Conversion of municipal solid waste to carboxylic acids using a mixed culture of mesophilic...

Bioresource Technology 97 (2006) 47–56

Conversion of municipal solid waste to carboxylic acidsusing a mixed culture of mesophilic microorganisms

Cateryna Aiello-Mazzarri a, Frank K. Agbogbo b, Mark T. Holtzapple b,*

a Universidad del Zulia, Facultad de Ingenierıa, Coordinacion de Ciclo Basico, Departamento de Quımica, Maracaibo, Venezuelab Texas A&M University, Department of Chemical Engineering, College Station, TX 77840-3122, USA

Received 22 October 2004; received in revised form 10 February 2005; accepted 11 February 2005

Available online 12 April 2005

Abstract

Waste biomass was anaerobically converted to carboxylate salts by using a mixed culture of acid-forming microorganisms. Muni-

cipal solid waste (MSW) was the energy source (carbohydrates) and sewage sludge (SS) was the nutrient source (minerals, metals,

and vitamins). Four fermentors were arranged in series and solids and liquids were transferred countercurrently in opposite direc-

tions, which allows both high conversions and high product concentrations. Fresh biomass was added to Fermentor 1 (highest car-

boxylic acid concentration) and fresh media was added to Fermentor 4 (most digested biomass). All fermentations were performed

at 40 �C. Calcium carbonate was added to the fermentors to neutralize the acids to their corresponding carboxylate salts. Iodoform

was used to inhibit methane production and urea was added as a nitrogen source. Product concentrations were up to 25 g/L, with

productivities up to 1.4 g total acid/(L liquid d). Mass balances with closure between 93% and 105% were obtained for all systems.

Continuum particle distribution modeling (CPDM) was applied to correlate batch fermentation data to countercurrent fermentation

data and predict product concentration over a wide range of solids loading rates and residence times. CPDM for lime-treated MSW/

SS fermentation system predicted the experimental total acid concentration and conversion within 4% and 16% respectively.

� 2005 Published by Elsevier Ltd.

Keywords: MixAlco process; Municipal solid waste; Sewage sludge; Fermentation; Carboxylic acids; Mixed acids

1. Introduction

Many energy experts are concerned about the deple-

tion of petroleum, the main source of liquid fuels and

many chemicals. Oil prices are volatile and supplies

are unstable. Oil combustion releases carbon dioxide,

the most important greenhouse gas implicated in global

warming (Sterzinger, 1995; Klass, 1998). In contrast,

biomass is a source of liquid fuels that does not resultin a net increase of carbon dioxide in the atmosphere

(Holtzapple et al., 1997; Hileman, 1999) because bio-

mass growth removes from the atmosphere the same

0960-8524/$ - see front matter � 2005 Published by Elsevier Ltd.

doi:10.1016/j.biortech.2005.02.020

* Corresponding author. Tel.: +1 979 845 9708; fax: +1 979 845 6446.

E-mail addresses: [email protected] (C. Aiello-Mazzarri), m-holtzapple@

tamu.edu (M.T. Holtzapple).

amount of carbon dioxide that biomass combustiongenerates (Bungay, 1981; Sterzinger, 1995).

Another environmental problem is the generation

and accumulation of the following wastes: Municipal

solid waste (MSW)—in the United States (US), ap-

proximately 220 million tons of MSW were generated

in 1998, four million tons more than in 1997 (EPA,

1998). Municipal solid waste contains about 65% biode-

gradable components, such as paper, food scraps, andyard waste (EPA, 1998). For the purpose of this

research, we will refer to MSW as the biodegradable

organic fraction, which excludes glass, metal, plastic,

ceramic, rock, or soil also found in MSW. Sewage

sludge (SS)—SS is the residual solids from conventional

aerobic or anaerobic sewage treatment. The Environ-

mental Protection Agency (EPA, 1999) estimated that

mailto:[email protected]



48 C. Aiello-Mazzarri et al. / Bioresource Technology 97 (2006) 47–56

6.9 million tons of dry SS were generated in 1998 in the

US of which about 60% were land applied, composted,

or used as landfill cover. The remaining 40% was dis-

carded with no attempt to recover nutrients.

MSW is an excellent energy source, but lacks nutri-

ents. SS has nutrients, but lacks energy-yielding carbohy-drates. In this research, MSW and SS were combined, so

they complement each other, making the process an

attractive alternative for managing two different streams

that are produced in every community.

Holtzapple et al. (1997) have developed a process to

obtain fuels and chemicals from biomass, the MixAlco

process (Fig. 1). In this process, biomass is first pre-

treated with lime, and then a mixed culture of acid-form-ing anaerobic microorganisms produces carboxylate salts.

These salts are subsequently concentrated and thermally

converted to mixed ketones and finally hydrogenated

to mixed alcohols. The advantages of this process

include the use of low-value substrates without the

use of a sterile environment or the need for enzyme addi-

tion.

