1(2)46-57 !!!!!!!!!!!!!!!!!!CONTACT&:&NUR&HIDAYATUL&ALAMI&&&&&&&Nh_daya@yahoo.com! ©!International!Journal!of!Applied!Biology! 46! Abstract& Biofertilizers! are! commonly! defined! as! microbial! inoculants! which! are! capable!of!mobilizing!important!nutrient!in!the!soil!from!nonAavailable!to! available!form!for!the!plants!through!their!biological!processes.!Hence!to! increase! the!productivity!of!the!soil,! the!use!of!biofertilizer! is!necessary.! Candida! yeast! as! a! phosphate! solubilizer! and! decomposer.! Azotobacter! bacteria!as!a!nitrogenAfixing!bacteria!and!Bacillus!as!a!phosphate!solubilizer.! The!combination!of!these!microorganisms!as!biofertilizer!needs!to!be!tested! its! effect! against! mustard! (Brassica' juncea! L.)! plant! growth.The! results! showed!that!the!combination!of!Bacillus!Candida!obtained!the!highest!yield! among!other!biofertilizer!treatments!with!a!value!of!13.6!cm!at!plant!height,! 4.96!grams!in!wet!weight!and!2.29!grams!in!dry!weight.!The!combination!of! Azotobacter!Candida!showed!the!highest!result!on!the!parameter!of!width! and!number!of!leaves!with!the!values!of!20.3!cm!and!5.3.!Combination!of! Azotobacter!Bacillus!Candida!showed!the!highest!yield!on!the!root!length! parameter!with!a!value!of!5.0!cm.!The!addition!of!biofertilizer!is!expected!to! reduce!pollution!on!the!soil!due!to!residues!produced!by!chemical!fertilizers.! ISSN!:!2580A2410! eISSN!:!2580A2119! ! ! ! Effect&of&Yeast&Based&Biofertilizer&combined&with&bacteria&on& Mustard&Plant&Growth& ! Nur&Hidayatul&Alami,&Silviya&Amaliya&Trisna&Ayu,&Nengah&Dwianita&Kuswytasari,&Enny& Zulaika,&and&Maya&Shovitri& & Department!of!Biology,!Institut!Teknologi!Sepuluh!Nopember,!Surabaya,!Indonesia! ! ! & & & & & & & & & & & & && & & Introduction& Biofertilizer!application!to!overcome!the!problem!of!land!degradation!due!to!the!use! of!chemical!fertilizers!has!been!the!focus!of!the!study!until!the!last!few!decades.!Compared! with!chemical!fertilizers,!biofertilizers!are!shown!to!have!a!better!ability!to!increase!crop! productivity! because! biofertilizers! are! enriched! by! microorganisms! and! plant! growth! promoters! with! a! beneficial! consortium! of! microorganisms! (Paszt! et' al.,! 2015).! The! biofertilizerAconsortia!microorganisms!may!include!groups!of!fungi,!bacteria,!or!protozoans! (CavalierASmith,!1998;!Lucy!et'al.,!2004;!Smith!and!Read,!2008;!Vessey,!2003)!which,!when! applied,!can!increase!the!availability!of!nutrients!from!biological!activity!and!can!improve!soil! quality.!Biofertilizer!is!able!to!provide!elements!of!N,!P!and!K!as!well!as!nutrients!for!plants! that!can!affect!the!growth!and!productivity!of!plants!!(Agamy!et'al.,!2013).! Study!on!the!production!of!biofertilizers!using!bacteria!and!yeast!has!been!widely! used,!Wani!et'al.!(2013)!state!that!Azotobacter!bacteria!are!able!to!fix!10!mg!of!nitrogen!from! &&&&&&&&OPEN&ACCESS& & & &&&&&&&&&&International&Journal&of&Applied&Biology& Keyword! Azotobacter' Bacillus' Biofertilizer' Candida' Mustard'Plant Article&History! Received!21!November!2017! !Accepted!29!December!2017!! International Journal of Applied Biology is licensed under a Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ! International Journal of Applied Biology, p-ISSN : 2580-2410 e-ISSN : 2580-2119. Journal homepage : http://journal.unhas.ac.id/index.php/ijoab International'Journal'of'Applied'Biology,'1(2),'2017' ! 47! oneAtenth!of!carbon!sources!conducted!in!vitro!(Amprayn!et'al.,!2012).!This!study!uses!a! consortium!of!Candida!yeast!and!Azotobacter!and!Bacillus!bacteria.!Some!Candida!species! are! able! to! solubilize! phosphate! and! degrade! organic! compounds,! Azotobacter! is! a! siderophore!bacteria!and!capable!of!fixing!nitrogen,!and!Bacillus!is!able!to!dissolve!phosphate! in!the!soil!