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Process Biochemistry 46 (2011) 1682–1687

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Process Biochemistry

j ou rna l ho me pag e : www.e l sev i e r. com/ loca t e /p rocb io

Short communication

Effect of inoculum/substrate ratio on mesophilic anaerobic digestion of bioethanol plant whole stillage in batch mode

Cigdem Eskicioglu ∗, Maryam GhorbaniSchool of Engineering, University of British Columbia Okanagan Campus, 3333 University Way, Kelowna, BC, Canada

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Article history:Received 31 December 2010Received in revised form 10 April 2011Accepted 26 April 2011

Keywords:Anaerobic digestionBioethanol plant residuesInoculum to substrate ratioMethane productionWhole corn stillage

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A study of anaerobic digestion of whole stillage from a dry-grind corn ethanol plant was conducted to

evaluate

the

possibility

of

replacing

fossil

fuel

input

of

large

ethanol

plants

by using

ethanol

residues

asdigester feedstock. The effect of inoculum to substrate ratio (ISR) on biogas/methane production rates andultimate yields was evaluated in mesophilic batch digesters. A rst order kinetic model was evaluated forboth volatile solids (VS) and total chemical oxygen demand (TCOD) removals. The results demonstratedthat in an ISR range of 3.67–0.46 g/g on VS basis, kinetic constants (k) for both VS and TCOD removalsdecreased signicantly, indicating an initial reactor overloading or substrate inhibition. However, despitethe slower biodegradable rates from the reactors with ISRs of 0.46 and 0.92, none of the reactors expe-rienced a chronic substrate inhibition. At the highest organic loading (ISR of 0.46 g/g), degradation wascomplete in 15–16 days. Biochemical methane potential assays indicated signicant digestion potential(691–788 mL biogas and 401–458 mL CH4 per g VSadded at 0 ◦ C, 1 atm) with organic removals between76% and 94% in batch mode. Future studies will verify the anaerobic digestion potential of full-strengthwhole stillage at larger scale.

© 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Countries throughout the world are looking for alternativesource of energy that can be generated locally. In recent years,mainly due to uncertain fuel supply and regulations to reducegreenhouse gas emissions, bioethanol has become one of the mostpromising technologies [1] . Furthermore, the corn based dry grindprocess is the most commonly used method in the North Americafor fuel ethanol production.

One of the major issues of bioethanol production is the disposalof stillage produced as a by-product. In a typical dry grind ethanolprocess, rst, corn grains are mixed with water and cooked duringthe liquefaction step.Then, resultinghammer corns are mixed withenzymes or bacteria and converted to fermentable sugars throughenzymatic hydrolysis process (saccharication). Hydrolyzed sub-strate is fermented with yeast to produce bioethanol and CO 2 .Bioethanol goes through distillation and evaporation process to

Portions of this paper were presented at IWA World Water Congress and Exhi-bitionheld in Montreal, Canada, September 19–24, 2010.∗ Corresponding author at: School of Engineering, The University of British

Columbia Okanagan Campus, 3333 University Way, Ofce: PBR 003, Kelowna, BC,Canada V1V 1V7. Tel.: +1 250 807 8544;fax:+1 250 807 9850.

E-mail addresses: cigdem.eskicioglu@ubc.ca , cigdem.eskicioglu@gmail.com(C. Eskicioglu), maryam.ghorbani@ubc.ca (M. Ghorbani).

be converted to fuel ethanol with high purities. The fermentationresidue, remainingafter bioethanol is removed, is called whole stil-lage with 12% total solids (TS). Then, whole stillage is centrifugedto produce wet cake and thin stillage with 6% TS. Usually, half of the thin stillage is evaporated to syrup and then mixed with cen-trifuged solids to produce Distiller’s Dried Grains with Solubles(DDGS) which can be sold as livestock feed. The remaining half isrecycled back to be used for corn saccharication.

There are very limited studies reported about methane poten-tial of whole stillage from bioethanol plants using corn as a feedstock for energy recovery/reuse while previous studies focused onanaerobic treatment of thin stillage [2] . Recent studies indicatedthat because of the high organic content, anaerobic digestion of thin stillage offers a prospect of nancial return from methaneproduction [3,4] . While biogas from thin stillage could be used forproduction of electricity and the waste heat for drying the wholestillage before it is used as animal feed, if corn to ethanol ramps up,the animal feed market will be quickly saturated. Whole stillage isgood cattle feed, however because it must be fed fresh daily, goodmanagement is needed. This is already creatingchallenges for largeethanol plants located in remote areas with no farming communi-ties nearby. Therefore alternative methods should be developed tohandle the whole stillage, a renewable resource with ve timeshigher TCOD than that of thin stillage.

