/ton steam byinstalling an economizer of 220m2 heating surface area eachon 2, 24 Kg/cm2, 45Tph boilers.Consequently saving 5,000 tonsbagasse per 140 days season.1.3 % steam consumption oncane by Increasing the area ofsuper-heater by 101m2 each on2, 24Kg/cm2, 45Tph boilers.Consequently saving 3400 tonsbagasse per 140 days season.

Automation andInstrumentationAutomation and instrumentationcan do nothing that humanbeing cannot do. All that is donethrough instrumentation is moreefficient. With automation andinstrumentation each function ofproduction is carried outaccording to a set of instructionsgiven to a machine by man. Theactions are taken withoutfeelings, emotions and humanlimitations thus tasks are carriedout with a uniform output anduninterrupted because ofabsence of human limitationslike fatigue boredom or lack ofconcentration.Due to these advantages thework is done more efficientlymore effectively and eliminatinghuman error. Rejection andreworking are reduced to a

minimum and productivity ismaximized. It consequentlyreduces the cost of productiondrastically. Automation andInstrumentation also reduces thedependence on the work forcethus reducing the man-hourcost. This does not mean thatinstrumentation and automationdo not require any attention.Timely and apt maintenance andcalibration by the work team aremost essential for effectiveworking of Instrumentation.Having new or maintainingalready installed Automation andInstrumentation is still veryexpensive in our country, thusbefore getting new automationor reconditioning of alreadyinstalled automation it’s returnon investment should becritically studied. Some loopsthat may lower the cost ofproduction in a sugar factory areAuto cane feeding system,Imbibition water temperaturecontrol, pH control, Evaporatorbrix control, Heaterstemperature control, Continuousand batch pan boiling, Sugar re-melting and boiler operation. Weat Matiari have installedAutomatic cane feeding to mills.The result is shown inAnnexure-3. Throughput hasincreased to above 3800 TCDfrom below 3400 TCD. Millextraction has Increased toabove 95.15 from below 94.1.Imbibition has also increased.Balancing, Modernization,Replacement and ExtensionFour important tasks areessential and are needed to becontinuously undertaken, tokeep the business viable andvibrant.


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i) Review and revamp the plant’sequipment capacities foroptimum operational output,revamp any equipment that hasa capacity lesser than required.At the same time try and reduceexcess capacity, this will keepmaterial losses at a minimumand help to utilize the plantcapacity to the fullest.ii) Keep the plant up to date;replace obsolete equipment withlatest efficient equipment thatapplies tried technology. All newequipment should be checked tohave the desired ROI. Reviewthe processes and change themwhenever a more viable processis found. This will increase theplants productivity and reducematerial and time loss,iii) Replace equipment that isless efficient less productive ordoes not fit into the processcapacity wise, this also reducesmaterial and time loss,iv) Extend the plant capacitywhen the optimum size ofproduction goes up.All these steps contribute inReducing Cost of Production. Allthis is termed as BMRE. Manysugar factories have periodicallytaken care of these four stepsand benefited. One of the manyexamples is Habib Sugar Millswhere the plant capacity hasbeen increased from 1500 TCDto 10,000 TCD, the plant hasbeen modernized many a times.It is still abreast with latesttechnology after 50 years of itsinception.

Human ResourceDevelopmentIt is an established fact thatorganizations having a formallytrained work team have an edge

over others. The trained workteam understands the processesand equipment operation betterand faster than untrained workforce. They are also morereceptive to change, thus theyhave a high productivity andthey cause fewer losses.Training should be considered tobe a regular component ofbusiness. Training and retrainingis therefore the only way to keepabreast of the developmentstaking place in and around anyspecific field of activity. It is alsoan established fact thatcompetence is not only arousedfrom the inborn creative abilitiesof a person but it is alsoachieved from fusion of ideasfrom different sources. Trainingsessions held by theorganization gives a very goodopportunity for doing this.Experience may teach what iswrong but what is right or new ina field can only be learned fromthe process of training andretraining. Training whencoupled with employee’s job andhealth security definitely resultsin lower production costs. Oneshould be mindful of the fact thatmore a person is trained andskillful more is his requirementfor recognition and reward. Toretain trained employees theyshould be offered incentivesschemes for high productivityworking. Career plans should bemade for each employee andtimely advancement in careershould be ensured. Socialbenefits such as education andhealth programs for family go along way in the efficient workingof an employee.

Inventory ManagementAll tangible items held for sale orthose under production for saleor those that may be consumedin production of salable items orhelp the production process arecalled Inventory. Inventoriesserve to uncouple all operationsand make them independentenough of each other. Thus it isnot only desirable, it is vital tolow cost manufacturing. We atpresent will be interested incontrol of raw material (cane),process material and plantspares. Effort should be made tokeep these three inventories atan optimum value. This willcontrol financial cost of inventoryand thus reduce the cost ofproduction. Holding too lesssugar cane may result in stockrun-out positions, this is morecommonly termed as ‘no cane’.This is undesired since itinterrupts the operation andreduces the plant capacityutilization. On the other hand iftoo much cane is procured thanstaleness of cane due to longwaiting time reduces the sugarrecovery consequentlyincreasing the cost ofproduction.A similar situation exists withspare parts and processconsumables. Too much stocksmeans a large inventory cost.Stock for these items should beheld in such quantities that theyare sufficient to avert a situationof stock run out during the timerequired for procurement andsupply of new stocks reachingthe plant. If this happens thenthe operation of certainequipment or a certain processmay stop. Expiry dates ofperishable items should also be


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considered while deciding uponquantity of stocks to be held. Oldstocks should definitely beconsumed before their expirydate reaches. FIFA Principleshould be followed forconsumption of stocks. Smallstocks should be kept for itemsthat can quickly get obsolete(such as computer spares).Good practices in physicalmaintenance and carefulkeeping of inventory reducematerial loss in storage.Similarly a proper documentedinventory system helps toprevent over stocking or run outof stocks, enables easy excessto each item when needed,authorizes issues and returns, italso enables identification ofredundant, leftover and obsoleteitems. The inventory decisionshave to be taken againstconflicting factors. Thus one isusually faced with the choicebetween the devil and the deepsea while dealing with theseissues. The available techniquesfor analysis are all indicative andthe final decision always has tobe made according to specificsituation and company policy. Ifa careful eye is kept oninventory management than theinventory cost can be minimizedand the cost of production will

also reduce. An important tip todeal with this situation is that20% of the total inventoryvolume carries 80% of thecapital value of inventory. Thuscritical consideration of this 20%stock will simplify the problemand lead to a better control ofthe entire inventory system.This process is inevitableanywhere. It is more rewardingwhen a change is brought aboutvoluntarily, in a planned andstructured manner, rather thanbringing it in a panic, whencompelled by prevalentconditions,. At this time the teamis unprepared for theinescapable change. Thesuccess in such cases isstaggering. Change within anorganization relating to reductionin cost of production may bebrought about to modernize orreplace inefficient equipmentand processes, use better rawand process materials, addresscompetitive issues, respond tomarket demand, enhanceemployees’ capabilities, improveworking environment, addresssafety issues, structural changesin management or any otherteam component. For planningdetermining and pin pointing theneed for a change, it isnecessary to periodically review

the company goals, profitabilityobtained compared to target,team performance processcapabilities, equipmentperformance, environmentaleffect of in-place processesmarket trends, customerdemands and new statutoryrequirements. Costeffectiveness of all changesshould always be consideredforemost. The most desiredchanges are those that require alow resource input and a highreturn. Excellent team leadersand team members are requiredfor the success of a change. Ifthe management feels that theteam is not strong enough or notsuitable to undertake thechange, than it should firstchange the team leaders orteam members or get themsuitably trained to take up theassignment. Then provide allrequired resources for thechange to a capable team Theremight be many other factors thatbring down the production costand have not been discussedhere; it can be claimed withconfidence that each of thefactors discussed here havetheir contribution in loweringproduction cost. Focusing onany or all of them will bringprolific results.