(Xiao!et'al.,!2013;!New!et'al.,!2013;!Fleet,!1992;!Ali!and!Vidhale,!2013;!Maheswar! and!Sathiyavani,!2012).!Biofertilizers!use!the!molasses!carrier!as!the!main!carbon!source!for! the! growth! of! the! isolates.! Molasses! were! selected! because! of! their! effectiveness! and! efficiency!as!carriers!for!controlling!microbial!growth.! Plants!that!have!the!potential!to!be!used!as!research!object! is!the!mustard!plant! (Brassica'juncea'L.).!Mustard!is!an!annual!plant!that!can!grow!in!lowland!to!upland!(Devi!et' al.,!2017),!able!to!grow!and!adapt!well!to!almost!all!soil!types,!have!a!short!harvest!age!(35! days),!and!leaves!are!used!by!Indonesian!people!as!vegetables!because!of!high!nutritional! value.!Based!on!the!ability!of!microorganisms,!it!is!necessary!to!study!more!about!the!effect! of!the!combination!!of!!yeast!and!bacteria!as!biofertilizer!on!mustard!plant!growth.! & Materials&and&Methods& Isolates& Isolates!used!in!this!research!were!Candida!W3.8!and!Candida!G3.2!and!isolates!of! Azotobacter!A10!and!Bacillus!spp.,!collection!of!Microbiology!and!Biotechnology!Laboratory,! Biology!Department,!Institut!Teknologi!Sepuluh!Nopember!Surabaya!(ITS).! & Preparation&of&Microbial&Suspension& Measurements!of!yeast!and!bacteria!growth!were!done!by!adding!each!culture!in! 0.85%! (w/v)! of! physiological! solution,! consisting! of! NaCl! and! aquades.! Absorbance! was! measured!using!a!UVAVis!spectrophotometer!with!OD!0.5!at!λ!600!for!yeast!and!OD!0.5!at!λ! 620!for!bacteria.!The!value!of!OD!is!based!on!the!approximate!number!of!yeast!and!bacterial! cell!densities!(about!107!CFU/ml).! ! Growth&of&&Yeast&and&Bacteria&in&Molasses&Medium& About! 10%! of! microbial! suspension! from! the! previous! step! then! inoculated! into! molasses!production!medium!and!CzapekDox.!Molasses!is!a!carrier!medium!which!has!been! known!as!microbial!growth!substrate.!CzapekDox!Broth!(g!/!L)!consisting!of!sodium!nitrate!(3! g);! Dipotassium! phosphate! (1! g);! Magnesium! sulphate! (0.5! g);! Potassium! chloride! (0.5);! Ferrous!sulphate!(FeSO4),!(0.01!g);!Aquadest!(H2O)!(1000!mL).!The!volumes!of!the!inoculum! was!added!to!the!carrier!medium!based!on!the!different!treatment!groups!(Table!1.)! ! Table&1.&&Volumes&of&Inoculum& Treatment! Suspension!Volume! Treatment!1!!!:! 5!ml!of!Candida!W2.8!+!5!ml!of!Candida!G3.2!+!90!ml!of!molasses!and!Czapek! Dox! Treatment!2!!!:! 10!ml!of!Azotobacter'A10!+!90!ml!of!molasses!and!Czapek!Dox! Treatment!3!!!:!!!! 10!ml!of!Bacillus!spp.!+!90!ml!of!molasses!and!Czapek!Dox! Treatment!4!!!:! 3,3!ml!of!Candida!W2.8!+!3,3!ml!of!Candida!G3.2!+!3,3!ml!of!Azotobacter'A10!+! 90!ml!of!molasses!and!Czapek!Dox! Treatment!5!!!:! 3,3!ml!of!Candida!W2.8!+!3,3!ml!of!Candida'G3.2!+!3,3!ml!of!Bacillus!spp.!+!90! ml!of!molasses!and!Czapek!Dox! Treatment!6!!!:! 2,5!ml!of!Candida!W2.8!+!2,5!ml!of!Candida!G3.2!+!2,5!ml!of!Azotobacter'A10!+! 2,5!ml!of!Bacillus!spp.!+!90!ml!of!molasses!and!Czapek!Dox! International'Journal'of'Applied'Biology,'1(2),'2017' ! 48! Each! combination! of! biofertilizer! was! homogenized! on! a! rotary! shaker! for! 7! days! (Anubrata!and!Rajendra,!2014).!Measurement!of!growth!was!done!daily!with!Total!Plate! Count! method! to! obtain! maximum! growth! day.! The! best! incubation! time! to! be! used! for! biofertilizer!production!was!based!on!the!total!number!of!inoculums!of!109!CFU/mL!