The growing interest on anaerobic digestion processes forenergy recovery created a need for an established procedure to

1359-5113/$ – seefront matter© 2011ElsevierLtd. All rightsreserved.

doi: 10.1016/j.procbio.2011.04.013

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1684 C. Eskicioglu, M. Ghorbani / Process Biochemistry 46 (2011) 1682–1687

Fig. 1. Effectof inoculum to substrateratio(ISR)on (a)specic biogasproduction rate, (b)ultimatebiogasyieldfrom whole corn stillage (data representthe mean anderrorbars represent absolute difference between mean and duplicates, respectively).

2.4. Statistical analysis

Standard statistical procedures were used, including standard deviation, meanaveraging and absolute difference. When signicant difference analysis wasrequired, a one-sided t -test was used, with p ≤ 0.05 considered signicantly dif-ferent.

2.5. Kinetic modelling

Anaerobic digestion is commonly expressed as a rst-order reaction. The sub-strate utilization rate, r su [mg/Ld], of anaerobic digesters in the BMP test can berepresented by rst-order kinetics (Eq. (1) ):

r su =dC

dt = − kC (1)

in which C is theamount of organics (mg TCOD/L or mg VS/L)and k is theanaerobicdegradation rate constant (d − 1 ). Integration and rearrangement of Eq. (1) yields:

Lt = Lu (1 − e − kt ) (2)

in which Lt is the amount of organics removed at time t (mg TCOD/L or mg VS/L)and Lu is ultimate biodegradable organics (mg TCOD/L or mg VS/L) in the sampleand t is thedigestion time (d). In this study,kinetic coefcient ( k) fordifferent ISRs,the amount of TCOD or VS removed with respect to time, Lt , was calculated fromthe biogas yield (biogas production per gram of TCOD or VS) of the digesters andcumulative biogas production. The coefcientof determination, R2 , was usedastheprimarydiscriminatorto evaluatethe adequacyof talong withthe visualjudgment.

3. Results and discussion

3.1. Anaerobic biodegradation rate and extent

Results from the BMP assays were used to evaluate the

mesophilic digestion rate and extent with different ISRs. The spe-cic biogas production rate in BMP bottles decreased signicantlywith decreasing ISR or with increasing substrate concentration intherst6 daysof digestion, indicating an initial reactor overloadingor substrate inhibition process ( Fig. 1a). The methane percentageinBMP bottles increasedfrom zero to nal valuesranging from 45%to 65%, depending on the ISR ratios ( Table 2 ). Despite the signi-cantly slower biodegradation rates ( p < 0.05) at lower ISRs between1 and 6 days of digestion ( Fig. 1a), all BMP bottles had similar spe-cic methane yields ( p >0.05) at the end of 22 day, indicating thatin an ISR range of 3.67–0.46g/g, ISRs did not affect the ultimatebiodegradability of corn whole stillage ( Fig. 1b). Despite similarultimate methane yields, Fig. 1b indicated statistically signicant( p < 0.05) differences among the ultimate biogas yields indicating

that batch bottles contained different biogas compositions at dif-

ferent ISRs. This conrms lower average methane percentages inthe biogas samples for the higher ISRs reported above.

Using the Microsoft Excel Solver tool which has a nonlinearoptimization code, k values from mesophilic batch digesters withISRs were estimated for both TCOD and VS removals ( Fig. 2). Thesquared correlation coefcient, R2 , was generally close to unity,being greater than 0.92 for all cases. For visual observation, pre-dicted TCOD and VS removals from digesters with different ISRswere also plotted along with the observed values ( Fig. 3a–d). Sim-ilar to specic biogas production rates, kinetic constants ( k) forTCOD and VS removals decreased signicantly with decreasing ISR (Fig. 2). This was also conrmed by poorer organic removal predic-tions by the rst order kinetics for reactors with ISRs of 0.46 and0.92 in Fig. 3c and d, respectively. Specically, k values decreased

more than 1.8 times when the ISR was reduced from 3.67 to 0.46.The reactors with an ISRof 0.46 experienced total volatile fatty acid(TVFA) accumulations and slight pH decrease while other reactorshad negligible TVFAs in the rst 8 days of digestion ( Table 2 ). Thisanalogous behaviour have been reported in several papers such as

Fig. 2. Variationof thebiodegradationkinetic constantsfor TCOD and VS removalsas a function of the inoculum to substrate ratio (ISR) (data represent arithmeticmean of duplicates and error bars represent the absolute difference between the

mean and thesamplevalues).