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Annexure 1 Comparison of Cane Crushing with respect to Fiber % Cane of Season 2014-15Date Cane Crushed (M.T) Fiber% Cane TCH Loss in Bagasse % Cane Date Cane Crushed (M.T)1 18.11.14 4102.98 14.65 173.36 0.75 18.02.15 3827.03 14.83 168.42 0.752 19.11.14 4140.92 14.67 173.14 0.75 19.02.15 3982.84 14.80 167.11 0.753 02.12.14 4059.81 14.71 170.34 0.74 20.02.15 3918.13 14.82 167.32 0.744 12.12.14 4084.13 14.64 173.18 0.74 22.02.15 3930.40 14.82 166.07 0.745 19.12.14 4090.78 14.53 170.45 0.72 24.02.15 33934.77 14.85 163.98 0.756 20.12.14 4006.27 14.60 170.48 0.72 25.02.15 3907.12 14.82 166.26 0.757 22.12.14 4127.93 14.59 172.00 0.72 27.02.15 3953.34 14.86 165.29 0.758 23.12.14 4112.65 14.23 171.36 0.72 28.02.15 3881.78 14.80 161.74 0.759 26.12.14 4180.44 14.01 174.79 0.71 07.03.15 3971.11 14.81 165.46 0.7510 31.12.14 4090.51 14.15 170.44 0.71 08.03.15 3987.40 14.81 167.30 0.7511 14.01.15 4087.73 14.20 172.11 0.74 09.03.15 3872.34 14.82 169.59 0.7612 16.01.15 4026.86 14.22 172.58 0.73 13.03.15 3874.42 14.81 166.04 0.7513 17.01.15 4059.13 14.26 170.31 0.73 14.03.15 3955.35 14.78 164.80 0.75

Annexure-2 Maximum 2/6/2015 Season 2014-15Sr. # Date N.S pol ratio Mol. Losses R1 Colour B.mass boiling time Hrs.1 30.10.14 32.70 1.444 0 0:002 31.10.14 32.52 1.448 0 8:003 01.11.14 32.60 1.447 0 5:304 02.11.14 32.54 1.447 0 6:125 03.11.14 32.54 1.442 40 4:486 04.11.14 32.39 1.449 35 4:567 05.11.14 32.26 1.441 35 5:348 06.11.14 32.28 1.437 34 5:039 07.11.14 32.28 1.438 33 4:1510 08.11.14 32.16 1.441 30 4:4511 09.11.14 31.91 1.438 29 4:3012 10.11.14 31.89 1.429 28 4:2713 11.11.14 31.64 1.443 29 4:5614 12.11.14 31.51 1.444 29 4:1715 13.11.14 31.22 1.420 27 3:55Average 32.16 1. 440 3 1.73 4:50

MinimumSr. # Date N.S pol ratio Mol. Losses R1 Colour B.mass boiling time Hrs.1 16.02.15 22.90 1.302 38 4:392 17.02.15 22.83 1.281 35 4:303 18.12.15 22.74 1.273 36 4:414 19.02.15 22.50 1.274 33 5:035 20.02.15 22.50 1.286 40 4:456 21.02.15 22.70 1.285 37 4:397 22.02.15 22.75 1.303 35 4:548 23.02.15 22.95 1.320 35 4:429 24.02.15 23.27 1.337 36 4:1510 25.02.15 23.40 1.355 38 4:3711 26.02.15 23.58 1.376 40 4:3612 27.02.15 23.74 1.392 37 4:2713 11.11.14 31.64 1.443 29 4:5614 12.11.14 31.51 1.444 29 4:1715 13.11.14 31.22 1.420 27 3:55Average 32.16 1.440 31.73 4:50


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MinimumSr. # Date N.S pol ratio Mol. Losses R1 Colour B.mass boiling time Hrs.1 16.02.15 22.90 1.302 38 4:392 17.02.15 22.83 1.281 35 4:303 18.12.15 22.74 1.273 36 4:414 19.02.15 22.50 1.274 33 5:035 20.02.15 22.50 1.286 40 4:456 21.02.15 22.70 1.285 37 4:397 22.02.15 22.75 1.303 35 4:548 23.02.15 22.95 1.320 35 4:429 24.02.15 23.27 1.337 36 4:1510 25.02.15 23.40 1.355 38 4:3711 26.02.15 23.58 1.376 40 4:3612 27.02.15 23.74 1.392 37 4:2713 28.02.15 23.77 1.394 36 4:3314 01.03.15 23.84 1.418 34 4:3315 02.03.15 23.90 1.423 35 5:07

Average 23.16 1.330 36 4:37

Matiari Sugar Mills LimitedAnexure3 Mill Performance before & after Auto Cane Feeding & Milling 5/6/2015# Description Year

Before 2007-08 2008-09 2009-10 2010-11 2011-12 2012-13 2013-14 2014-151 TCH 3374 3861 3885 3918 3823 38.43 3993 3944 39342 Loss in Bagasse 0.8334 0.7081 0.7092 0.6652 0.6847 0.701 0.708 0.7228 0.73973 Milling Loss % 5.9 4.8 4.66 4.55 4.77 4.63 4.71 4.92 5.054 Mill Extraction 94.09 95.19 95.34 95.45 95.22 95.36 95.28 95.15 95.125 Imbibition % Cane 42.69 44.14 49.24 47.42 49.04 46.04 43.89 44.06 43.796 Bagasse Pol 2.62 2.18 2.14 2.09 2.13 2.1 2.16 2.21 2.23


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ISOLATION AND CHARACTERIZATION OFNITROGEN-FIXING BACTERIA FROM

SUGARCANE RHIZOSPHEREK. M. Alam, T. Zhang2, Y. Yan2, W. Zhang2, M. Lin2 and W. Lu2

1Bangladesh Sugarcane Research Institute, Ishurdi-6620, Pabna, Bangladesh2Biotechnology Research Institute, Chinese Academy of Agricultural Sci., Beijing 100081, China

ABSTRACTThe seven nitrogen fixing bacterial strains were isolated from rhizosphere soil and surface sterilized roots ofsugarcane. The aimed of this study were to isolate nitrogen-fixing bacteria, determine the nitrogen-fixingability and test the indole acetic acid production. It was found that the five isolated species were distributedin the two genera as Klebsiella and Burkholderia based on 16S rRNA sequence. The PCR amplification ofnifH gene showed that seven produced the expected 360-bp amplification product. The molecularidentification results from 16S rRNA analyses of these bacteria were also corroborated with themorphological and biochemical data. The nifH gene sequences from the strains belonging to Klebsiellashowed that the strains clustered with Klebsiella sp but the nifH gene for Burkholderia was not detected.The ability to fix nitrogen was verified by the acetylene reduction assay and the variation of nitrogenaseactivities were 1.29+0.4 to 29.63+0.3 nmol C2H4/h/mg protein. The highest nitrogenase activity found inGp47, the type strain Klebsiella pneumoniae strain sctcc295; was 29.63+0.3 nmolC2H4/h/mg protein.Diazotrophic strains were assessed for plant-growth-promoting trait such as indole acetic acid production.The highest indole acetic acid production was found in Gp10, the type strain Klebsiella sp. strain zmmowhich was 99.0+7 µg/ml.

Keywords: Sugarcane, Rhizospher, Diazotrophs and Nitrogen fixation

INTRODUCTIONA group of bacteria can be foundin the rhizosphere, at rootsurfaces and in association withroots of plant. Diazotrophicbacteria isolated from therhizosphere, rhizoplane and theinterior of the roots of grasses,cereals and food crops (Franckeet al. 2009). Roots of importantcrops like rice, wheat andsugarcane are frequentlycolonized by nitrogen fixing,plant growth-promoting bacteria(Desnoues et al. 2003).Diazotrophic bacteria have beenlinked with the high nitrogenfixation reported particularly insugarcane where these bacteriawere found in high numbers(Boddey et al. 1995). N2-fixationis one of the possible biologicalalternatives to N-fertilizers andcould lead to more productiveand sustainable agriculturewithout harming the environment(Dobereiner and Urquiaga

1992). The contribution ofbiological nitrogen fixationthrough nitrogen-fixing bacteriato sugarcane cultivar rangedfrom 50 to 80% of the totalnitrogen required by the caneplant (Boddey et al. 1991). Manynitrogen fixing bacteria such asAcetobacter, Bacillus,Azotobacter, Enterobacter,Erwinia, Klebsiella, andAzospirillum are associated withsugarcane.The N2-fixation requires theinteraction of several geneproducts including thenitrogenase structural proteinslike nifD, nifK and nifH. Thephylogeny based on nifH geneshas been shown to resemble the16S rRNA phylogeny (Zehr et al.2003); thus nifH is an idealphylogenetic gene marker forinvestigating N2-fixing organismsin natural environments. Theidentification of nitrogen fixingbacteria and measurement of

their nitrogenase activity hasrevealed that a number ofdiazotrophic species areassociated with grasses andcereals, either as colonizers ofplant rhizospheres orintercellular spaces within thexylem vessel of plants (Jamesand Olivares 1997). Thus thisexperiment was undertaken toisolate and identify the nitrogen-fixing bacteria, determine thenitrogen-fixing ability and testthe plant growth promoting traitof the strains.

MATERIALS AND METHODSIsolation of bacteriaThe rhizosphere soil and rootssamples were collected formsugarcane (variety: Taitan)cultivating area at ShaoguanCity (1130N, 220E), GuangdongProvince, China. Approximately10g of soil sample was takeninto the 250 ml conical flask with90 ml sterilized water. Several


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glass bids were added. Samplemixed thoroughly with the waterand mixture was shaking on ashaker for 200 rpm at 300C. 1 mlsuspension was taken in a vialcontained with 9 ml sterile water.For root sample, 1g of healthyroots were weighed, washedwith distilled water to removeadhering soil particles and againwashed with 3% H2O2 for 3minutes for surface sterilization.Then the roots washed 6-7times with sterile water. Theroots were finely ground with thepestle and motor with 99 mlsterile water and the mixturewere vortex 2 to 3 minutes. Forboth samples, all procedureswere done separately. Serialdilutions of the suspension weremade up and formed dilutionseries of 10-2 to 10-7. These wererepeated for three times. Then0.1 ml of each suspension werespread in nitrogen free (NFb)minimal lacted media. Thestrains were sub-cultured on N-free LGIM media (Estrada et al.2002) and PCAT media(Burbage and Sasser 1982).Single colonies were visible afterculturing for 48 h. They wereisolated, tested for purity andstored at -800 C.

