1. Proceedings of the 3rd Annual FEP Honors Research Symposium
Copyright, 2011, Johnson,BAJ. Pleasedo not usethematerials without expressed permission oftheauthors.
Johnson, BAJ 1
Identification and Quantification of Sugars in Algae
Blake Johnson
Ralph E. Martin Department of Chemical Engineering
Mentors:
Ching-Shuan Lau
Department of Biological and Agricultural Engineering
Danielle Julie Carrier, Ph.D.
Department of Biological and Agricultural Engineering
Abstract
With gas priceson the rise, ethanol andother alternative fuelsare increasingindemand.
However, the mainsource of starch feedstockfor ethanol production, corn, is increasing inprice
and causing foodshortagesinparts ofthe world. Inorder to fix this problem, new sources of
feedstock for ethanol production are necessary. Non-starchfeedstock, such as lignocellulose, like
swichgrass, poplar andalgae canserve as a source of sugars that can be convertedto ethanol.
Acquiringlignocellulose from cheapandabundant sources wouldalsogreatlybenefit ethanol
production, as some sources canbe costlyifnot producedandharvested bythe production plant
making the ethanol. Determiningifalgae is a source of lignocellulose carbohydrates, as well as
refining the pretreatment process ofproducingethanol, are the goals for our research.
1. Motivation
Oil isa resource thatisharmingour economyandecosystem.If we,humanity,wishtosurvive and
prosper,we have tofindwaysto breakaway fromour dependencyonoil andgasoline.One solutionto
our oil dependencyisusingalternativefuels,and one of the mostcommonlyusedalternative fuelsis
ethanol. Currently,the mainsource of ethanol isfromcornstarch. Making ethanol fromcorn has caused
the price of corn to rise.Ithas alsocaused foodshortagesincertainparts of the world. Lignocellulosic
feedstockspresentanalternativesugarsource thatcan be fermentedtoethanol. Examplesof biomass
include switchgrass,poplar,cornstover, or sweetgum.Thismaterial canbe deconstructedintotheir
monomeric(simple)sugars, suchasthose that we extractfrom corn, whichcan be fermentedinto
ethanol. The use of lignocellulose material wouldnotonlyeliminate the needforcorninthe production
of ethanol,butalsowouldcreate more jobsinthe country.
2. Background Information
The rate withwhichwe have usedgasoline hascausedthe price togo up, compoundedby the fact that
it isan politicallyunreliable source of fuel thatwilleventuallyrunout. We mainlydependonoil from
international sourcesandoil wellsthatwillnotlastmore thana few more decades. Inordertoreplace
gasoline,effortshave beeninvestedintothe productionof ethanol,adomesticgrown,plant-based
motor fuel.However,currentlythe mainsource of ethanol isfromcorn,whichhascauseditsprice to go
up (1) and the quantityof corn in parts of the country to go down,causingfoodshortages.

2. Johnson, BAJ 2
Recently,the EPA (EnvironmentalProtectionAgency)increasedthe percentageof ethanol thatcanbe
mixedwithgasoline to15% (2).The EPA alsoreducedthe amountof corn that isallowedtobe usedin
the productionof ethanol. These twoeventshave increasedthe demandforethanol,aswell as
increasingthe incentive touse methodsof producingethanol otherthancorn.
Research isbeingdone toextractthe sugarsfromlignocellulose materialsasopposedto extractingit
fromcorn. Lignocellulosemakesupthe cell wallsof plants,andis composedof complicatedanddurable
strandsof sugars,like celluloseandhemicellulose.The mainissue withthisisthatthe cost of producing
ethanol fromthese well wovensugars issubstantiallyhigherthanbothgasoline andcornbasedethanol.
Anotherissue isthat the processesinvolvedinthe release of these sugars are alsomuchmore delicate.
The firstpart of the processis pretreatment,where the cropinuse isplacedindilute acid.The dilute
acid pretreatmentissimplyanexampleasthere are variouspretreatmenttechnologies. Inthisprocess,
the hemicellulose breaksdownintoxylose, leavingbehindthe cellulose andlignin.The solid,cellulose
and lignin,getshydrolyzedwithenzymesintoglucose.The releasedsugars, mainly glucose, are then
fermented toproduce ethanol. Inthe pretreatment,inwhichwe will mainlybe involvedintesting,if the
solidmassto sulfuricacidratioisnot right,then twodifferentextremescouldoccur.If the acid ratiois
too low,thenthe sugars,mainlyxylose, won’tbe fully released,whereasif the ratioistoohigh,then
there will be toomuchbyproductformation.
Our researchisto quantifythe amountof sugarsreleasedinalgae bypretreatingthe driedbiomasswith
dilute acidandhydrolyzingthe pretreatedpelletwithcellulase enzymes.The released- sugarswill be
quantifiedbyhighpressureliquidchromatography(HPLC).We were fortunate tohave accesstoa series
of samplesthatwere obtainedfromthe NolandWaste WaterTreatmentFacility(NWWTF) locatedin
Fayetteville,AR. Representativemonthlygreenalgae sampleswereobtained fromMayto September.
3. Objectives
The objectivesforthisresearchwill be todetermine if the (NWWTF) algae containscarbohydrate that
can laterbe fermentedtoproduce ethanol.If itisdeterminedthatthisalgae containscarbohydrates,we
will quantifyandidentifythe differentcarbohydrates.We will alsodetermine if the algae containsoil
that can be usedto produce biodiesel.Finally,we will testif the oil andcarbohydrate contentof the
algae variesas a functionof the summerseason isdependentonthe seasons,asthe algae’sgrowth
couldbe affected bydifferenttemperaturesandseasons.
4. Process
The part of the extractionprocessthatwe are dealingwithiscalledthe pretreatmentprocess.Itbegins
by measuringthe algae andplacingitina stainlesssteel reactortube with 1% dilute sulfuricacid.The
reactorsare thenheatedina sand bath to 160o
C for one hour.Afterwards,the reactorsare flashcooled
inwater thencooledina coldroom.Once the reactorsand the product inside are cool,theyare
removedandseparatedintotheirsolidandliquidportions.The solidmasscanthenbe treatedwith
enzymesandthenanalyzedforsugarafterwards.The liquidportionwill be mixedwithcalcium
carbonate,inorderto stabilize the pHto around6. Once the pH has stabilized, the samplesare placed
on the HPLC instruments,,whichwill tell usexactlywhatsugarsandbyproductsare presentinthe
sample,aswell ashowmuch of each. We had to run the exudatesontwoseparate HPLCs:one was
configuredforcarbohydrate analysiswhilethe otherwasset-uptoanalyze forby-productcompounds.