A countercurrent fermentation (Fig. 2) allows theleast reactive biomass to contact the lowest carboxylic

acid concentration, which in batch fermentations could

not be digested because of accumulating carboxylic acid.

As the solids are transferred from one fermentor to the

next upstream fermentor (i.e., from F1 to F2, F2 to F3,

and F3 to F4), the biomass becomes less reactive and the

carboxylate salt concentration drops. This flow arrange-

ment reduces the inhibitory effect from the accumulating

WasteBiomass

Pretreatment Fermentation Concentration

ThermalConversion

HydrogenationMixed Alcohols

Fig. 1. MixAlco process.

F1

Fresh Biomass

ProductLiquid

F2F1

Fig. 2. Four-stage countercurrent system fermentation (F1: Fermento

carboxylate salts by adding fresh liquid to the most di-

gested biomass. Both high conversions and high product

concentrations are possible by using countercurrent

operation.

Long residence times are associated with countercur-

rent fermentations. Obtaining a wide range of operatingconditions experimentally would be tremendously time

consuming. Mathematical modeling of the counter-

current fermentations was performed using continuum

particle distribution modeling (CPDM) developed by

Loescher (1996). CPDM can predict the operating con-

ditions for the countercurrent fermentations from batch

fermentation data.

2. Methods

2.1. Fermentor

Each fermentor consisted of a 1-L plastic centrifuge

bottle (Beckman # 355676, Fisher, PA), which was

capped with a rubber stopper with a glass tube insertedthrough the stopper (Ross, 1998). The glass tube was

capped with a rubber septum for gas sampling and vol-

ume measurement. A large hole was drilled in the centri-

fuge bottle cap, so that when the cap was replaced it

could fit over the glass tube, and still be tightened to

hold the stopper in the bottle. Inside the bottle there

were two stainless steel (0.25-in. welded 304) tubes with

welded ends that mixed the components of the fermenta-tion. The fermentors were placed on a Wheaton Modu-

lar Cell Production Roller Apparatus (Model III,

Fisher, PA) located in an incubator and rotated at

1 rpm.

2.2. Substrates

2.2.1. Municipal solid waste (MSW)

To ensure uniform substrate composition through all

the MSW experiments, 136 kg of MSW were prepared.

To simulate the organic fraction of landfill waste (Holtz-

ProductSolid

Fresh Liquid

F3 F4

r 1, F2: Fermentor 2, F3: Fermentor 3, and F4: Fermentor 4).

C. Aiello-Mazzarri et al. / Bioresource Technology 97 (2006) 47–56 49

apple et al., 1992). The components of MSW were col-

lected (Bryan, College Station, TX), sun-dried, ground

and passed through a 10-mm screen in a hammer mill

(Forest Science Research Laboratory, Texas A&M Uni-

versity). Once combined, the MSW was ground and

passed through a 6-mm screen to ensure a uniform mix-ture. Fats and oils were not added to the MSW to pre-

vent spoiling during storage.

2.2.2. Sewage sludge

Aerobically treated sewage sludge was obtained from

Bryan Wastewater Treatment Plant Number 3 (Bryan,

TX). The sewage had undergone activated sludge treat-

ment in which the incoming sewage stream was sent toan aeration basin for approximately 15 d. After leav-

ing the aeration basin, the sludge was digested aero-

bically for approximately 40 d. Upon removal from

the digestor, the sludge was coagulated, dried for 10 d,

and ground in a hammer mill fitted with a 3-mm screen.

2.3. Lime treatment

The components of lignocellulose are complex and

include solubles, cellulose, hemicellulose, lignin, and

ash. As biomass digests, the remaining fraction is less

reactive (Gizjen et al., 1988), which is logical considering

that biomass is heterogeneous; some components are

highly digestible whereas others are almost inert. To

enhance digestibility, lime treatment was performed at

100 �C for 1 h with loadings of 0.1 g Ca(OH)2/g dry bio-mass and 10 mL of distilled water/g dry biomass. After

treatment, CO2 was bubbled through the biomass slurry

to neutralize the lime and the slurry was dried at 105 �C.

Detailed procedures of the lime treatment are available

in Aiello-Mazzarri (2002).

2.4. Media and nutrients

The fermentation media consisted of deoxygenated

distilled water, 0.28 g sodium sulfide/L distilled water,

and 0.28 g cysteine hydrochloride/L distilled water.

Dry nutrients were added to the fermentations. The

dry nutrient mixture used in all the experiments corre-

sponds to the modified Caldwell and Bryant medium

(Caldwell and Bryant, 1966).