Morphology of the strainsBacterial growth fromnitrogenase-positive vials, with awhite or yellowish pellicle 1-4mm below the surface, werestreaked on LGIM agar platesand incubated for 72 h. The finalstreaking and purification wereperformed on Burkholderiagenus-specific PCAT andPseudomonas agar F. Theflagella and morphology wereobserved on a transmissionelectron microscope. The gramstain reactions were observedon an optical microscope.

Physiological andbiochemical testsBacterial motility tested bygrowth in a semisolid 0.3%

mannitol motility test medium.Oxydase and catalase weredetermined using commerciallyavailable discs. Utilization ofcarbon sources were examinedusing the Biolog system. Allstrains were tested three timeswith GN2 microplates accordingto the Manufacturer’srecommendation, with reactionsobserved after 24 or 48 h. Forthe enzymeatic activity of theisolates, api ZYM test kit wasused (BIOMERIEUX, Inc).Phenotypic characterizations ofthe strains were performedaccording to the Bergey’sManual of DeterminativeBacteriology (Buchanan andGibbons 1984) and Manual ofCommon SystematicDeterminative Bacteriology(Dong and Cai 2001).Acetylene reduction assay andcolorimetric estimation ofindoleacetic acid productionThe nitrogenase activity of theisolates was carried out byacetylene reduction assay. The10 fold serial dilutions of thesuspension were used toinoculate N-free semisolid LGIMmedia in triplicate (Estrada et al.2002). 1% acetylene gas wasinjected. After 96 h ofincubation, vials were assayedfor acetylene reduction activity(Mascarua-Esparza et al. 1988).Nitrogenase positive isolateswere only used formorphological, physiological andbiochemical tests. For proteinconcentration, the bacterialmixture was determined bystandard method (Bradford1976). Indoleacetic acidproduction was estimated bygrowing the isolates in NFbmedium supplemented withNH4Cl and 100 µg/ml DL-tryptophan at 30°C with shakingfor 72 h in the dark. Five milliliterof each culture was centrifuged(20 min, 6,000×g), andindoleacetic acid production wasmeasured as indolic compoundsin 2 ml of supernatant by mixing

with 2 ml of Salkowski reagentand following the absorbance at535 nm after 30 min in the dark(Glickman and Dessaux 1995).A standard curve was used forcalibration.

DNA extractionFor the genomic DNA extraction,the isolates were grown innutrient agar medium at 280C for24 h and centrifuged at12,300×g. The pellets werewashed with TE buffer andresuspended in 10 ml of TE (1×)containing 3 ml of 5% SDS in TE(1×) and 3 ml of proteinase K(2.5 mg/ml). The suspensionswere incubated 370 C for 1 h.After 1 h, the cleaned lysateswere extracted with phenol:chloroform: isoamyl alcohol(25:24:1). DNA wereprecipitated by adding 0.1volume of 3 M sodium acetate(pH 5.2) and 2.5 volumes ofethanol to the supernatant. Thedried pellets were dissolved inTE (1×) buffer (Govindarajan etal. 2007).

PCR amplification andsequencing of 16S rRNAgenesThe 16S rRNA genes wereamplified by PCR usinguniversal primers F27 (5′-AGAGTTTGATCATGGCTCAG-3′) and R1492 (5′-TACGGTTACCTTGTTACGACTT-3′). The PCRamplifications were carried outin 50 µl reaction volumes withddH2O, 10×buffer, 2.5 mmol/ldNTP, 10µmol/l primer, DNAtemplate and Pyrobest TM DNApolymerase. The reaction wasset up as follows: initialdenaturation at 95°C for 4 min.Each of the 30 cycles iscomposed of a denaturationstep of 94°C for 30 s, anannealing step of 56°C for 40 s,and an extension step of 72°Cfor 2 min, and the last cycle wasfollowed by a final extension at72°C for 6 min. Products were


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visualized on a 1.5% agarosegel in 1×TBE buffer. Theamplicons were purified with aPCR purification kit. The purifiedPCR products were sequencedbi-directionally with F27 andR1492 by TsingkeBiotechnology, Beijing. TheBLAST at the National Centrefor Biotechnology Information(NCBI) was used for alignmentanalysis of the nucleotidesequences with the informationfrom GenBank/EMBLdatabases.PCR amplification andsequencing of the nifH genesThe sequences of the nifHforward and reverse primerswere, PolF-I 5′-TGCGAICCSAAIGCIGACTC-3′and AQERGC 5′-CGCCCGCCGCGCCCCGCGCCCGGCCCGCCCGACGATGTAGATYTCCTG-3′ (Diallo et al. 2004),respectively. Amplificationreactions were performed in atotal volume of 25 µl. Thereaction mixture contained 2.5µl10×PCR buffer, 2.5µl each of 2mMdNTP, 3µl of each forwardand reverse primer (30 ng), 1µlof template DNA (10 ng), and0.3µl of Taq DNA polymerase (3units/µl), to a final volume of25µl with milli-Q water. Thestep-up PCR procedure includeddenaturation at 940C for 1 min,560C for 30 sec, and 720C for 30sec, followed by 30 cycles of940C for 3 min, 720C for 5 min.Amplification products wereelectrophoreses on a 1.5%agarose gel in 1×TBE buffer.The amplicons were purifiedwith a PCR purification kit. Thepurified PCR products weresequenced by TsingkeBiotechnology, Beijing. Theobtained sequences werecompared with the sequences inthe database of GenBank (www.ncbi.nlm.nih.gov) using BLASTsoftware to acquire the nifHgene sequence of similar typestrains.Cloning

The PCR products wereseparated by electrophoresis in1.5% (w/v) agarose gel(Invitrogen, UK); the band wasexcised and purified using aQIAquick® Gel Extraction kit(QIAGEN, Germany). Therecovered DNA was cloned intothe pGEM-T Easy Vector(Promega, USA) according tothe manufacturer’s protocol.Four white colonies werechosen at random, transferred toa new plate, and incubatedovernight. Small aliquots fromfour white colonies from eachtransformation experiment werechosen and transferred to thePCR mixture containing thevector primers T7 and SP6.Clones containing the desiredinsert DNA were identified byagarose electrophoresis.Plasmids containing theappropriate inserts were isolatedfrom 2 ml of bacterial culturesusing the QIAprep® SpinMiniprep kit, according to themanufacturer’s instructions(QIAGEN). Purified plasmidswere commercially sequenced(Eurofin MWG, Germany) inboth directions. The nucleotideidentity of all the sequencedclones were compared to theGenBank database(http://blast.ncbi.nih.gov) onlineto obtain the representative thatrelated most closely to 16SrRNA and nifH genes in thedatabase.DNA sequencing andphylogenetic analysisDNA sequences were alignedusing the multiple alignmentsCLUSTAL W software(Thompson et al. 1994) with 16SrRNA gene sequences of typestrains involved in the samegenus of the isolates as a resultof previous GenBank sequencecomparisons. The evolutionarydistance was determined byconstruction of a phylogenetictree using the MaximumComposite Likelihood method in

the MEGA 4 program (Tamuraet al. 2007).Statistical analysesAll assays were performed in atleast triplicate. Basic statisticalparameters and analyses ofvariance (ANOVA) wereperformed using commercialstatistical software (MSTAT).Differences with P values of<0.05 were consideredstatistically significant.Nucleotide sequenceaccession numbersThe nucleotide sequencesdetermined in this study hasbeen deposited in the GenBankdatabase under the followingaccession numbers: 16S rRNA(KC907121, KC907122,KC907123, KC907124,KC907125, KC907126 andKC907127).RESULTSMorphological characteristics ofN2-fixing bacteria After 72 hoursof incubation of diazotrophicbacteria, colonies on LGIM agarplates were large, yellow andround with entire margin.Colonies on PCAT agar plateswere white, small, round,smooth and convex with entiremargin.Physiological andbiochemical characteristicsThe strains of all were capableof fermenting lactose and gaveoxydase negative and catalasepositive results (Table 1).The strains were non motile,reduced nitrate and indole testgave negative. The strains werecapable of lysin decarboxylation.The system for the reactions ofenzymeatic activities of theisolates was positive andnegative in the presence ofdifferent substrates (Table 2).The data from physiologicalcharacterization and Biologsystem were compared withstandard species using Bergey’sManual of DeterminativeBacteriology. Based on thesephysiological and biochemicalcharacteristics, the isolates were