3. Johnson, BAJ 3
The other part of our researchisextractingoil.Todo this,we measure oursample andplace itin a
cellulosepaperthimble,whichwillholdouralgae while allowingliquidandoil topass through.We then
measure out200 mL of hexane,a6 carbon alcohol,whichisplacedinaflaskthat goesunderthe algae.
The entire apparatus,or Soxhlet,issealedwiththe exceptionof asmall hole atthe top, to prevent
pressure buildup.The hexane isboiledandevaporated,whereittravelsupthe Soxhletandcondenses
inthe topchamber,where coldwateron the outside of the Soxhletcoolsthe hexane toliquid
temperature.Itthendripsintothe chamberwiththe algae,where itcyclesbackintothe bottomflask,
takingsome of the oil withit.Thisprocesscontinuesfor twohours,cyclinghundredsof times.Afterthe
twohours,the hexane isevaporated,leavingjustthe oil behind. Becausewe hadcarefullyweighedthe
emptyflaskandthe lipidcontainingflask,we were able todetermineviamassdifferencesthe massof
oil.
5. Results
Mass % of Sugar Obtained
Mg presentin 2000 mg of Algae May June July August September
LiquidFraction Glucose 69.69 24.63 24.47 - 24.21
After dilute acid Xylose 33.63 43.96 40.83 - 40.15
pretreatment FormicAcid 120.45 75.07 82.00 42.67 169.40
AceticAcid 55.03 64.34 35.94 15.37 88.13
Furfural 1.83 1.42 0.89 0.20 1.50
Hydroxyl methyl furan
(HMF) 1.98 2.25 1.52 0.70 2.97
SolidFraction Glucose 37.79 47.75 36.57 - 45.74
After enzymatic Xylose 2.33 35.30 27.42 - 32.12
hydrolysis
Total Glucose 107.48 72.38 61.04 - 69.96
Xylose 35.96 79.25 68.25 - 72.27
FormicAcid 120.45 75.07 82.00 42.67 169.40
AceticAcid 55.03 64.34 35.94 15.37 88.13
Furfural 1.83 1.42 0.89 0.20 1.50
Hydroxyl methyl furan
(HMF) 1.98 2.25 1.52 0.70 2.97
Yield(gsugar / g algae) Glucose 5.4% 3.6% 3.1% - 3.5%
Xylose 1.8% 4.0% 3.4% - 3.6%

4. Johnson, BAJ 4
Mass % of Oil Obtained
May June July August September
Mass of oil (mg) in recoveredin3
gram of algae
30.00 62.60 ± 10.75 21.00 51.30 ± 0.42 40.50 ± 7.78
% Oil Recovered/ gram algae 1.0% 2.1% 0.7% 1.7% 1.3%
6. Discussionsand Conclusion
Driedalgae samplescontainedxyloseandglucose inverysmall concentration(<10% of the overall
mass).
Algae samplesharvestedinJune andJulycontainedoil,asshownbythe yellowishcolorof hexane
solvent.The oil contentvariedbetween0.7% and2.1% of the driedalgae mass.
The concentrationof formicacid wasunusuallyhighandwouldbe problematicinthe fermentationstep
as formicacid isdocumentedtoinhibitthe fermentationstep.
The oil contentin driedalgae islessthan 3% massof the overall algae mass,whichislowerthanalgae
usedforbiofuel thattypicallycontains30% oil. Itisimportantto note that the algae contain30% oil is
usuallyculturedinbioreactorsandnotinalgae raceways,like thatof the (NWWTF) situation.
Basedon the amountof oil,formicacid and sugarextracted,thisparticulartype of algae wouldnot
serve asan ideal source of biomassforbiofuelsproduction.
7. WorksCites
(1) http://www.nytimes.com/2007/01/05/business/05ethanol.html?_r=1
http://chemicallygreen.com/corn-ethanol-higher-food-prices/
(2) http://www.epa.gov/otaq/regs/fuels/additive/e15/