(Noel! et.al.,'2016)!in!each!treatment!group.! ! Planting&and&Maintenance&of&Mustard& Mustard!seeds!were!soaked!with!water!for!24!hours,!then!seeds!that!were!submerged! in!water!were!selected.!Seeds!were!allowed!to!grow!in!seedling!medium!at!least!until!two! leaves!were!formed!or!14!days!after!planting.!Application!of!biofertilizer!was!done!in!2!stages,! the!first!given!at!14!days!before!the!seed!was!transferred!to!planting!medium,!and!the!second! at!14!days!after!planting.!Biofertilizer!is!composted!by!watering!250!ml!into!polybags.!Plants! are!grown!for!4!weeks.!Measurements!of!growth!include!plant!height,!leaf!area,!number!of! leaves,!root!length!and!biomass!(dry!weight).! ! Plant&Height&Measurement& Plant! height! is! measured! from! ground! level! to! the! highest! leaf.! Plant! height! measurement!was!done!once!a!week!for!5!weeks.! ! Measurement&of&Leaf&Area& The! leaf! area! is! calculated! by! millimeter! paper! method! by! putting! the! leaves! on! millimeter!paper!and!leaf!pattern!followed.!The!leaf!area!is!estimated!based!on!the!number! of!squares!contained!in!the!leaf!pattern.!The!measurement!of!leaf!area!is!done!once!a!week! for!5!weeks.! ! Number&of&Leaf&Calculation& The! number! of! leaf! calculation! was! done! on! leaves! that! had! developed! perfectly.! Counting!the!number!of!leaves!were!done!once!a!week!for!5!weeks.! ! Root&Length&Measurement& The!measurement!of!root!length!with!destruction!method!was!done!by!removing!the! whole!plant!and!measuring!the!root!length!from!root!base!to!root!tip!by!using!ruler.!Root! length!measurements!were!made!at!harvest!time.! & Biomass&Calculation& Biomass!is!obtained!from!the!dry!weight!of!the!plant.!Before!weighing!the!plant!is! cleaned!with!water!and!dried!wind.!The!dry!weight!of!the!plant!was!carried!out!by!drying!at! 60!°!C!for!4!days,!then!was!weighed!using!analytical!balance.!Biomass!measurements!were! made!during!harvest!time.! ! Research&Design& The!design!of!this!study!used!a!completely!randomized!design!with!6!treatments!for!5! weeks!and!3!repetitions.!Research!design!are!shown!in!the!figure!1.! ! ! ! ! International'Journal'of'Applied'Biology,'1(2),'2017' ! 49! & & & Figure&1.&Research&Design,&ConT1:&Negative&control&repetition&1;&C1:&Biofertilizer&of&Candida& repetition&1;&Con+&2:&Positive&control&repetition&2;&A1:Biofertilizer&of&Azotobacter& repetition& 1;& CA2:& Biofertilizer& of& Candida& and& Azotobacter& repetition& 2;& CAB3:& Biofertilizer&of&Candida,&Azotobacter,&and&Bacillus&repetition&3;&ConT2:&Negative& control&repetition&2;&CB1:&Biofertilizer&of&Candida&and&Bacillus&repetition&1;&Con+&1:& Positive& control& repetition& 1;& A2:& Biofertilizer& of& Azotobacter& repetition& 2;& B1:& Biofertilizer& of& Bacillus& repetition& 1;& CB3:& Biofertilizer& of& Candida& and& Bacillus& repetition&3;&C3:&Biofertilizer&of&Candida&repetition&3;&B3:&Biofertilizer&of&Bacillus& repetition& 3;& CB2:& Biofertilizer& of& Candida& and& Bacillus& repetition& 2;& CAB2:& Biofertilizer& of& Candida,& Azotobacter,& and& Bacillus& repetition& 2;& Con+3:& Positive& control& repetition& 3;& B2:& Biofertilizer& of& Bacillus& repetition& 2;& ConT3:& Negative& control&repetition&3;&CA3:&Biofertilizer&of&Candida&and&Azotobacter&repetition&3;& CA1:&Biofertilizer&of&Candida&and&Azotobacter&repetition&1;&CAB1:&Biofertilizer&of& Candida,&Azotobacter,&and&Bacillus&repetition&1;&A3:&&Biofertilizer&of&Azotobacter& repetition&3;&C2:&Biofertilizer&of&Candida&repetition&2.& ! Data&Analysis& The!data!analysis!used!ANOVAAOne!Way!for!combination!of!yeast!and!bacteria!as! biofertilizer!against!plant!growth.! !! Results&and&discussion& The!preparation!of!biofertilizer!should!consider!the!substance!of!carrier!media!and! biological!agents.!Carrier!media!should!also!contain!an!important!component!to!support!the! viability!and!growth!of!inoculated!microbes.!One!of!the!carrier!media!for!the!biofertilizer!is! molasses!which!have!been!known!as!the!microbial!growth!substrate.!Molasses!were!selected! because!of!their!effectiveness!and!efficiency!as!carriers!for!the!growth!of!biofertilizer!agent.! Molasses!can!be!used!as!an!alternative!substrate!because!it!contains!complex!nutrients!that! was!taken!by!microbes!for!metabolism!process,!it!contains!not!less!than!46%!total!sugars! (Duraisam!et'al.,!2017),!including!a!water!content,!nitrogen!and!minerals!that!are!used!for! microbial!growth.!