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identified to the genera ofKlebsiella and Burkholderia.Nitrogenase activity andindole acetic acid (IAA)productionAfter 96 hours of incubation,vials were assayed for acetylenereduction. The nitrogenaseactivities of the strains were1.29+0.4 to 29.63+0.3 nmolC2H4/h/mg protein (Table 3).The strains were further testedfor IAA production. All strainsgrown in the minimal mediumsupplemented with tryptophanexhibited considerableproduction of IAA with amountranging from 41.5+0.5 to 99.0+7µg IAA/ml (Table 3).Phylogenetic position of theN2-fixing bacteriaPCR amplification of the 16SrRNA gene allowed thenucleotide sequence of about1450 bp of the 16S rRNA genefrom each of the seven isolatedstrains to be determined. ABLASTN search of the 16SrRNAsequences database oftype strains implemented by theEzTaxon server revealed theclosest type strain affiliated toeach of the studied strains(Table 3).Among the isolated strains ofbacteria, all of them wereincluded into two genera suchas Klebsiella, and Burkholderia.The 16S rRNA gene nucleotidesequences of three isolates(Gp7, Gp12 and Gp22) showedhigh similarity (97.34-97.86%) tothe 16S rRNA gene sequencesof type strain Klebsiellapneumoniae strain VD (Table 3)but other one (Gp10) showedhigh similarity (97.89%) to thetype strain Klebsiella sp. strainzmmo and the another one(Gp47) gave high similarity(97.89%) to the type strainKlebsiella pneumoniae strainsctcc295. In Burkholderia, thestrain (Gp31) showed highsimilarity (96.36%) to the 16SrRNA gene sequences of typestrain Burkholderia cepacia

isolate WS11.7. The phylogene-tic position based on the 16SrRNA gene sequences of theisolates was shown in Fig. 1.nifH gene characterizationand phylogenetic analysisPCR amplification of nifH geneshowed that isolates Gp7, Gp10,Gp12, Gp22, Gp40 and Gp47produced the expected 360 bpamplification band. These bandswere a coding gene of Fe-protein of nitrogenase, whichproved that these bacteriabelonged to nitrogen-fixingbacteria. The nifH sequence ofthe six isolates had beensubmitted to the GenBankdatabase of NCBI. Thephylogenetic tree constructed bynifH sequence of the isolates(Gp7, Gp10, Gp12, Gp22, Gp40and Gp47) and the high similarsequences in the GenBank,were illustrated in the fig. 5.Sequence similarities of theisolates of nifH genes were asfollows: Gp7 of Klebsiella sp.CRPV0611a, 98.35%; Gp10 ofKlebsiella sp. CRLI0728,99.00%; Gp12 of Klebsiella sp.CRPV0611a, 99.34%; Gp22 ofKlebsiella sp. CRPV0611a,99.01%; Gp40 of Klebsiella sp.CRLI0728, 99.32% and Gp47 ofKlebsiella sp. CRLI0728,99.32%. A phylogenetic genetictree constructed based on nifHgene nucleotides revealed thatnifH gene sequences fromisolates belonging to Klebsiellaspecies were clustered withKlebsiella sp. CRPV0611a andKlebsiella sp. CRLI0728 (Fig. 2).DISCUSSIONIsolation and morphologicalcharacteristics of strainsAfter incubation of the N2-fixingbacteria, it were found thatcolonies on LGIM agar plateswere large, yellow and roundwith entire margin. Colonies onPCAT agar plates were white,small, round, smooth andconvex with entire margin. Onthe basis of colony observation,our results were supported by

Govindarajan et al. (2007). Inmicroscopic observation, strainswere gram negative and rodshaped. The diazotrophicbacteria count (MPN) wascarried out. The numbers ofbacteria in the rhizosphere soilwere of 1.3-2.2×103 CFU/g ofdry weight and then the valuesfound roots sample were of 0.1-1.5×103 CFU/g of dry weight.Interestingly, it was showed thatmicrobiological activity wasmuch greater in sugarcanerhizosphere (Freitas et al. 1981)as well as at that time bacteriahave been reported insugarcane roots (Patriquin et al.1980). The concept of biologicalnitrogen fixation has led toinvestigate a variety of free livingnitrogen-fixing bacteria capableof association with the rootsystem of graminaceous plants,such as Klebsiella pneumoniae,Azotobacter vinelandii, andAzospirillum brasilense(Elmerich and Newton 2007). InEgypt, Hegasi et al. (1979)found that Klebsiella spp.abundant in the rhizosphere ofsugarcane, this finding isconsistent with our result. Onthe other hand, the physiologicalactivity and genetic diversity ofendophytic Burkholderia spp.isolated from sugarcane rootswas grown in the field in Brazil(Luvizotto et al. 2010). Caballeroet al. (2004) reported thatBulkhorderia unamae isolateshave been recovered fromsugarcane plants in Mexico anda few Bulkhorderia tropicaisolates have been found fromwithin sugarcane stemscultivated in Brazil (Reis et al.2004) but our study Bulkhorderiacepacia was isolated fromsugarcane root.Physiological andbiochemical characteristicsBased on physiological andbiochemical tests, almost allisolates of rhizosphere and rootsfrom sugarcane were identifiedas Klebsiella and Bulkholderia.


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In accordance withGovindarajan et al. (2007), whoworked on diazotrophic bacteriaKlebsiella sp. GR9 of India,Klebsiella and Burkhoderia aresaid to be gram negative,catalase positive and able tocatabolize D-glucose andcarbohydrates. The strains werereduced the nitrate. Nitrate canserve as the sole nitrogensource for plants, fungi andbacteria (Janine et al. 1993).Kiebsiella pneumoniae can usenitrate and nitrite as solenitrogen sources during aerobicgrowth. Nitrate can also serveas an electron acceptor foranaerobic respiration inenterobacteria (Stewart 1988).In Burkhoderia cepacia NH-17,the conversion of NO2

- to N2Oand NO3

- occurredconcomitantly with a decrease inNO2

- under aerobic conditions ingrowing culture.Nitrogenase activity andindole acetic acid productionKlebsiella isolates were differedin their ability to reduceacetylene in pure culture.Acetylene reduction assays(ARA) of the isolates weretested and it was recorded from8.57+0.4 to 29.63+0.3 (Table 3).The isolate Gp47, the Klebsiellapneumoniae strain sctcc295showed the highest nitrogenaseactivity of 29.63+0.3. The typestrain Klebsiella variicola strainHUB-IV-005 (Gp40) showed thelowest nitrogenase activity of8.57+0.4. In our study, amongthe Klebsiella species strainsGp7 and Gp10 showedstatistically similar nitrogenaseactivity. On the other hand,strains Gp12, Gp22 and Gp 40showed similar nitrogenaseactivity but different from otherstrains. Klebsiella naturallyoccurs in the soil, and about30% of strains can fix nitrogen inanaerobic conditions (Postgate1998). Kielo et al. (1983) statedthat anaerobic conditions (pO2value of < 0.002) were required

for maximum nitrogenaseactivity with the facultativeanaerobic bacteria Klebsiellapneumoniae and the maximumnitogenase activities (expressedas micromoles activity: milligramof bacterial protein/hour) notedduring the exponential growththat was 2.41. Another reportfrom Liu et al. (2011) stated thatthe acetylene reduction assay ofK. pneumoniae NG14 was 47.56nmol C2H4/ml/h, which wasisolated on the root surface ofrice. Measurement ofnitrogenase activity of Gp31, thetype strain Burkholderia cepaciaisolate WS11.7 was 1.29+0.4(Table 3). Burkholderia could fixatmospheric nitrogen in legumesand cereal crops (Chen et al.2006); and Burkholderia cepaciagot entry inside the root,indicating endophyticcolonization pattern (Sharma etal. 2008). Details regarding theextent of Burkholderia cepacianitrogen fixation in relation to thecolonization peculiarities of thesugarcane roots are still lackingbut the acetylene reduction bythe various isolates ofBurkholderia vietnamiensis wereconstant (44 to 68 nmol/ha inacetylene production rate) in softgel medium containing 0.2%sucrose (Sui et al. 2010). Ourfindings might be the first reportabout nitrogen fixing abilities ofBurkholderia cepacia isolatedfrom sugarcane roots.The nitrogen fixing isolates usedin this study were capable of IAAproduction. Growth media fromall of the isolates containedsubstantial amounts of thephytohormone indoleacetic acid.Considerably higher amounts ofIAA were produced in thepresence of tryptophan (100µg/ml). Isolate Klebsiella sp.strain zmmo (Gp10) producedthe highest amount of IAA was99.0+7 IAA µg/ml (Table 3), ithas more similarity with strainsGp22 (95.0+1 µg/ml) and Gp40(96.0+2 µg/ml). The three

strains Gp12, Gp47 and Gp31produced the statistical identicalIAA of 88.0+0, 88.0+2 and85.0+1µg/ml, respectively andleast IAA produced by Gp7 was41.5+0.5 µg/ml. According to theIAA production, it was found thatthe capacity to synthesize IAA iswide-spread among soil plant-growth-promoting bacteria suchas Azospirillum spp. Klebsiellasp. and Enterobacter cloace(Spaepen et al. 2007). It hasbeen reported that theproduction of IAA play a role inregulating plant development,including processes thatdetermine root architecture,such as root pole establishmentduring early embryogenesis,root meristem maintenance, rootgravitropism and lateral rootorganogenesis (Kramer andBennett, 2006). IAA produced bybacteria in host plant, enhancesroot branching and rootelongation, which helps touptake of soil water andminerals of the plant(Steenhoudt and Vanderleyden2000). In recent advances it wasfound in transcriptome analysesin A. brasilense revealed thatthe IAA biosynthesis led totranscriptional changes in thebacteria and suggesting IAA isan important signalling moleculeinvolved in the plant-PGPRcommunication process (VanPuyvelde et al. 2011; Santi et al.2013).Phylogenetic analysis of nifHgenePCR amplification of nifH genesconfirmed that the presence ofthe structural nitrogenase genein all of the isolates. The highlyconserved nature of the nifHgene makes it an idealmolecular tool to determine thepotential for biological nitrogenfixation (Zehr and Capone1996). Designing of PCRprimers that amplified the nifHgene and sequencing theamplified genes can identify theassemblage of microorganisms