2.5. Inocula

Rumen fluid was used as the main inoculum. Rumen

contents were removed from a fistulated steer (Univer-

sity Nutrition and Field Laboratory, Texas A&M Uni-

versity), filtered through four layers of cheesecloth into

1-L propylene centrifuge bottles, transported to the lab-

oratory, and inoculated (5% v/v) into each fermentorwithin 1 h after collection to ensure microorganism via-

bility. Other sources of microorganisms included swamp

material from Bee Creek Park (College Station, TX),

and compost material from domestic and commercial

piles. The compost samples were taken from the middle

of the pile to ensure a large quantity of anaerobic micro-

organisms. To minimize exposure to oxygen, the swamp

material and compost were collected into bottles filledwith deoxygenated distilled water.

2.6. Methanogens inhibitor

Iodoform (CHI3) was used as a methanogen inhibitor

in all fermentations. An iodoform solution (20 g CHI3/L

ethanol) was added individually to each reactor contin-

uously throughout the fermentations. Iodoform is lightand air sensitive, so the solution was kept in amber-

colored glass bottles and special care was taken to

replace the cap immediately after use.

2.7. Batch experiments

Batch experiments were performed to determine

experimental reproducibility, and to collect the neces-sary data for continuum particle distribution modeling

(CPDM). These data were used to develop a model rate

equation for the countercurrent fermentation. The pro-

cedure involved operating five different fermentors with

different initial substrate concentrations (20, 40, 70,

100 g dry substrate/L liquid). An additional fermentor

with high substrate concentration (100 g dry substrate/

L liquid) and high carboxylic acid concentration(approximately 20 g carboxylic acids/L liquid) was eval-

uated. Samples were taken by removing the fermentor

from the incubator, collecting the gas, removing the

stopper, purging with nitrogen, measuring pH, centri-

fuging for 10 min, collecting a liquid sample under nitro-

gen purge, resealing the fermentor, and placing it back

in the roller apparatus. For the CPDM experiments,

the resulting carboxylic acid concentrations were con-verted into acetic acid equivalents to be used in the

modeling equations developed by Loescher (1996).

The batch carboxylic acid concentrations can be con-

verted to acetic acid equivalents (a)

a ðmol=LÞ ¼ acetic ðmol=LÞ

þ 1:75 � propionic ðmol=LÞ

þ 2:5 � butyric ðmol=LÞ

þ 3:25 � valeric ðmol=LÞ

þ 4:0 � caprioc ðmol=LÞ

þ 4:75 � heptanoic ðmol=LÞ ð1Þ

On mass basis, the acetic acid equivalent can be ex-

pressed as

Ae ðg=LÞ ¼ 60:05 ðg=molÞ � a ðmol=LÞ ð2Þ


2.8. Countercurrent fermentations

In countercurrent operation, liquid and solids flow in

opposite directions in four-fermentor trains. In the labo-

ratory scale, the fermentors operate in a semi-continuous

manner, whereas on an industrial scale, the fermentationwould be continuous. Countercurrent fermentations were

initiated as batch cultures under anaerobic conditions by

adding the substrates, calcium carbonate, urea, nutrients,

and inocula to deoxygenated water media in each fer-

mentor. The experiments were conducted as batch fer-

mentations until the culture was established (7–10 d).

Countercurrent operation was initiated with the transfer

of liquid and solids occurring every 1, 2, or 3 d. Counter-current fermentations were conducted at varying liquid

residence times (LRT) and volatile solid loading rate

(VSLR). The operating parameters for each fermentation

with lime-treated MSW/SS are shown in Table 1.

2.9. Reaction conditions

The fermentations were performed under anaerobicconditions at 40 �C. Every 3 d, after each liquid/solid

transfer, 2.0 g calcium carbonate was added to each fer-

mentor to neutralize carboxylic acids. To maintain

anaerobic conditions, nitrogen from a high-pressure

liquid-nitrogen cylinder (Praxair, Bryan, TX) was flushed

whenever the fermentors were open to the atmosphere.

The solid and liquid transfer procedures are detailed in

Aiello-Mazzarri (2002).

2.10. Analytical methods

The fermentor broth was analyzed by gas chromatog-

raphy to measure the concentration of carboxylic acids.

Table 1

Operating parameters for lime-treated MSW/SS countercurrent

fermentations

Fermentation trains A B C D E

LRT (d) 20.7 19.0 22.7 20.7 17.0

VSLR (g VS/(L liquid in

all fermentors d))

3.0 4.0 8.1 3.1 6.4

VS feed at each

transfer (g VS)

11.6 19.3 24.1 11.6 15.4

Solid feed at each

transfer (g dry)

14.4 24.0 30.0 14.4 19.2

Liquid feed to F4 at

each transfer (L)

0.15 0.20 0.10 0.15 0.10

Frequency of transfer Every 3 d

Iodoform addition rate

(mg iodoform added/L

liquid fed to F4)

5.33 8 8 5.33 8

Nutrients addition rate

(g dry nutrients added/L

liquid fed to F4)

1.33 1.00 2.00 1.33 2.00

Urea addition rate

(g urea added/L

liquid feed to F4)

1.0 0.75 1.5 1.0 1.5

The broth was mixed with equal parts of an internal

standard (4-methyl-n-valeric acid) and 3-M H3PO4.