& Molasses! used! in! this! research! is! a! source! of! carbon! (C)! for! the! microorganism,! molasses!can!be!combined!with!the!medium!Czapekdox!broth,!with!a!composition!of!Sodium! nitrate! (NaNO3)! as! a! source! of! protein,! Dipotassium! phosphate! (KH2PO4),! Magnesium! sulphate!(MgSO4.7H2O),!Potassium!chloride!(KCl),!and!Ferrous!sulphate!(FeSO4)!which!is!a! source!of!salts!and!minerals!for!the!growth!of!biofertilizer!agent.!Carrier!media!with!the! ConA!1! C!1! Con+!2! A!1! CAB!3!! ConA!2! CB!1! Con+!1! B!1! CB!3! C!3! B!3! C!2!A!3!CAB!1!CA!1! ConA!3!B!2!Con+!3!CAB!2! CA!3! CB!2! A!2! CA!2! International'Journal'of'Applied'Biology,'1(2),'2017' ! 50! content!of!organic!and!mineral!are!expected!to!make!the!microorganisms!can!compete!with! microorganisms!in!the!soil.!Thus!able!to!dominate!around!plant!roots.& The!biofertilizer!should!be!contain!the!minimum!population!of!the!total!bacteria!of!≥! 109!cfu!/!ml!or!9!log!cfu!/!ml!for!the!positive!influence!of!microbes!in!the!biofertilizer!(Masso! et'al.,!2015).!Therefore,!the!consortium!formulation!of!biological!agents!and!carrier!media!is! necessary!to!enhance!plant!growth!and!productivity.& The!Calculation!of!microorganism!was!performed!before!the!biofertilizer!was!applied! to! mustard! for! 7! days.! Observations! started! from! day! 0! of! inoculation! to! day! 7! of! the! incubation!period.!The!microbial!growth!results!are!shown!in!Figure!2.& & & & & & & & & & & & & & Figure&2.&Growth&of&Yeast&and&Bacteria&Consortium&on&Molasses&Medium& & Figure!1!shows!that!the!growth!increased!gradually!until!the!7!days!of!incubation!time.! This!suggests!that!yeast!and!bacterial!of!biofertilizer!was!able!to!increase!in!numbers!during! the! application! of! the! soil! so! that! the! microbes! in! the! biofertilizer! potentially! increasing! nutrient!uptake!by!plants!.! The!increase!in!the!population!of!consortium!in!a!test!of!viability!indicates!that!the! agent!biofertilizer!used!consisting!of!a!single!colony!which!include!yeast!candida,!bacteria! Azotobacter!and!Bacillus,!a!consortium!of!the!yeast!Candida!and!Azotobacter,!yeast!Candida! and!Bacillus,!as!well!as!a!consortium!of!yeast!Candida,!Azotobacter!and!Bacillus!able!to!grow.! On!day!0!shows!an!indication!of!metabolic!activity!performed!by!all!inoculants.!Yeasts!and! bacteria! utilize! nutrients! in! the! medium! for! growth.! According! to! (Brock,! 1991),! in! the! adaptation!phase,!an!increase!in!cell!size,!the!cell!does!not!divide!or!slightly!division.!This! condition,! characterized! by! an! increase! in! the! macromolecular! components,! metabolic! activity,!and!the!number!of!cells!that!slowly!began!to!increase.! In!all!treatments!on!the!first!day!until!the!seventh!day!there!is!an!increase!in!the! number!of!cells.!This!is!because!Yeast!and!bacteria!are!in!the!logarithmic!phase.!Cells!will! continue! to! multiply! by! utilizing! molasses! as! the! main! source! of! nutrients.! According! to! (Paulton,! 1991)! In! the! exponential! or! logarithmic! phase,! the! period! and! volume! of! cells! increase!and!and!the!speed!of!increase!can!be!expressed!with!exponential!function.! N u m b er &o f& C o lo n y& (L o g& C FU /m l) Day CAB CB CA B A C International'Journal'of'Applied'Biology,'1(2),'2017' ! 