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capable of nitrogen fixation inany environment. In our study,according to the nifH genes, theisolated sequences formed twodistinct clusters, one clusterincludes the sequences ofGp10, Gp40, Gp47 nifH and thesequences of Gp7, Gp12, Gp22were assigned to the othercluster, these findings weresupported by Liu et al. (2011).Klebsiella pneumoniaecontained a total of 20 nif geneslocated on the bacteria'schromosome in a 24 kb regionand nif genes as nifH, nifK andnifD encoded the nitrogenase’ssubunits of the bacteria Thephylogenetic analyses indiazotrophs use as query theamino acid sequence of thewell-studied Mo-dependentnitrogenase reductase alsoknown as iron protein coded bynifH (Cummings et al. 2009).Genes coding for nifH orproteins similar to nifH havebeen found in all knowndiazotrophs with sequencedgenomes. The strict requirementof nifH in biological nitrogenfixation and its universalpresence in diazotrophs hasresulted in this protein servingas a sequence tag for theidentification of nitrogen fixers.Phylogenetic position of theN2-fixing bacteriaOur study reported that sevennitrogen fixing strains wereisolated; six as Klebsiella fromrhizosphere soil and one asBulkholderia from roots ofsugarcane. Analysis of a largenumber of prokaryotic genomeshas shown that evolutionaryrelationships among prokaryotesare generally represented by16S rRNA gene sequences(Stackebrandt et al. 2002). 16SrRNA gene sequences

contained hyper variable regionsthat can provide species-specificsignature sequences useful forbacterial identification. As aresult, 16S rRNA genesequencing has becomeprevalent in as a rapid, accurate,alternative to phenotypicmethods of bacterialidentification (Clarridge 2004).Although it was originally usedto identify bacteria, 16S rRNAsequencing was subsequentlyfound to be capable ofreclassifying bacteria intocompletely new species, or evengenera. In the present study, thediazotrophic isolates wereclassified based on phylogeneticposition of their 16S rRNA genesequences, as members ofKlebsiella pneumoniae,Klebsiella sp., Klebsiellavariicola and Burkholderiacepacia species. The presenceof nifH genes and their ability fornitrogen-fixation, as judged bythe ARA assay, confirmed thegrouping of all the isolates asdiazotrophs. In respect ofphylogenetic position of thebacteria showed that the 16SrRNA gene nucleotidesequences of isolate Gp47exhibit a striking similarity(>97%) to the 16S rRNA genenucleotide sequences of theKlebsiella pneumoniae strainsctcc295 and Klebsiella sp.strain zmmo, while the 16SrRNA gene nucleotidesequences of the isolate Gp10exhibit a divergence close to thethreshold (97-98.7% identity) forassigning two organisms to thesame species (Stackebrandt etal. 2002; Stackebrandt andEbers 2006) but the constructedphylogenetic tree of 16S rRNAgene clearly shows that the 16SrRNA gene of the isolate Gp10

cluster in a separate branch(Fig. 1). The phylogenetic treesof 16S rRNA gene suggests thatisolate Gp10 may represent anew species of the genusKlebsiella, adding one more tothe increasing the list ofnitrogen-fixing Klebsiella species(Doty et al. 2009; Mehnaz et al.2009; Yim et al. 2009; Venierakiet al. 2011).CONCLUSIONIt was established that membersof the genus Klebsiella andBurkholderia predominate in thenitrogen-fixing bacterialcommunity associated with therhizosphere and roots of field-grown sugarcane. Bergey’sManual of DeterminativeBacteriology and 16S rRNAgene sequence analysis, thenitrogen fixing strains werebelonged to the Klebsiella andBurkholderia. Presence of nifHgenes and measurement ofnitrogenase activity proved thatKlebsiella bacteria can fixnitrogen but Burkholderia wasnot prominent. The highestnitrogenase activity found inGp47, the type strain Klebsiellapneumoniae strain sctcc295;was 29.63+0.3 nmolC2H4/h/mgprotein. Diazotrophic strainswere assessed for as indoleacetic acid production. Thehighest indole acetic acidproduction was found in Gp10,the type strain Klebsiella sp.strain zmmo which was 99.0+7µg/ml. Hopefully, the nitrogen-fixing bacteria isolated in thisstudy will be used in our furtherresearch for improvingassociation between crops anddiazotrophs and for developingefficient biofertilizers to reducethe need for chemical Nfertilizer.


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REFERENCESBoddey, R.M., O.C. Oliveira de, S. Urquiaga, V.M. Reis, F.L. Olivares, V.L.D. Baldani, and J.Dobereiner. 1995. Biological nitrogen fixation associated with sugarcane and rice: contributions andprospects for improvement. Plant Soil. 174: 195–209.Boddey, R.M., V.M. Uruiaga, and J. Dobereiner. 1991. Biological nitogen fixation with the sugarcane.Plant Soil. 137: 111-117.Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities ofprotein utilizing the principle of protein-dye binding. Anal Biochem. 72: 248-254.Buchanan, R.E., and N.E. Gibbons. 1984. Bergey’s Manual of Determinative Bacteriology. 8th edition, p345-347, Science and Technology Press, Beijing.Burbage, D.A., and M. Sasser. 1982. A medium selective for Pseudomonas cepacia. Phytopathol.72:706.Caballero, S., Xavier, and A. Boch. 2004. Rotavirus virus-like particles as surrogates in environmentalpersistence and inactivation studies. Appl Environ Microbiol. 1-76.Chen, W.M., E.K. James, T. Coenye, J.H. Chou, E. Barrios, S.M. Faria de, G.N. Elliott, S.Y. Sheu, J.I.Sprent, and P. Vandamme. 2006. Burkholderia mimosarum sp. nov., isolated from root nodules ofMimosa spp. from Taiwan and South America. Int J Syst Evol Microbiol. 56: 1847-1851.Clarridge, J.E. 2004. Impact of 16S rRNA gene sequence analysis for identification of bacteria onclinical microbiology and infectious diseases. Clin Microbiol Rev. 17 (4): 840-862.Cummings, S.P., P. Gyaneshwar, and P. Vinuesaet. 2009. Nodulation of Sesbania species byRhizobium (Agrobacterium) strain IRBG74 and other rhizobia. Environ Microbiol. 11: 2510-2525.Desnoues, N., M. Lin, X. Guo, L. Ma, R. Carren˜ o-Lopez, and C. Elmerich. 2003. Nitrogen fixationgenetics and regulation in a Pseudomonas stutzeri strain associated with rice. Microbiology. 149: 2251-2262.Diallo, M.D., A. Willems, N. Vloemans, S. Cousin, T.T.V anderckhove, P. Lajudie de, W. Vyverman, M.Gillis, and K. Van der Gucht. 2004. Polymerase chain reaction denaturing gradient gel electrophoresisanalysis of the N2-fixing bacterial diversity in soil under Acacia tortillis ssp. raddiana and Balanitesaegyptiaca in the dryland part of Senegal. Environ Microbiol. 6: 400-415.Dobereiner, J., and S. Urquiaga. 1992. Soil biology and sustainable agri. An Acad Bras Sci 84: 127-133.Dong, X.Z., and M.Y. Cai. 2001. Manual of common systematic determinative bacteriology, p 353-386.Sci & Technol Press, Beijing.Doty, S.L., B. Oakley, G. Xin, J.W. Kang, G. Singleton, Z. Khan, A. Vajzovits, and J.T. Staley. 2009.Diazotrophic endophytes of native black cotton and willow. Symbiosis. 47: 23-33.Elmerich, C., and W.E. Newton. 2007. Associative and endophytic nitrogen fixing bacteria andcyanobacterial associations. Springer, Dordrecht, The Netherlands.Estrada, P., P. Mavingui, B. Counoyer, F. Fontaine, J. Balndreau, and J. Cabellero-Mellado. 2002. Anitrogen fixing endophytic Burkholderia sp. associated with maize plants cultivated in Maxico. Can JMicrobiol. 48:285-294.Francke, C., K. Lindstrom, and C. Elmerrich. 2009. Nitrogen-fixing bacteria associated with leguminousand non-leguminous plants. Plant Soil. 321:35-59.Freitas, JR., A.P. Ruschel, and P.B. Vose. 1981. Radiorespirometry studies as an indication of soilmicrobial activity in relation to root system in sugarcane comperision with other species. In AssociativeN2-fixation, eds. P.B.Vose, and A.P. Ruschel, Vol II, CRC Press, Boca Raton, Florida, 141-144.Glickman, E., and Y. Dessaux. 1995. A critical evaluation of the specificity of Salkowski reagent forindole compounds produced by phytopathogenic bacteria. Appl Environ Microbiol. 61:793-796.Govindarajan, M., S.W. Kwon, and H.Y. Weon. 2007. Isolation, molecular characterization and growth-promoting activities of endophytic sugarcane diazotroph Klebsiella sp. GR9. World J MicrobiolBiotechnol. 23: 997-1006.Hegazi, N.A., N. Eid, R.S. Faraq, and M. Monib. 1979. Assymbiotic N2-fixation in the rhizosphere ofsugarcane planted under semi-arid condition in Egypt. Rev. Ecol. Biol. Sol. 16: 23-37.James, E.K., and F.L. Olivares. 1997. Infection and colonization of sugarcane and other graminaceousplants by endophytic diazotrophs. Crit Rev Olant Sci. 17: 77-119.Janine, T.L., S.G. Barry, and S. Valley. 1993. Structures of genes nasA and nasB, encodingassimilatory nitrate and nitrite reductases in Klebsiella pneumoniae M5al. J Bacteriol. 175: 2370-2378.