The analysis was performed using an Agilent 6890 series

gas chromatograph (Agilent Technologies, Palo Alto,

CA) equipped with a flame ionization detector (FID)

and a 7683 series injector. A 30-m fused-silica capillarycolumn (J&W Scientific, Model # 123-3232 CX, Agilent

Technologies, CA) was used. The column head pressure

was maintained between 90 and 103 kPa (13–15 psig).

At every sample injection, the gas chromatograph tem-

perature program allowed the temperature to rise from

50 �C to 200 �C at a 20 �C/min rate. The temperature

was subsequently held at 200 �C for 10 min. Helium

was used as carrier gas, and the total run time per sam-ple was 17 min. The fermentation broth consists of a

mixture of carboxylate salts and carboxylic acids. This

analytical procedure converts all salts to their corre-

sponding acids, allowing product concentrations to be

reported as g carboxylic acid/L.

Gases produced during fermentation were accumu-

lated within the reactor. Every sampling day, the volume

of gas produced since the last transfer session was mea-sured. The volume was measured by displacing water in

an inverted glass graduated cylinder apparatus that was

filled with 30% CaCl2 solution. The CaCl2 minimized

microbial growth in the water tank, and reduced water

evaporation. The CaCl2 solution had an acidic pH

(5.6), which prevented CO2 adsorption. To check meth-

anogen inhibition, every 2 or 3 d the gas from the

fermentation was analyzed for CH4 by gas chromatog-raphy. A 5-mL sample was taken through the reactor

septum and analyzed using an Agilent 6890 series gas

chromatograph. The chromatograph was equipped with

a thermal conductivity detector (TCD). A 4.6-m stain-

less steel packed column with 2.1-mm ID (60/80 Carbo-

xen 1000, Supelco� 1-2390 U, Agilent Technologies,

CA) was used. Samples were injected manually. The

inlet temperature was fixed at 230 �C, and the detectortemperature was set at 250 �C. The oven temperature

was maintained at 225 �C for 5 min. Helium was used

as carrier gas. The total elution time for a sample was

5 min.

Volatile solids in the initial substrates and solid fer-

mentation residues were determined by first drying the

material at 105 �C and then ashing the material at

550 �C for at least 3 h. Volatile solid determinationin the liquid fermentation broth followed the same

heating procedure above; except prior to drying, the

liquid was mixed with lime to ensure that the carbox-

ylic acids would not volatize and alter the measure-

ment.

2.11. Mass balance

For all the countercurrent fermentation experiments,

a complete mass balance was obtained on the entire


train during a steady-state period. Closure is defined as

follows:

closure ¼ mass out

mass in þ water of hydrolysisð3Þ

closure ¼ undigested VS þ dissolved VS þ carboxylic acids produced þ biotic CO2 þ CH4

mass in þ water of hydrolysisð4Þ

To calculate the water of hydrolysis, it was assumed that

the biomass could be represented as cellulose, which has

a monomer weight of 162 g/mol. When cellulose ishydrolyzed, it gains one molecule of water per mono-

mer; therefore, the water of hydrolysis is calculated as

water of hydrolysis ¼ VS digested � 18

162ð5Þ

2.12. Operational parameters

The liquid residence time determines how long the

liquid remains in the system, which affects the final prod-

uct concentration. Long liquid residence times allow

high product concentrations whereas shorter liquid

residence times allow lower product concentrations

(Holtzapple et al., 1999). Liquid residence time is calcu-lated as

liquid residence time ðLRTÞ ¼ TLV

Qð6Þ

where, Q = flowrate of liquid out of the fermentor set(L/d), TLV = total liquid volume.

TLV ¼X

i

ðKi � wþ F iÞ ð7Þ

where, Ki ¼ average wet mass of solid cake in Fermentor

i ðgÞ; w = average liquid fraction of solid cake inFermentor i (L liquid/g wet cake); F i ¼ average

volume of free liquid in Fermentor i ðLÞ.The volatile solids loading rate is calculated as

volatile solids loading rate ðVSLRÞ ¼ VS fed=d

TLVð8Þ

At a low VSLR, the solid residence time increases,allowing for more complete digestion.

Biomass is composed of volatile solids (VS) and ash,

and except for the lignin most VS are reactive. The

digestion process converts part of the VS into gas and

liquid products, with some solids remaining undigested.