51! The!results!of!plant!height!measurements!of!the!mustard!are!shown!in!Figure!3.!In! Figure!3!it!shows!that!plant!height!increased!every!week!during!the!planting!time!up!to!28! days!after!planting.The!increasing!of!plant!height!can!be!affected!by!the!availability!of!various! nutrients!on!the!soil!that!has!been!provided!by!biofertilizer.!OneAway!ANOVA!results!of!the! week!4!(harvest!time)!show!that!probability!value!is!less!than!0.05!ie!0.001,!so!the!biofertilizer! gives!effect!to!the!height!of!the!mustard!so!that!Tukey!test!was!done!at!95%!(α!=!0,05)!using! Minitab!software.!Tukey!test!results!are!shown!in!Table!2.& ! Figure&3.&Result&of&Plant&Height& & Table&2.&Height&of&mustard&plants&at&harvest&time& Treatment& Plant&high&(cm)& Positive!control! 16,167a! Bacillus'Candida' 13,600ab! Bacillus' 12,467abc! Azotobacter' 12,100abc! Azotobacter'Bacillus'Candida' 11,833bc! Azotobacter'Candida' 11,400bc! Candida' 11,000bc! Negative!control! 8,833c! *Figures!followed!by!different!letters!show!significant!differences!based!on!the!Tukey!test!at! the!95%!confidence!level!(α!=!0.05).! ! Based!on!Table!2!it!can!be!seen!that!the!influence!of!biofertilizer!on!the!height!of!the! mustard! in! Bacillus! Candida! treatment! has! the! highest! effect! when! compared! with! the! treatment! of! the! addition! of! another! biofertilizer.! Bacillus! is! able! to! synthesize! growth! hormone!IAA,!gibberellins!and!cytokinins!(Park!et'al.,!2017),!and!dissolves!phosphates!that! are!not!available!in!the!soil!by!the!mechanism!of!organic!acid!secretion.!Phosphate!is!an! important!macronutrient!for!plants!for!high!plant!growth!(Wang!et'al.,!2011)!The!ability!of! Bacillus!is!combined!with!Candida!which!has!the!potential!to!dissolve!organic!and!inorganic! phosphate,!acting!as!a!bioAactivator!for!the!breakdown!of!organic!matter!that!will!contribute! nutrient!supply!for!soil!to!plant!height!.! ! P la n t& H ei gh t& (c m ) Treatment Week!1 Week!2 Week!3 Week!4 International'Journal'of'Applied'Biology,'1(2),'2017' ! 52! Plant!height!in!positive!control!is!highest!because!the!positive!control!(NPK!fertilizer)! contains!nutrients!and!minerals!that!can!be!utilized!directly!by!plants,!so!the!plant!can!take! the!nutrition!quickly.!Mustard!plants!have!a!short!harvest!age,!thus!the!available!uptake!of! nutrients!is!also!fast.!But!the!continued!use!of!chemical!fertilizers!can!leave!residues!thereby! destroying!soil!and!the!environment.!Treatment!of!negative!control!has!the!lowest!effect!on! the!high!of!mustard.!This!is!because!the!negative!controls!in!this!study!only!use!soil!as!a! planting!medium!without!any!addition!of!fertilizers!in!which!contain!more!nutrients!needed! for!plant!growth!height,!so!the!roots!will!only!absorb!the!macro!and!micro!essential!elements! available!in!the!soil.! The!results!of!the!measurements!of!the!leaf!number!and!leaves!area!are!shown!in! Figure.!4!and!5.!The!leaf!number!and!leaves!area!has!increased!each!week.!The!result!of!oneA way!ANOVA!showed!that!the!probability!value!of!the!area!and!the!number!of!leaves!was!less! than!0.05!ie!0.003!and!0.000!respectively!so!that!the!biofertilizer!had!an!effect!on!the!leaf! area! and! number,! so! that! The! Tukey! test! was! done! at! 95%! (α! =! 0.05)! By! using! Minitab! software.!Tukey!test!results!are!shown!in!Table!3.! & Figure&4.&Result&of&Leaf&Area& & & & & & & & & & & & & & & & Figure&5.&Result&of&Leaf&Number& Le af &A re a& (m m 2 ) Treatment Week!1 Week!2 Week!3 Week!4 N u m b er &o f& Le af Treatment Week!1 Week!2 Week!3 Week!4 International'Journal'of'Applied'Biology,'1(2),'2017' ! 53! Table&3.&Leaf&Area&and&Number&of&Leaf&at&Harvest&Time& Treatment! Leaf&Area!(cm)! Number&of&Leaf! Positive&Control! 