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Kielo, H., K. Kirsti, and S. Veronica. 1983. Nitrogenase activity (acetylene reduction) of root-associatedcold climate Azospirillum, Enterobacter, Klebsiella, and Pseudomonas species during growth on variouscarbon sources and at various partial pressures of oxygen. App Environ Microbiol. 45: 563-570.Kramer, E.M., and M.J. Bennett. 2006. Auxin transport: a field in flux. Trends in Plant Science 11: 382-386.Liu, Y., H. Wang, X. Sun, H. Yang, Y. Wang, and W. Song. 2011. Study on mechanisms of colonizationof nitrogen-fixing PGPB, Klebsiella pneumoniae NG14 on the root surface of rice and the formation ofbiofilm. Curr Microbiol. 62: 1113–1122.Luvizotto, D.M., J. Marcon, F.D. Andreote, F. Dini-Andreote, A.A.C. Neves, W.L. Araujo, and A.A.Pizzirani-Kleiner. 2010. Genetic diversity and plant-growth related features of Burkholderia spp. fromsugarcane roots. World J Microbiol Biotechnol. 26: 1829–1836.Mascarua-Esparza, M.A., R. Villa-Gonzallez, and J. Caballero-Melado. 1988. Acetylene reduction andindolacetice acid production by Azospirillum isolates from cactaceous plants. Plant Soil. 106: 91-95.Mehnaz, S., B. Weselowski, F. Aftab, S. Zahid, G. Lazarovits, and J. Iqbal. 2009. Isolation,characterization, and effect of fluorescent pseudomonads on micropropagated sugarcane. Can JMicrobiol. 55: 1007-1011.Patriquin, D.G., L.A. Graciolli, and A.P. Ruschel. 1980. Nitrogenase activity of sugarcane propagatedfrom stem cutting in sterile vermiculite. Soil Biol Biochem. 12: 413-417.Postgate, J. 1998. Nitrogen Fixation, 3rd Edition. Cambridge University Press, Cambridge UK.Reis, V.M., P. Estrada-de los Santos, and S. Tenorio-Salgado et al. 2004. Burkholderia tropica sp.nov., a novel nitrogen-fixing, plant-associated bacterium. Int J Syst Evol Microbiol. 54: 2155-2162.Santi, C., D. Bogusz, and C. Franche. 2013. Biological nitrogen fixation in non-legume plants. Annals ofBotany. 1-25.Sharma, S, S. Sharma, R.K. Singh, and A. Vaishampayan. 2008. Colonization behavior of bacteriumBurkholderia cepacia inside the Oryza sativa roots visualized using green fluorescent protein reporter.World J Microbiol Biotechnol. 24: 1169–1175.Spaepen, S, J. Vanderleyden, and R. Remans. 2007. Indole-3-acetic acid in microbial andmicroorganism-plant signaling. FEMS Microbiol Rev. 31: 425-448.Stackebrandt, E., and J. Ebers. 2006. Taxonomic parameters revisited: tarnished gold standards.Microbiol Today. 33: 152-155.Stackebrandt, E., W. Frederiksen, G.M. Garrity, P.A.D. Grimont, P. Kämpfer, M.C.J. Maiden, and X.Nesme et al. 2002. Report of the ad hoc committee for the revaluation of the species definition inbacteriology. Int. J. Syst. Evol Microbiol. 52: 1043-1047.Steenhoudt, O., and J. Vanderleyden. 2000. Azospirillum, a free-living nitrogen-fixing bacterium closelyassociated with grasses: genetic, biochemical and ecological aspects. FEMS Microbiol Rev. 24: 487-506.Stewart, V. 1988. Nitrate respiration in relation to facultative metabolism in enterobacteria. MicrobiolRev. 52: 190-232.Sui, Y.T., H. Shintaro, M. Lulie, J.G. Kah, and H. Yasuyuki. 2010. Burkholderia vietnamiensis isolatedfrom root tissues of Nipa palm (Nypa fruticans) in Sarawak, Malyasia, proved to be its major endophyticnitrogen fixing bacterium. Biosci Biotechnol Biochem. 74: 1972-1975.Tamura, K., J. Dudley, M. Nei, and S. Kumar. 2007. MEGA4: Molecular Evolutionary Genetics Analysis(MEGA) software version 4.0. Mol Biol Evol. 24: 1596-1599.Thompson, J.D., D.G. Higgins, and T.J. Gibson. 1994. CLUSTAL W: improving the sensitivity ofprogressive multiple sequence alignment through sequence weighting, position-specific gap penaltiesand weight matrix choice. Nucleic Acids Res. 22: 4673–4680.Van Puyvelde, S., L. Cloots, and K. Engelen et al. 2011. Transcriptome analysis of the rhizospherebacterium Azospirillum brasilense reveals an extensive auxin response. Microbial Ecology. 61: 723-728.


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Table-1 Physiological and biochemical characteristics of the isolated strainsCharacteristics Gp7 Gp10 Gp12 Gp22 Gp31 Gp40 Gp47Lactose fermentation + + + + + + +Indole test - - - - - - -Methyl Red-VogesProskauer test + + + + + + +Lysin decarboxylase + + + + + + +Motility - - - - - - -Nitrate reduction + + + + + + +Production of oxydase - - - - - - -Production of catalase + + + + + + +Growth on:D-Fructose + + + + + + +D-Galactose + + + + + + +α-D-Glucose + + + + + + +α-D-Lactose + - + + - + -Maltose + + + + - + +D-Mannitol + + + + - + +L-Rhamnose + + + + + + +Sucrose + + + + + + +Glycerol + + + + + + +D-Sorbitol + + + + + + +

Table-2 Test of enzymatic activities of the four isolatesNo. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Gp7 1 3 2 5 1 5 3 2 1 1 4 3 1 1 0 5 4 1 1 0Gp10 1 3 2 4 1 5 3 2 0 1 4 5 1 1 1 5 3 1 0 1Gp12 1 3 2 5 1 5 3 2 1 1 4 3 1 1 0 5 4 1 1 0Gp22 1 3 2 5 1 5 3 2 1 1 4 3 1 1 0 5 4 1 1 0Gp31 1 1 1 1 1 5 2 1 1 1 2 2 1 1 1 3 1 1 1 1Gp40 1 3 2 4 1 5 3 2 1 1 4 5 1 1 1 5 3 1 0 1Gp47 1 2 2 4 1 5 3 2 1 1 5 5 1 1 1 5 4 1 1 11-20 indicated enzyme assayed with substrates (api ZYM REF 25 200, BIOMERIEUX, inc.). 0 and 1 indicatednegative reactions while 2, 3, 4 and 5 as positive reactions.

Table-3 Bacterial strains isolated from rhizosphere and roots of sugarcaneStrains Isolated

partsType strainsa Highest

mach (%)nmol C2H4 /h/ mg

proteinIAA µg/ml

Gp7 Soil Klebsiella pneumoniae strain VD 97.69 15.02+2.0 b 41.5+0.5 cGp10 Soil Klebsiella sp. strain zmmo 97.85 15.14+1.0 b 99.0+7 aGp12 Soil Klebsiella pneumoniae strain VD 97.34 10.02+1.0 c 88.0+0 bGp22 Soil Klebsiella pneumoniae strain VD 97.86 9.53+3.2 c 95.0+1 aGp31 Root Burkholderia cepacia isolate WS11.7 96.36 1.29+0.4 d 85.0+1 bGp40 Soil Klebsiella variicola strain HUB-IV- 005 96.31 8.57+0.4 c 96.0+2 aGp47 Soil Klebsiella pneumoniae strain sctcc295 97.89 29.63+0.3 a 88.0+2 baIdentification based on cultural characteristics, colony morphology and 16S rRNA gene sequence.