In the liquid products, VS consist of carboxylic acids,

extracellular proteins and energy-storage polysaccha-

rides (Ross, 1998). The following terms are usedthroughout this paper.

conversion ðxÞ ¼ VS digested

VS fedð9Þ

yield ðyÞ ¼ total carboxylic acids produced

VS fedð10Þ

total acid selectivity ðsÞ

¼ total carboxylic acids produced

VS digestedð11Þ

total acid productivity ðpÞ

¼ total carboxylic acids produced

L liquid in all reactors � timeð12Þ

2.13. Statistical analyses

The statistical analyses were performed using Excel

software. Analysis of variance for fermentations A and

D were performed using the student t-test at 5% level

of significance. The mean and standard deviations of

the total acid concentrations for fermentations A and

D were determined from the steady-state operational

data from days 126 to 257. Correlations between volatile

solid loading rate (VSLR) and acid productivity (p),selectivity (s), yield (y), and conversion (x) were ob-

tained by fitting experimental results to linear equations

using Excel software.

3. Results and discussion

3.1. Batch reproducibility

For the following experiments, simulated MSW and

SS were combined in an 80:20 ratio. To determine the

reproducibility of MSW/SS batch experiments, two sets

of three fermentors were operated simultaneously under

identical conditions. Untreated MSW/SS was used as

the substrate for one set, whereas lime-treated MSW/

SS was used for the other. The fermentors containedthe substrate at a concentration of 100 g dry substrate/

L liquid. So that the microorganisms would already be

adapted to the substrate, the inoculum used in these

experiments was inocula from countercurrent experi-

ments with the same substrate.

The initial pH for all fermentations was approximately

6.5. During the fermentations, the pH varied from 5.4 to

6.5. The off-gas was analyzed for methane every otherday, but no methane was found. The combination of

0

2

4

6

8

10

12

14

16

18

0 2 4 6 8 10 12 14 16 18 20Time (days)

Tot

al c

arbo

xylic

aci

d co

ncen

trat

ion

(g/L

)

Treated MSW/SSUntreated MSW/SS

Fig. 3. Average carboxylic acid for untreated MSW/SS and treated

MSW/SS batch fermentations at 100 g substrate/L liquid, error bars ±1

standard deviation.


low pH and iodoform addition effectively inhibited meth-

anogenesis. Average total carboxylic acids concentra-

tions for the untreated and lime-treated MSW/SS are

shown in Fig. 3 along with one-standard-deviation errors

bars. The variation was less as the beginning andincreases as the fermentation progresses. The largest

standard deviation was 0.46 g/L untreated MSW/SS

and 0.62 g/L for treated MSW/SS. This experiment dem-

onstrated that batch experiments using lime-treated or

untreated simulated MSW/SS as substrate system were

very reproducible.

3.2. Countercurrent fermentations

The results from the lime-treated countercurrent fer-

mentations are shown in Table 2. Fermentations A and

D were initiated using the same substrate (80% lime-

Table 2

Results for lime-treated MSW/SS countercurrent fermentations

Fermentation trains A

Average pH in all fermentors 5.96 ± 0.15

Total carboxylic acid concentration (g/L) 16.90 ± 0.77

Acetic acid (wt%) 39.57 ± 4.72

Propionic acid (wt%) 15.70 ± 3.57

Butyric acid (wt%) 23.31 ± 5.00

Valeric acid (wt%) 8.04 ± 1.41

Caproic acid (wt%) 9.47 ± 2.49

Heptanoic acid (wt%) 1.61 ± 0.86

Conversion (g VS digested/g VS fed) 0.432

Yield (g total acids/g VS fed) 0.276

Selectivity (g total acids/g VS digested) 0.639

Total carboxylic acid productivity (g total acids/(L liquid d)) 0.832

Biotic CO2 productivity (g CO2/(L liquid d)) 0.390

Methane productivity (g CH4/(L liquid d)) 0.002

Mass balance closure (g VS digested/g VS in) 0.96

Note: All errors are ±1 standard deviation.

ND = not determined.

treated MSW/20% SS) and the same inocula mixture.

Both fermentations were started on the same day and

were operated identically to determine the reproducibil-

ity of the countercurrent fermentations.

In Fig. 4, the total carboxylic acid concentration as a

function of time during fermentations A and D are pre-sented. Both fermentations have the same behavior

through the operation time (258 d). A t-test was con-

ducted over the steady-state operation (days 126–257),

and the results showed that there were no significant

differences between these data set at 5% level of sig-

nificance. The results show excellent reproducibility for

countercurrent fermentations.