25,100a! 6a! Azotobacter'Candida' 20,300ab! 5,3b! Bacillus'Candida' 18,867ab! 5,0b! Azotobacter'Bacillus'Candida' 15,967abc! 5,0b! Candida' 14,333!abc! 5,0b! Azotobacter' 11,533bc! 5,0b! Bacillus' 9,867bc! 5,0b! Negative&Control! 5,167c! 5,0b! *Figures!followed!by!different!letters!show!significant!differences!based!on!the!Tukey!test!at! the!95%!confidence!level!(α!=!0.05).! ! Based!on!Table!3!it!can!be!seen!on!the!biofertilizer!treatment!of!Azotobacter!and! Candida!consortium!showed!the!highest!yield.!The!most!needed!nutrient!in!the!leaf!area!and! the! number! of! leaves! is! the! Nitrogen! (Ng’etich,! 2013).! The! potential! of! Azotobacter! in! nitrogen!fixation!is!combined!with!Candida!in!dissolving!phosphate!into!the!forms!available! for!plants.!This!is!consistent!with!(Plaster,!2003)!!that!nitrogen!is!more!necessary!in!support! of!vegetative!growth!than!generative!and!important!for!vegetable!crops!consumed!by!the! canopy.! The!positive!control!has!the!highest!result,!this!is!the!same!as!the!previous!parameter! indicating! positive! control! treatment! has! the! highest! value.! This! is! because! the! chemical! fertilizer!(NPK!fertilizer)!provided!an!inorganic!nitrogen!element!that!is!ready!for!use!for!leaf! area!and!the!number!of!leaves.!In!the!negative!control!showed!the!lowest!result.!This!is!in! accordance!with!soil!nitrogen!content!test!results!indicating!that!the!nitrogen!content!in!the! soil!with!the!addition!of!NPK!fertilizer!amounted!to!0.21%,!in!the!addition!of!biofertilizer!of! 0.14%!and!the!media!of!garden!soil!only!0.02%.! Root!length!measurement!results!are!shown!in!Figure!6.!Based!on!figure!6!it!can!be! seen! that! the! positive! control! effect! on! long! root! growth! with! the! highest! value! when! compared!with!treatment!of!biofertilizer!and!negative!control.!OneAway!ANOVA!results!show! that!the!probability!value!is!less!than!0.05,!so!the!biofertilizer!gives!effect!to!the!root!length! of!the!mustard!so!that!The!Tukey!test!was!done!at!95%!(α!=!0.05!)!By!using!Minitab!software.! Tukey!test!results!are!shown!in!Table!4.! ! ! ! ! & & & & & & & & & Figure&6.&Result&of&Root&Length& Le n gt h &R o o t& (c m ) Tratment International'Journal'of'Applied'Biology,'1(2),'2017' ! 54! Table&4.&Root&Length&at&Harvest&Time& Treatment& Root&length&(cm)& Positive!Control! 7,333a! Azotobacter'Bacillus'Candida' 5,000ab! Candida' 5,000ab! Bacillus'Candida' 4,867ab! Azotobacter'Candida' 4,233ab! Bacillus' 4,000ab! Azotobacter' 3,333b! Negative!Control! 2,933b! *Figures!followed!by!different!letters!show!significant!differences!based!on!the!Tukey!test!at! the!95%!confidence!level!(α!=!0.05).! ! Based!on!Table!4!it!can!be!seen!on!the!biofertilizer!treatment!of!Azotobacter!Bacillus! Candida!consortium!and!single!culture!Candida!showed!the!highest!result.!Candida!is!able!to! decompose! the! organic! compounds! in! the! soil! and! phosphate! solubilizer,! Azotobacter! potential! for! nitrogen! fixation! and! also! have! siderophores,! are! complexing! or! specific! compounds!of!chelating!Fe!3+!or!iron!to!bind!elemental!iron!in!the!rhizosphere,!so!it!is!not! available!for!the!development!of!pathogenic!microbes.!Bacillus!sp.,!potential!as!a!phosphate! solubilizer!and!root!biological!controller.!It!is!also!appropriate!with!the!test!results!of!the! phosphate!(P2O5)!shows!that!the!content!of!phosphate!in!the!soil!with!the!addition!of!NPK! fertilizer!is!0.48%,!biofertilizer!is!0.41%!and!garden!soil!media!is!0.31%.! The!dry!weight!of!mustard!plants!in!this!study!is!indicated!by!weighing!the!total!dry! weight!of!the!harvested!crops!(roots,!stems!and!leaves).!The!results!of!the!measurement!of! the! dry! weight! of! plants! can! be! seen! in! Figure! 