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Fig. 1 A phyllogenetic tree based on the 16S rRNA gene sequence of 17 isolates and other typesstrains of Bacteria.Bootstraps values (percentage of 1000 replicates) are shown at the nodes. Bar denotes 0.02substitutions per nucleotides position.

Gp47Klebsiella pneumoniae str. sctcc295(HQ622341)Klebsiella sp. str. zmmo

(U31075)

Gp10Gp40Klebsiella variicola str. HUB-IV-005

(JN848785)Klebsiella pneumoniae str. VD(HQ857583)

Gp12Gp7Gp22

Klesiella pneumoniae(X87276)Klebsiella rhinoscleromatis

(AF009169)Burkholderia sp. SWF66044(FJ648692)Burkholderia gladioli

(X67038)Burkholderia mallei str. ATCC 23344 (AF110188)

Burkholderia vietnamiensis(U96928)Burkholderia cepacia str. ATCC25416(AF097530)

Gp31Burkholderia cepacia iso. WS11.7

(AF311972)

97

76

93

68

90

92

88

100

48

62

52

41

75

32

34

0.02


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Fig. 2 A phyllogenetic tree based on the nifH gene sequence of 6 isolates and other types strains ofKlebsiella spp.Bootstraps values (percentage of 1000 replicates) are shown at the nodes. Bar denotes 0.005substitutions per nucleotides position

Klebsiella pneumoniae str. NG14(HQ40430)

Klebsiella variicola str. 801(AY367395)

Klebsiella pneumoniae 342(AY242355)

Klebsiella sp. CRLI0728(FJ593762)

Gp40

Gp10Gp47

Klebsiella sp. CRPV (FJ593768)

Gp7

Gp12

Gp22

90

100

52

99

63

93

775

9

0.005


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SUGAR INDUSTRY ABSTRACTS

1.2.3.4.5.6.7.8.9.10.11.12.13.14.15.16.17.18.19.20.21.22.

FACTORY REMOTE ACCESS USING SMART DEVICES

GM Peatey, JF Knight, I Messenger

Maryborough Sugar Factory (MSF) runs a low cost milling operation. We continually strive to achievechanges in cost competitiveness by significantly improving the way employees go about their normal dayto day work through the use of technology. Investigations were undertaken to identify how the existingcomputer network could be adapted to provide remote access throughout the majority of the mill site, andthen how this same access could be extended to off-site usage. This included the selection and locationof WiFi devices, smart devices and software. The software had to provide remote access to the factorycontrol system, but not interrupt normal factory operations. Any mode of access had to employ a highlevel of security so that only selected staff and suitably trained maintenance personnel could use thesystem. The introduction of factory remote access using smart devices has given mill staff the ability toview factory operations without the need to interrupt operators from their normal duties, particularly whenthere is a problem. It also provided quicker after-hours support as staff could diagnose problems andoffer a solution, where before they had to travel to the mill before determining the problem and thenecessary solution. Commissioning of new equipment, control wiring and advanced fault finding havebeen improved since remote access has been implemented.

CONSIDERATIONS FOR THE DESIGN OF MODERN-DAY LABORATORIES

TJ Prange

Laboratory buildings are an essential part of every sugar mill. In Maryborough Sugar Factory’s case the building was inpoor condition, dated, and well below required minimum laboratory design standards. The poor state of the buildingand unacceptable working conditions led to the development of a plan to build a modern purpose-built laboratory. Thebasis of the paper will outline the methodology, developed design and difficulties associated with the design andconstruction of modern day sugar mill laboratory. The paper gives particular emphasis to design briefs, buildingapprovals, juice sampling, Sugar Industry Act and cane payment, modes of construction, and project management, allaimed at developing and implementing a successful plan to construct a modern-day laboratory.


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MANAGING MILLING TRAIN OPERATION AND PERFORMANCE

GA Kent

This Paper Discusses the main milling train management tasks necessary for maintaining goodextraction performance through a season. The main activities discussed are making week by weekdecisions about shredder and mill setting adjustments, and selecting preseason mill settings. To maintainsatisfactory milling train extraction performance, the main factors affecting extraction should beexamined: cane preparation with pol in open cells or shredder torque, delivery nip compaction throughthe load or torque controller outputs such as roll lift, feed chute flap position or pressure feeder to millspeed ratio, and added water rate. To select mill settings for the coming season, delivery nip compactionand feed chute exit compaction can be inferred from the previous seasons.

SOME INTERESTING BEHAVIOURS OF PREPARED CANE AND FINAL BAGASSE

F Plaza

There are opportunities to decrease bagasse moisture from a milling unit, with substantial financial benefits, forexample, from co-generation. Also, there has been some recent renewed interest in the valuing of the contents ofbagasse and the assessment of the milling performance of new cane varieties. Previous laboratory investigations haveshown that juice flow through prepared cane and bagasse obeys Darcy’s permeability law, that the grip of the roughsurface of the grooves on the bagasse can be represented by the Mohr-Coulomb failure criterion for soils, and that theinternal mechanical behaviour of the bagasse is critical state behaviour similar to that for sand and clay. However, theextension from this laboratory fundamental work in order to describe and improve the design and operation of factoryunits remains unrealised. This paper presents some interesting behaviours of prepared cane, first bagasse, and finalbagasse that have mostly been previously unreported, with a focus in change in volume of the materials underchanges in stress, the resulting reduction or increase in juice pressure and its potential effects on the removal of juice.Details of the experimental geometry and procedure are presented which are relevant to the drainage and griprequirements of mill rollers. With reference to the same equipment, the last part of the paper links the testing of canevarieties to provide a unified story of compaction and feeding behaviour in a milling unit.


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MODIFICATION OF THE DISTRIBUTOR AIR DUCTING, No. 3 BOILER PIONEER MILL

A Wallwork, L Franklin

NO. 3 boiler was installed in 2004–2005 during the co-generation project undertaken by CSR at PioneerSugar Mill. Since commissioning, the bagasse distribution on the grate of this boiler had favoured oneside of the furnace. Over 10 seasons many different possible causes were explored with no explanationfound. Modifications were made to the distributor bagasse chute outlets into the furnace to reduce build-

up along one wall, but this had little effect on the skewed bagasse distribution. In 2013 another attempt todetermine the root cause was made with some rudimentary experimentation conducted to determinewhether there was a consistent flow of air into the furnace across each distributor air nozzle. Thisshowed that there was a significant pressure differential across each distributor air chute causing thebagasse to be propelled into the furnace at a measurable angle. The root cause of the problem wasdiscovered to be the upstream distributor air ducting causing a pressure differential across the distributornozzles. To rectify the issue two different ducting designs were analysed with CFD modelling whichshowed an improvement in pressure distribution. The preferred design was manufactured and installed inthe 2014 maintenance season resulting in a marked improvement in bagasse distribution during the 2014crushing season.

MARIAN SECONDARY JUICE HEATER CONDENSATE SYSTEM IN 2014

Paul Stuart, Ken Griffin, Brett Bampton, Mauro Giannangelo

IN 1991, Marian Mill was upgraded to handle cane from its historical cane area and that of Cattle Creek, moving from450 tch to a nominal 700 tch. The secondary juice heater system was expanded to provide seven parallel heaters.These heaters are all situated very low to the ground floor level, leaving only 1000 mm height from the floor to the bodyof the heaters. The heaters’ condensate drains were piped to two condensate mains each leading to a separatecondensate vessel and pump. In subsequent years, the heaters frequently suffered operational problems and multiplechanges were subsequently made to the condensate system. Over the period 1991 to 2013, the direct results of thepoor performances included low secondary juice temperatures, reduced condensate return to the boiler feedwatersystem and condensate discharged to the floor juice reclaim system. In 2014, a number of simple changes were madeto the condensate piping and the condensate collection vessels which largely eliminated all those problems. Theresults of the change include improved secondary juice temperature control, reduced evaporator loading, surplusquality condensates and a lower steam on cane.


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TRACTIVE EFFORT MODELLING AND DESIGN OF HYBRID POWERTRAIN FOR A DIESEL-HYDRAULIC CANE HAUL LOCOMOTIVE

BR Sebastian, OP Kenny, R Situ, LG Santarossa

Wilmar owns and operates a mixed fleet of diesel hydraulic locomotives that operate on two foot narrowgauge rail in its Queensland operations. This network serves as an effective transport system, haulingapproximately fifteen million tonne of cane annually. An assessment of the practicality of convertingWilmar’s diesel locomotives to a hybrid powertrain design provides an opportunity for improving energyefficiency of cane transport and requires the duty cycle performance of the locomotive to be assessedprior to further work. This paper presents a novel method to determine the tractive effort and energyrequirements for the duty cycle, using track dependant physical equations ofpropulsion dynamics, as theconventional methods of dynamometer car measurements and empirical equations are restricted tobroad-gauge rail. A simulation model was developed in MATLAB to determine the performanceparameters of a modifiable real– time diesel-hydraulic train configuration. Additionally, the performancerequirements of the hybrid powertrain in comparison to the diesel-hydraulic powertrain was demonstratedby developing an optimisable real-time model of a series-hybrid locomotive in SIMSCAPE, an interactivetool for physical systems simulation in MATLAB. Testing and validation using field data obtained from on-board sensors, the locomotive’s Programmable Logic Controller (PLC) and a track profile extractionmethod exclusively developed for this research, demonstrated that the model was capable of accuratelypredicting the performance parameters and energy consumption of the diesel-hydraulic locomotive for aparticular duty cycle. The hybrid model developed in SIMSCAPE exhibited superior traction performanceand significant improvements in fuel efficiency and emissions compared to the diesel-hydrauliclocomotive for the same duty cycle.