Fermentation C had the highest total carboxylic acidconcentration obtained for lime-treated MSW/SS

(26.0 g carboxylic acids/L liquid) which surpassed the

economic goal of 22 g carboxylic acid/L liquid (Holtzap-

ple et al., 1999). This fermentation was operated at pH

5.7, LRT = 22.7 d, and VSLR = 8.1 g VS/(L liquid d)

for 403 d, and also had the highest acid productivity

(1.36 g total acids/(L liquid d)). The conversion was

0.302 g VS digested/g VS fed, the yield was 0.18 g car-boxylic acid/g VS digested, and the selectivity was

0.577 g VS digested/g VS fed. Fermentation D (pH

5.9, LRT = 20.7 d, and VSLR = 3.1 g VS/(L liquid d))

had the highest conversion (0.449 g VS digested/g VS

fed). The total carboxylic acid productivity was 1.12 g

total acids/(L liquid d), the yield was 0.264 g carboxylic

acid/g VS digested, and the total carboxylic acid produc-

tivity was 0.577 g total acids/(L liquid d).The correlations between volatile solid loading rate

(VSLR) and acid productivity (p), selectivity (s), yield

(y), and conversion (x) are shown in Figs. 5–8. The re-

sults show that at higher volatile solids loading rates,

higher total acid productivities are obtained. Conver-

sion, selectivity, and yield decrease with increasing

B C D E

5.79 ± 0.02 5.68 ± 0.06 5.93 ± 0.20 5.85 ± 0.11

16.29 ± 1.52 25.99 ± 1.46 17.07 ± 0.99 18.62 ± 0.97

40.55 ± 4.30 40.40 ± 3.10 45.06 ± 4.48 43.89 ± 4.40

14.18 ± 2.10 14.17 ± 1.40 17.81 ± 2.39 12.24 ± 2.21

21.33 ± 3.71 22.15 ± 2.99 18.69 ± 3.63 22.08 ± 3.31

7.77 ± 1.44 6.97 ± 0.96 7.35 ± 1.49 7.25 ± 1.01

12.33 ± 2.96 11.09 ± 2.54 6.98 ± 2.22 9.35 ± 2.82

2.64 ± 1.27 1.41 ± 0.74 1.70 ± 1.12 1.45 ± 0.83

0.366 0.302 0.449 0.392

0.221 0.175 0.264 0.237

0.603 0.577 0.589 0.558

0.886 1.360 0.829 1.125

0.251 0.372 0.290 ND

0.001 0.007 0.002 ND

101.9 100.3 0.93 ND

0

5

10

15

20

25

10 30 50 70 90 110 130 150 170 190 210 230 250 270

Time (days)

Tot

al c

arbo

xylic

aci

d co

ncen

trat

ion

(g/L

)

Fermentation A

Fermentation D

Steady-state operation

Switch to 4 mg iodoform/L fed to F4

8 mg iodoform/L fed to F4

Iodoform/Bromoform8 mg/L fed to F4

Fig. 4. Total acid concentration for lime-treated MSW/SS fermenta-

tion A and D.

0

0.5

1

1.5

2

0 2 4 6 8 10

VSLR (g VS/(L liquid in all fermentors·d))

Tot

al a

cid

prod

ucti

vity

(g

tota

l aci

ds/g

/(L

liqu

id·d

))

Experimental data

Best Line Fit

Fig. 5. Correlation of total acid productivity with volatile solid

loading rate.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 2 4 6 8 10


Sele

ctiv

ity

(g

tota

l aci

ds/g

VS

dige

sted

)

Experimental data

Best line fit

Fig. 6. Correlation of selectivity with volatile solid loading rate.

0

0.1

0.2

0.3

0.4

0 2 4 6 8 10


Yie

ld (

g to

tal a

cids

/g V

S fe

d)

Experimental data

Best line fit

Fig. 7. Correlation of yield with volatile solid loading rate.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 2 4 6 8 10


Con

vers

ion

(g

VS

dige

sted

/g V

S fe

d)

Experimental data

Best line fit

Fig. 8. Correlation of conversion with volatile solid loading rate.


VSLR. These correlations are described by the following

equations:

p ¼ 0:0965VSLR þ 0:5317 ð13Þ

s ¼ �0:0090VSLR þ 0:6274 ð14Þ

x ¼ �0:0263VSLR þ 0:5037 ð15Þ

y ¼ �0:0178VSLR þ 0:3086 ð16ÞThe substrate system, lime-treated MSW/SS, is a hetero-

geneous lignocellulosic material in which some of thecomponents are more digestible by the microorganisms

(Ross, 1998). MSW is the source of carbon and energy,

and SS is the source of micronutrients. At high VSLR,

more substrate is available for the microorganisms,

which means that more nutrients and easily digested

substrate are present. The mixed culture of microorgan-

isms digested the primarily accessible fraction of the

substrate; however, less carboxylic acids were producedper unit of digested biomass. Part of the digested sub-

strate is non-acid soluble volatile solids (dissolved VS).

When substrate and nutrients are abundantly available,

the microorganisms may produce energy-storage com-

pounds that can be used in periods of starvation as sug-

gested by Domke (1999).