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Table 2Reactor characteristics during batch mesophilic digestion.

Inoculum to substrateratios (gVS/gVS)

0.46 0.92 1.83 3.67

Ultimate CH 4 percentages (%) 65.4 (0.5; 2) a 59.3 (1.2; 2) 54.8 (1.2; 2) 45.7 (0.5; 2)Ultimate VS removal (%) 83 (1; 2) 87 (2; 2) 86 (1; 2) 94(1; 2)Ultimate TCOD removal (%) 86 (3; 2) 88 (0; 2) 76 (11; 2) 86 (11; 2)Volatile fatty acids (mg/L) 1435 (38; 2) 452 (24; 2) 201 (2; 2) 117 (21; 2)Reactor pH (–) b 6.7 (0.0; 2) 7.0 (0.4; 2) 7.0 (0.2; 2) 7.1 (0.1; 2)

a Data representarithmetic mean of replicates (standard deviations; number of data points). VS:volatile solids; TCOD: total chemical oxygen demand.b Arithmetic average of data collected in therst 8 days of digestion.

for anaerobic digestion of wastewater from a cellulosic pulp pro-duction from wheat straw [20] , anaerobic digestion of sunoweroil cake in batch reactors, untreated molasses [21] , and two-phaseolive mill efuents [14,22] in batch reactors. However, Fig. 1b alsosuggests that, despite of slower biodegradation rates for reactorswith ISRs of 0.46 and 0.92, none of the reactors experienced achronic substrate inhibition. At the highest organic loading (ISR of 0.46g/g), degradation was complete in 15–16 days; an additionalweek of digestiondid not display any signicant changes in organicremovals although digesters continued producing small amounts

of biogas ( Fig.1 a). At the endof 22 days, digesters achieved 83–94%VS removals and 76–86% TCOD removals under mesophilic batchmode ( Table 2 ).

The production amount and characteristics of stillage changesignicantly depending on the feedstock as well as ethanolproduction process. Literature suggests that the stillage produc-tion can vary from 1.5L/L ethanol (barley and rice spirits) to20L/L ethanol (beet and cane molasses) [2] . Even within thecellulosic feedstock group, signicantly different stillage produc-tion values have been reported, such as 6–15L/L ethanol froma steam exploded timothy grass and 16.7L/L ethanol from apine ( Pinus radiata ) after diluted acid treatment/fermentation [2] .Other studies reported 3.92L/L and 8L stillage/L ethanol valuesfor thin and whole stillage from corn-based bioethanol plants[4,16] . Comparison of anaerobic or aerobic treatment perfor-mances of conventional and cellulosic stillage is presented inTable 3 . Anaerobic treatment of ethanol stillage from conven-tional feedstock (corn, cane molasses, barley and sweet potato)has been cited as effective and economical treatment optionwith very high and comparable organic removal efcienciesranging from 78% to 97% VS or TCOD [4,16,23,26] . Anaero-bic methane yields from these studies ranged from 0.43 to0.75L/gVS removed with thermophilic digesters often achievinghigher than 0.60L/gVS removed (Table 3 ). Finally, the limited datareporting on anaerobic digestion of stillage from cellulosic feed-stock are also comparable to conventional stillage results with

high (85–87%) TCOD removal efciencies and methane yields(0.23–0.3 L/gTCOD removed ).

3.2. Energy analysis

The generated methane from thin or whole stillage digestionwillpartially or completely replace fuel,often natural gas,as energyinput in a bioethanol plant. Using an average methane yield of 0.43 ± 0.03 L/gVS added (or 49 ± 5L/L whole stillage) from Fig. 1b,mesophilic anaerobic digesters would have a methane production

of approximately 52millionm 3 from a 133 million L/year ethanolfacility. The whole corn stillage production from this ethanol facil-itycan be estimatedas 1064 million L/per year with 12% TS content.This also represents 1.94E + 15Joule/year of heating potential forthis facility with unit conversion factors of 35,310 British ther-mal unit (BTU)/m 3 methane and 1056 Joule/BTU. From literature,conventional ethanol plants with stillage evaporation requiresaround 8.3E + 6 Joule/L ethanol, while plants without stillage evap-oration would require 6.1E+ 6Joule/L of ethanol produced [4] .These correspond to energy consumptions of 1.11E+ 15Joule/yearand 8.13E + 14Joule/year for this facility with and without stillageevaporation. Therefore energy output from methane production(1.94E+ 15Joule/year) will achieve an energy balance of 2.4:1 asratio of output to input energy unit for this ethanol facility without