7.! Based! on! the! oneAway! ANOVA! test,! biofertilizer!has!an!effect!on!the!dry!weight!of!the!plant.!This!is!indicated!by!the!value!of!pA value!(P!<0.05)!for!dry!weight!is!0.002.!Then!proceed!with!Tukey!test!to!know!which!treatment! has!a!significant!influence!on!the!treatments!that!have!been!done.!The!average!result!of!dry! weight!measurement!can!be!seen!in!Table!5.! & Figure&7.&Result&of&Biomass& & & & & & B io m as s& (g ra m ) Treatment International'Journal'of'Applied'Biology,'1(2),'2017' ! 55! Table&5.&Biomass&at&Harvest&Time& Treatment& Dry&weight&(gram)& Positive!Control! 3,573a! Bacillus'Candida' 2,290ab! Azotobacter'Candida' 2,060b! Candida' 1,810b! Bacillus' 1,713b! Azotobacter'Bacillus'Candida' 1,766b! Azotobacter' 1,463b! Negative!Control! 1,433b! *Figures!followed!by!different!letters!show!significant!differences!based!on!the!Tukey!test!at! the!95%!confidence!level!(α!=!0.05).! ! Table!5!shows!that!treatment!of!Candida'Bacillus!consortium!has!the!highest!value!on! the! dry! weight! parameters! when! compared! with! other! biofertilizer! treatment! and! not! significantly!different!from!the!positive!control!treatment!with!the!addition!of!NPK.!This!is! because!Bacillus!has!the!potential!as!a!phosphate!solubilizer!by!releasing!a!number!of!organic! acids!and!Candida!has!the!potential!to!decompose!organic!matter!in!the!soil.! Dry!weight!is!also!associated!with!carbon!compounds!that!build!plant!organs.!This! correlates!with!the!content!of!C!in!soil.!The!result!of!the!research!is!in!accordance!with!the! amount!of!soil!C!content!that! indicating!the!content!of!Carbon!element!on!soil!with!the! addition!of!NPK!fertilizer!is!2.05%,!the!addition!of!biofertilizer!is!1,98%!and!the!garden!soil!is! 1,62%.! & Conclusions& The!results!showed!that!all!combination!of!yeast!and!bacteria!as!biofertilizer!effect!on! mustard! growth! when! compared! with! the! treatment! without! biofertilizer.! Candida! and! Bacillus!consortium!has!the!highest!value!of!13,6!cm!at!plant!height.!The!Azotobacter!Candida! consortium!on!the!parameters!of!the!leaf!area!and!number!of!leaves!showed!the!highest! yields!with!values!of!20.3!cm!and!5.3,!respectively,!and!the!Azotobacter!Bacillus!Candida! consortium!showed!the!highest!value!of!5.0!cm!on!the!root!length!parameter.!The!Bacillus! Candida!consortium!showed!!the!highest!value!of!2.29!grams!in!dry!weight.! & Acknowledgment&& This!work!was!conducted!as!part!of!“Superior!and!Commercial!Biofertilizer!of!Marine! Yeast!and!Bacteria”.!This!research!was!supported!by!grants!from!Pemula!Research!2017! awarded!by!ITS!local!fund!2017.!! & References& Agamy,!R.,!Hashem,!M.!&!Alamri,!S.!2013.!Effect!Of!Soil!Amendment!With!Yeasts!As!BioA Fertilizers!On!The!Growth!And!Productivity!of!Sugar!Beet.'African'Journal'of'Agricultural' Research,!8(1).! Ali,S.S! &! Vidhale,! N.N.! 2013.! Bacterial! Siderophore! and! their! Application:! A! review.! Int.J.Curr.Microbiol.App.Sci,!2(12):303A312.! International'Journal'of'Applied'Biology,'1(2),'2017' ! 56! Amprayn,!K.O.,!Rose,!M.T.,!Kecskes,!M.,!Pereg,!L.,!Nguyen,!H.T.!&!Kennedy,!I.R.!2012.!Plant! Growth! Promoting! Characteristics! of! Soil! Yeast! (Candida' tropicalis! HY)! and! its! Effectiveness!for!Promoting!Rice!Growth.!Journal'Of'Applied'Soil'Ecology,!61:!295–!299.! Anubrata,!P.!&!Rajendra,D.!2014.!Isolation,!Characterization,!Production!of!Biofertilizer,!and! Its!Effect!on!Vegetable!Plants!with!and!without!Carrier!Materials.!International'Journal'of' Current'Research,!6:7986A7995! Brock,! T.D.! &! Madigan,! M.T.1991.! Biology! of! Microorganisms.! Sixth! ed.! Prentice! Hall! International,!Inc! CavalierASmith,!T.!1998.!A!Revised!SixAKingdom!System!Of!Life.!Biological'Reviews,!73:203– 66.! Devi,M.,!Sharma,H.K.,!Rana,!K.!