However, at low VSLR, acid productivity was lower,

but conversion, selectivity, and yield were higher. Atlower VSLR, less substrate was added, so fewer nutri-

ents and less easily digestible fractions of the substrate

were available for the microorganisms. As a conse-

quence, the microorganisms not only would consume

the more-available fractions, but also the less-available

fractions of the substrate. In natural environments, such

as rumen, substrate concentrations are low, and there-

fore not enough for the microorganisms to reach maxi-mum growth rates (Russell and Baldwin, 1979). At these

conditions, per unit of digested biomass, the micro-

organisms may produce more carboxylic acid and less

energy-storage components.

The CPDM method developed by Loescher (1996)

has been used to predict the product acid concentration

and conversions for countercurrent fermentation sys-

tems (Ross, 1998; Domke, 1999; Thanakoses, 2002;

0

5

10

15

20

25

30

35

0 0.2 0.4 0.6 0.8 1

Conversion (g VS digested/g VS fed)

Tot

al c

arbo

xylic

aci

d co

ncen

trat

ion

(g/L

)

23456812

10

15

2530

35

LRT (days)

VSLR (g/(L·d))

Fig. 9. CPDM ‘‘map’’ for lime-treated MSW/SS countercurrent

fermentation (200 g VS/L liquid).


Chan, 2002; Aiello-Mazzarri, 2002). CPDM utilizes

batch data to predict the results of the countercurrent

fermentations.

The acetic acid equivalents (Ae) from each of the five

batch experiment was fit to the equation

Ae ¼ aþ bt1 þ ct

ð17Þ

where t is the time (d) of fermentation, and a, b, and c

are constants fit by least squares analysis. The rate r

was obtained from Eq. (18).

r ¼ dðAeÞdt

¼ b

ð1 þ ctÞ2ð18Þ

The specific rate, r (g Ae produced/(g VS d)) was deter-

mined from Eq. (18) by dividing it by the initial amount

of substrate concentration, S0 (g VS/L) in each of thefive fermentors

r ¼ rS0

ð19Þ

The predicted rate, rpred, was obtained from Eq. (20);

where the rate of acid production depends on volatilesolids conversion (x) and product concentration (Ae)

rpred ¼ eð1 � xÞf

1 þ g½/Aehð20Þ

Least square analysis was used to determine the empir-ical parameter constants e, f, g, and h for rpred (Eq.

(20)) from the specific rate r (Eq. (19)). Ae was converted

back to carboxylic acid concentration by / (the ratio of

total grams of actual acids to grams of Ae). The (1 � x)

term in the numerator of Eq. (20), is the conversion pen-

alty function by South and Lynd (1994).

The conversion, x was calculated using

xðtÞ ¼ Ae � TLV

M0 � rð21Þ

where, r = selectivity (g Ae produced/g VS digested);

M0 = the initial substrate mass (g VS).

The selectivity r, for Eq. (21) was calculated from theselectivity s (g total acids produced/g VS digested) deter-

mined in the countercurrent experiment.

s ¼ /r ð22ÞThe selectivity (s) used in the CPDM Mathematica pro-

gram was 0.60 g total acids/g VS digested, the average

value of selectivity for the lime-treated MSW/SS coun-tercurrent fermentations with terrestrial inocula. Selec-

tivity slightly decreases with increasing VSLR (Fig. 6)

and was described by Eq. (14). Eq. (20) was used in

a Mathematica program (Aiello-Mazzarri, 2002) to

predict acetic acid equivalent concentration (Ae) and

conversion (x) for the countercurrent fermentation at

various VSLR and LRT. Ae was converted back to

carboxylic acid concentration by multiplying by /.

From the CPDM Mathematica program, the values

of the predicted total carboxylic acid concentration

and conversion for the countercurrent fermentation at

various LRT and VSLR were obtained. CPDM predic-

tions for conversions and product concentrations for the

lime-treated MSW/SS countercurrent fermentations(substrate = 200 g VS/L liquid) are presented in the

‘‘map’’ in Fig. 9. The experimental carboxylic acid con-

centrations and conversions from the lime-treated

MSW/SS countercurrent fermentations are compared

with the predicted values from the CPDM ‘‘map’’

shown in Fig. 9. As shown in Table 3, the average abso-

lute error between the experimental and predicted

total carboxylic acid concentration and conversionfor lime-treated fermentations was 4.4% and 15%,

respectively.

The error between the predicted conversion, xpred and

the experimental conversion, xexp as a function of selec-

tivity is shown in Fig. 10. Linear regression of the data

gave the following correlation:

Error ¼ xpred � xexp

xexp

¼ 2:663s� 1:418 ð23Þ

Future improvements to the CPDM model should

incorporate a varying selectivity, r, to better describethe countercurrent fermentation process.