INSIGHT INTO DEVELOPING DURABLE ENGINEERING DESIGNS FROM AN AUTOMOTIVE PERSPECTIVE

CM Downing

Designed and developed in Australia, built in Thailand, South Africa and Argentina,and sold in over 100 countries, theFord Ranger is an example of a truly international product. This paper outlines some of the considerations in makingthe product durable enough for the global market, particularly the collection and application of ‘road load data’ (RLD) inthe design and product development process. The definition of load profiles is essential to conducting detailedmechanical strength and fatigue life design refinement, whether the component in question is an automobile frame, aharvester steering link, wagon coupling, mill roll shaft or a steam turbine blade.


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DEGRADATION STUDY OF A LIGNIN MODEL COMPOUNDDJ Cronin, Wos Doherty

than that reported in the literature was proposed for the NaOH-catalysed degradation of PEB to phenoland styrene. The organic phase (i.e. oil) obtained was characterised using gas chromatography—massspectrometry (GC-MS) and Fourier Transform-Infrared (FT-IR) spectroscopy. The highest yield of henol(55 wt%) was obtained when a PEB concentration of 2.4 wt% and a reaction time of 60 min were used,whereas at the higher concentration of 6.8 wt% PEB and a reaction time of 60 min, the maximum tyreneyield was obtained (21 wt%).The information obtained will be used in the study for the conversion of lackliquor derived from bagasse to chemicals.

LIMITATIONS OF A LABORATORY SCALE MODEL IN PREDICTING OPTIMAL PILOT SCALE CONDITIONS FORDILUTE ACID PRETREATMENT OF SUGARCANE BAGASSE

AA Greenwood, TW Farrell, Z Zhang, IM O’hara

Pilot and industrial scale dilute acid pretreatment data can be difficult to obtain due to the significant infrastructureinvestment required. Consequently, models of dilute acid pretreatment by necessity use laboratory scale data todetermine kinetic parameters and make predictions about optimal pretreatment conditions at larger scales. In order forthese recommendations to be meaningful, the ability of laboratory scale models to predict pilot and industrial scaleyields must be investigated. A mathematical model of the dilute acid pretreatment of sugarcane bagasse haspreviously been developed by the authors. This model was able to successfully reproduce the experimental yields ofxylose and short chain xylooligomers obtained at the laboratory scale. In this paper, the ability of the model toreproduce pilot scale yield and composition data is examined. It was found that in general the model over predicted thepilot scale reactor yields by a significant margin. Models that appear very promising at the laboratory scale may havelimitations when predicting yields on a pilot or industrial scale. It is difficult to comment whether there are anyconsistent trends in optimal operating conditions between reactor scale and laboratory scale hydrolysis due to thelimited reactor datasets available. Further investigation is needed to determine whether the model has some efficacywhen the kinetic parameters are re-evaluated by parameter fitting to reactor scale data, however, this requires thecompilation of larger datasets. Alternatively, laboratory scale mathematical models may have enhanced utility forpredicting larger scale reactor performance if bulk mass transport and fluid flow considerations are incorporated intothe fibre scale equations. This work reinforces the need for appropriate attention to be paid to pilot scale experimentaldevelopment when moving from laboratory to pilot and industrial scales for new technologies.


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INTERNATIONAL EVENTS CALENDAR

November 30-December-4

ISSCT Co-Products Workshop MauritiusISSCT Co-Products Workshop

February 1-3

Louisiana Division of American Society of Sugar Cane Technologists (ASSCT) Lafayette, LA USAASSCT.org

May 15-18Sugar Industry Technoligists 75th Conference, New York, NY USA SIT.org

February 21 - 24

Sugar Processing Research Institute (SPRI) Walnut Creek, CA USA SPRI.org

December 5-8

XXIX International Society of Sugar Cane Technologists' (ISSCT) Congress, Chiang Mai,Thailand ISSCT Thailand 2016


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STORY OF SWEETS

i. Gulab Jaman

IngredientsEgg 1Plain flour 1 cupMilk powder ½ cupAlmonds 10 to 12Baking powder 1 tspSemolina 2 tbspOil 2 tbspOil for fryingFor syrup:Small cardamoms 8Water 1 cupYellow color A pinch ofSugar 2 ½ cup

Cooking DirectionsMix plain flour, milk powder, semolina, bakingpowder, egg and oil and knead into fine dough.

For syrup, add sugar, water, small cardamomsand a pinch of yellow color to a cooking potand cook.

Now grease your hands with some oil and forma portion of the dough into shape of a gulabjaman, placing an almond in the middle.

Heat oil in a wok for frying, fry gulab jamansuntil they turn golden brown.

Place syrup on a low flame and dip goldenbrown gulab jamans in the syrup before youserve.

ii. Sheer Khurma (Vermicelli Pudding)

IngredientsVermicelli Noodles(Seviyan) 1 & 1/4 cupMilk 1 literDates (dry) 7-8Almond 10Pistachio 12Black raisin 1/2 cupGhee 2 1/2 to 3 tbspGreen cardamom 6Clove 4Sugar 10-11 tbsp

Cooking DirectionsSoak the dates in water overnight and thenslice cut them.Take milk and add sugar.Cook it well.Put a pot on stove and add ghee.Then add green cardamom & clove.Cook them for a while.Then add almonds, pistachio and fry them.Take them out of the pan and put aside forlater use.With hands, crush the siwaiyan and add in panto cook till golden brown.Cook the siwaiyan on low heat and be veryattentive whiles this.Add black raisins and cook on slow-mediumheat.Add milk, almonds, pistachio and black raisinsin the above mixture.Cook it for a while and add roh kewra forfragrance.Cook for 15-20 minutes.Cool down the dish and serve.Tip you can use condensed milk instead ofnormal milk.


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Venieraki A., M. Dimou, E. Vezyri, I. Kefalongianni, N. Argyris, G. Liara, P. Pergalis, I. Chatzipavlidis, andP. Katinakis. 2011. Characterization of nitrogen-fixing bacteria isolated from field-grown barley, oat andwheat. J Microbiol. 49: 525-534.

Yim, W.J., S. Poonguzhali, M. Madhaiyan, P. Palaniappan, M.A. Siddikee, and T. Sa. 2009.Characterization of plant-growth promoting diazotrophic bacteria isolated from field-grown chinesecabbage under different fertilization conditions. J Microbiol. 47: 147-155.

Zehr J.P., and D.G. Capone. 1996. Problems and promises of assaying the genetic potential for nitrogenfixation in the marine environment. Microb Ecol. 32: 263-281.

Zehr J. P., B.D. Jenkins, S.M. Short, and G.F. Steward. 2003. Nitrogenase gene diversity and microbialcommunity structure: a cross-system comparison. Environ Microbiol. 5: 539–554.


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GUIDELINES FOR AUTHORS

Dear Fellow Author(s),

Pakistan Sugar Journal (PSJ) offers research, analysis, and reviews to keep its local andinternational readership up to date with latest developments in the sugar industry. PSJtakes into account the application of research and focuses on areas in agriculture related tosugar, milling and processing.

In order to have your articles published in the PSJ, you are requested to adhere to thebelow instructions and prerequisites to enable timely review of your submissions by theeditorial board:

I. Write the title of your article in CAPITAL LETTERS in the center of the page.II. Write the complete name of all authors with their addresses – it is compulsory in the

text. References should be cited by author and years as, for one, two or moreauthors (Hammer, 1994, Hammer and Rouf, 1995; Hammer et al., 1993),respectively.

III. Write HEADINGS in bold letters and in the center of the page.IV. Type your article only in TIMES NEW ROMAN format.V. Send TABLES and FIGURES on separate page with bold title and mark its numbers

correctly.VI. Observe the following rule for REFERENCE, for one author: Hussain, K. 1991 for

two authors; Khan, M. and A. Habib 1995, for more than two; Ali, K., A.Hussain and S. Nasir, 1990.

VII. Always send two soft copies and one hard copy of CD. Please do not use FLOPPYDISK for this purpose.

VIII. Send copies on an A-4 size page, preferable LASER PRINT in word documentIX. Papers published in the PSJ are free of charges (for authors).X. Send your papers to following address by mail or email:

Dr. Shahid AfghanEditor-in-Chief, Pakistan Sugar JournalShakarganj Sugar Research Institute, Jhang (Pakistan)Phone: +92 47 763 1001-5 | Ext. 602, 603Email: [email protected]

apr.-jun., 2016ssri.pk/psj/psj apr-jun-2016.pdf · trashing of cane in the field or whatever is...

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