stillage evaporation. However, additional energy may

be requiredto recover make-up water from anaerobic digester efuent. Also,complete loss or considerable lower mass of produced animalfeed from stillage have not been considered in our calculations.Previously, a low-rate thermophilicanaerobicdigestion of thinstil-lage was reported to have an energy input reduction of 43% atthe bioethanol plant [4] . Another study on thermophilic anaer-obic sequencing batch reactors digesting thin corn stillage alsoestimated 43% energy input reduction due to methane recoverywithout accounting mass reductions in animal feed [3] . A higherenergy input reduction (60%) was anticipated by another study[28] due to methane recovery after incorporating available energy

Table 3Comparison of anaerobic or aerobic treatment performance of conventional and cellulosic stillage in literature a .

Feedstock Reactor type Temperature Methane yield Organic removal References

Corn ethanol whole stillage Batch reactor Anaerobic/35 ◦ C 0.50 ± 0.04 L/gVS removed 83–94% VS This studyCorn ethanol w hole s tillage Batch r eactor Anaerobic/55 ◦ C 0.60 ± 0.08 L/gVS removed 82–97% VS [16]Corn ethanol thin stillage CSTR Anaerobic/55 ◦ C 0.63–0.75 L/g VS removed 82.5–89.8% VS [4]Corn ethanol thin stillage Sonicated-CSTR Anaerobic/55 ◦ C 0.65–0.73 L/g VS removed 82.6–88.5% VS [4]Wheat stillage Batch reactor Aerobic/45 ◦ C n/a b 71.4% TCOD [23]Potato stillage Batch reactor Aerobic/20–63 ◦ C n/a 77.6–89.1% TCOD [24]Barley UFB reactor Anaerobic/53 ◦ C 0.27 L/g TCODremoved 78% TCOD [25]Barley and sweet potato UASB-2 phase reactor Anaerobic/37 ◦ C 0.28 L/g TCODremoved 80% SCOD [26]Wood ethanol stillage UASB reactor Anaerobic/37 ◦ C 0.302 L/g TCOD removed 86% TCOD [25]Eucalyptus wood stillage FFR Anaerobic/35 ◦ C 0.27 L/g TCODremoved 86.6% TCOD [27]Eucalyptus wood stillage FFR Anaerobic/55 ◦ C 0.26 L/g TCODremoved 84.4% TCOD [27]Eucalyptus wood stillage CSTR Anaerobic/35 ◦ C 0.23 L/g TCODremoved 85.5% TCOD [27]

a CSTR: continuous stirred tank reactor; FFR: xed lm reactor; TCOD; SCOD: total and soluble chemical oxygen demands; UFB: upow uidized reactor; UASB: upowanaerobic sludge blanket; VS: volatile solids.

b n/a: not available.

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Fig. 3. Comparison between experimental and theoretical organic removal values predicted by Eq. (2) f or ISRs of (a) 3.67g/g, (b) 1.83g/g, (c) 0.92g/g, and (d) 0.46g/g (datarepresentarithmetic mean of duplicatesand error bars representthe absolute difference between themean and thesample values).

from waste heat by not evaporating the thin stillage. Comparingthe results, anaerobic digestion of whole stillage presents morefavourable energy balance compared to digestion of thin stil-lage.

4. Conclusions

In an ISR range of 3.67–0.46g/g, rst order biodegradationrate constant of whole corn stillage decreased signicantly withdecreasing ISR, indicating an initial reactor overloading or sub-strate inhibition process. However, ISRs did not affect the ultimatebiodegradability of corn whole stillage with similar specicmethane yields at the end of 22 days of mesophilic digestion. TheBMP assays indicatedthat whole cornstillageis a suitable substratefor anaerobic digestion due to high organic removals achieved.

However follow-up studies should pursue the continuous-ow

anaerobic digesters at larger scales to verify the potential of thistechnology for the full-scale applications.

Acknowledgments

Theauthors thank Mr. Juan Marin and Dr. Kevin J. Kennedyat theUniversity of Ottawa, ON for their contributions duringdata gener-ation. This study was funded by the University of British ColumbiaOkanagan Internal Individual Research and British Columbia Min-istry of Labour and Citizens, Student Led Research Grants.

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