Higher substrate concentrations would be allowed if

the process were done on a large scale (Holtzapple

et al., 1999). A higher VS concentration should result in

higher total carboxylic acid concentrations. CPDM

was used to predict the conversion and product concen-

trations for a VS concentration of 300 g VS/L liquid; the

CPDM ‘‘map’’ is shown in Fig. 11. As observed in theCPDM ‘‘map,’’ total acid concentrations as high as

40 g/L can be reached at LRT of 35 d and VSLR of

12 g/(L d). Also, conversions as high as 82% can be

achieved at LRT of 10 d and VSLR of 2 g/(L d). Both,

high conversions (>70%) and high product concentra-

tions (>30 g/L) can be achieved at LRT of 35 d and

VSLR between 2 and 3 g/(L d).

Table 3

Comparison of experimental and predicted carboxylic acid concentration and substrate conversion for lime-treated MSW/SS countercurrent

fermentations A–F

Fermentation trains A B C D E F Averagea (%)

LRT (d) 20.7 19.0 22.7 20.7 17 19.7

VSLR (g VS/L d) 3.0 4.0 8.1 3.1 6.4 5.7

Experimental carboxylic acid concentration (g/L) 16.90 16.29 25.99 17.07 18.62 20.66

Predicted (CPDM) carboxylic acid concentration (g/L) 16.12 17.54 23.53 17.10 18.39 19.97

Errorb (%) �4.6 7.7 �9.5 0.2 �1.2 �3.3 4.4

Experimental conversion 0.43 0.37 0.30 0.45 0.39 0.33

Predicted (CPDM) conversion 0.54 0.49 0.29 0.56 0.36 0.38

Errorb 20.3 24.3 3.3 24.3 �7.7 15.1 15.8

a Average absolute error ¼P

ðjðpredicted � experimentalÞ=experimentaljÞ=6.b Error = ((predicted � experimental)/experimental) · 100.

0

5

10

15

20

25

30

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

Selectivity (g total acid/g VS digested)

Err

or (

%)

Fig. 10. Correlation of error between experimental and predicted

conversion and selectivity for lime-treated MSW/SS fermentations

with terrestrial inocula.

0

5

10

15

20

25

30

35

40

45

0 0.2 0.4 0.6 0.8 1


Tot

al c

arbo

xylic

aci

d co

ncen

trat

ion

(g/L

)

2 3

4 56 812

10

15

2530

35

LRT (days)

VSLR (g/(L·d)

Fig. 11. CPDM ‘‘map’’ for lime-treated MSW/SS countercurrent

fermentation for substrate concentration 300 g VS/L liquid.

0

10

20

30

40

50

60

0 0.2 0.4 0.6 0.8 1


Tot

al c

arbo

xylic

acid

con

cent

rati

on (

g/L

)

2 3

4 56 812

10

15

2530

35

3 46

Mesophilic

-------- Thermophilic

VSLR (g/(L·d))

LRT (days)

Fig. 12. CPDM ‘‘maps’’ for lime-treated MSW/SS four-stage coun-

tercurrent fermentation under mesophilic and thermophilic conditions

(80/20 MSW to SS ratio, and 300 g VS/L liquid for both conditions.

Thermophilic data from Chan, 2002).


The CPDM ‘‘maps’’ for lime-treated MSW/SS coun-

tercurrent fermentations under mesophilic and thermo-

philic conditions (Chan, 2002) at 300 g substrate/Lare compared in Fig. 12. For the same LRT and VSLR

lower than 8 g VS/(L d), using mesophilic conditions

(40 �C) higher acid concentrations and conversions can

be achieved than with thermophilic conditions (55 �C).

At high VSLR using thermophilic conditions, acid con-

centrations as high as 60 g acids/L can be obtained, but

conversions may be lower than 40%.

4. Conclusions

The batch and countercurrent fermentations using

lime-treated simulated MSW/SS as substrate were very

reproducible. MSW/SS countercurrent fermentationshave been well characterized, and countercurrent fer-

mentations can be operated for extended periods of time

under steady-state conditions. Addition of 4–8 mg iodo-

form/(L liquid added to F4) inhibited methane genera-

tion. The highest total carboxylic acid concentration

obtained for lime-treated MSW/SS with terrestrial in-

ocula was 26.0 g carboxylic acids/L liquid, which sur-

passed the economic goal of 22 g carboxylic acid/Lliquid (Holtzapple et al., 1999). The fermentation was

operated at pH 5.7, LRT = 22.7 d and VSLR = 8.1 g

VS/(L liquid d) for 403 d. Industrial-scale fermentors

with higher substrate concentrations would achieve

higher product acid concentrations and conversions.

The CPDM model predicts product concentrations

and conversions within 4% and 16%, respectively. The

CPDM model was a useful tool in predicting productconcentration and conversion. The predicted values


can be used to estimate the costs and process economics

of industrial-scale fermentors.

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http://www.epa.gov/oigearth/ereading_room/BIOSOLIDS_FINAL_REPORT.pdf

http://www.epa.gov/oigearth/ereading_room/BIOSOLIDS_FINAL_REPORT.pdf

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