&!Mehta,D.K.!2017.!Studies!on!Flower!Biology!and!Pollination! in!Mustard!(Brassica'juncea'L.).!International'Journal'of'Science'and'Nature,!8(1):35A39.! Duraisam,R.!Salelgn,K.!&!Berekete,!A.K.!2017.!Production!of!Beet!Sugar!and!BioAethanol!from! Sugar!beet!and!it!Bagasse:!A!Review.!International!Journal!of!Engineering!Trends!and! Technology!(IJETT).!43(4)!:!222A233.! Fleet!G.H.!1992.!Spoilage!Yeasts.!Critical'Reviews'in'Biotechnology,!12(2):1A20.! Lucy,! M.,! Reed,! E.! &! Glick,! B.R.! 2004.! Application! of! Free! Living! Plant! GrowthAPromoting! Rhizobacteria.!Antonie'Van'Leeuwenhoek,!86:!1–25.! Maheswar,!N.!U.!&!Sathiyavani,!G.!2012.!Solubilization!of!Phosphate!by!Bacillus!Sps,!from! Groundnut!Rhizosphere.'Journal'of'Chemical'and'Pharmaceutical'Research,!4(8):4007A 4011! Masso,!C.,!Ochieng,!J.R.A.!&!Vanlauwe,!B.!2015.!Worldwide!Contrast!in!Application!of!BioA Fertilizers!for!Sustainable!Agriculture:!Lessons!for!SubASaharan!Africa.!Journal'of'Biology,' Agriculture'and'Healthcare,!5!(12).! New,M.T.,!Yu,S.S.!&!Latt,!Z.K.!2013.!Study!on!Phosphate!Solubilization!of!Salt!Tolerant!Soil! Yeast!Isolates!and!Effects!on!Maize!Germination!and!Growth.!International'Journal'of' Advances'in'Applied'Sciences,'2(3):157A164.! Ng’etich,!Niyokuri,!O.K.A.N.,!Rono,!J.J.,!Fashaho,!A.!&!Ogweno!J.O.!2013.!Effect!of!Different! Rates! of! Nitrogen! Fertilizer! on! the! Growth! and! Yield! of! Zucchini! (Cucurbita' pepo' cv.' Diamant'L.)!Hybrid!F1!in!Rwandan!High!Altitude!Zone.'International'Journal'of'Agriculture' and'Crop'Sciences.!5!(1)!:!54A62.! Noel,! D.D.,! Nafan,D.,! Souleymane,! S.,! JeanALuc,! M.A.,! Jesus,! F.I.! &! Lamine,! B.M.! 2016.!! Combination!of!Rizhobacteria!and!Foliar!BioAFertilizer!Accelerating!Maize!and!Soybean! Crop! Plants! Growth! Process! in! Arid! Soil.! Academia' Journal' of' Agricultural' Research,! 4(7):446A456.! Park,!Y.,!Mun,!B.,!Kang,!S.,!Hussain,!A.,!Shahzad,!R.,!Seo,!C.,!Kim,!A.,!Lee,!S.,!Oh,!K.Y.,!Lee,D.Y.,! Lee,!I.!&!Yun,!B.!2017.!Bacillus'aryabhattai'SRB02!Tolerates!Oxidative!and!Nitrosative! Stress! and! Promotes! the! Growth! of! Soybean! by! Modulating! the! Production! of! Phytohormones.!Plos'One,!12(3).! Paszt,! L.S.,! Derkowska,! E.,! Trzciński,! P.,! Przybył,! M.! &! Weszczak,! K.! 2015.! Influence! of! Biofertilizers! on! Plant! Growth! and! Rhizosphere! Microbiology! of! GreenhouseAGrown! Strawberry!Cultivars.!Acta'Sci.'Pol.'Hortorum'Cultus,'14(6):83A96.! Paulton,!R.J.L.!1991.!The!Bacterial!Growth!Curve.!J'Biol'Educ,'25:92–93.! Plaster,!E.!J.!2003.!Soil!Science!and!Mangement.!Delmar!Learning!Inc.!4th!ed!United!States.! Smith,!S.E.!&!Read,!D.J.!2008.!Mycorrhizal!Symbiosis,!3rd!ed.!New!York,!London,!Burlington,! San!Diego:!Elsevier!and!Academic.! International'Journal'of'Applied'Biology,'1(2),'2017' ! 57! To&cite&this&article:&& Alami,! N.H.,! Ayu,! S.A.T.,! Kuswytasari,! N.D.,! Zulaika,! E! &! Shovitri,! M.! 2017.! Effect! of! Yeast! Based! Biofertilizer!combined!with!bacteria!on!Mustard!Plant!Growth.!International'Journal'of'Applied'Biology.! 1(2):46A57.! & Vessey,!J.K.!2003.!Plant!Growth!Promoting!Rhizobacteria!as!Biofertilizers.!Plant'Soil,!255:!571– 86.! Wang,!X.,!Pan,!Q.,!Chen,!F.,!Yan,!X.!&!Liao,!H.!2011.!Effects!of!CoAinoculation!with!Arbuscular! Mycorrhizal!Fungi!and!Rhizobia!on!Soybean!Growth!as!Related!to!Root!Architecture!and! Availability!of!!N!and!!P.!Mycorrhiza.'21:173–181.! Wani,!S.A.,!Subhash,!C.!&!Tahir,!A.!!2013.!Potential!Use!Of!Azotobacter'Chroococcum!in!Crop! Production.!Current'Agriculture'Research'Journal,!1(1).! Xiao,!C.,!Chi,!R.,!Pan,!X.,!Liu,!F.!&!He,!J.!2013.!Rock!Phosphate!Solubilization!by!Four!Yeast! Strains.'Annals'of'Microbiology,!63:173A178! ! ! ! ! ! ! ! !