HUNGAR~JOURNAL 

OF INDUSTRIAL CHEMISTRY 
VESZPREM 

Vol. 30. pp. 19-25 (2002) 

POSSIBLE SUBSTRATES FOR ACETONE-BUTANOL AND ETHANOL 
FERMENTATION BASED ON ORGANIC BY-PRODUCTS 

E. VARGA, Z. KADAR, K. C. SCHUSTER1, J. R. GAPES 1, Z. SZENGYEL and K. RECZEY* 

(Department of Agricultural Chemical Technology, Budapest University of Technology and Economics, Szt.Gellert ter 4, 
Budapest, H-1521, HUNGARY 

1
Institute for Chemical Engineering, Fuel and Environmental Technology, Vienna University of Technology, 

Getreidemarkt 91159 Vienn<t, A-1060, AUSTRIA) 

Received: February 27, 2001 

An assessment study was carried out on available biomass by-products in Hungary, and also several fruit juice wastes 
were evaluated for their accessibility to enzymatic hydrolysis. Among the fruit juice residue samples examined the best 
results were obtained with apple, peach, apricot and tomato. It can be shown that using intensive stirring, higher 
conversions can be reached. Highest conversion of 98 % - based on carbohydrate content - was obtained with frozen and 
thawed apple residues. 
ABE fermentation of apple pulp hydrolysate supplemented with glucose yielded high solvent concentrations up to 22.5 
gil. For growth and solvent production supplementation by additional nutrients was not essential, however, this 
accelerated the process. Ethanol fermentation by baker's yeast was carried out on apple pulp hydrolysate supplemented 
with glucose. This also gave reasonable yields of about 90 % - based on glucose consumption. 

Keywords: hydrolysis, biomass, fruit pulp, alternative substrate of ABE fermentation 

Introduction 

The problem of environmental change caused by 
greenhouse gas emissions mainly from fossil fuels has 
motivated research in the utilisation of renewable energy 
sources. Biomass of various origins is a great resource 
for energy carriers, not only by direct combustion, but . 
also by biological conversion such as biogas production, 
ethanol fermentation, and acetone- butanol (ABE) 
fermentation [7]. In the traditional ABE process, the 
high cost of conventional "starch" (maize, wheat, millet, 
rye, etc.) or sugar (molasses) substrates was a major 
factor affecting the economic viability of the process 
[7]. ABE fermentation, following its success in the 40's 
and 50's, and its nearly complete disappearance in the 
60's once again became of interest in the 70's after the 
first oil crisis, when much emphasis was placed on the 
use of renewable raw materials. This interest and the 
ability of sugar-fermenting clostridia to utilise many 
different carbohydrates have stimulated research into the 
use of alternative cheaper substrates [7]. 

The use of whey and whey permeate has been 
studied successfully [8]. Prescott and Dunn [14] have 
used a number of non-cellulosic, starch-based materials 

(including potato, rice, jawari, bajra) as substrates with 
varying success. Cheap low-grade potatoes have been 
used in the ethanol industry for a long time and proved 
to be an excellent substrate for batch ABE fermentation 
after minor pretreatment with starch-liquefying enzyme 
[3, 12], and could also be used in a continuous process 
[17]. As early as 1922, Robinson [15] studied the 
fermentation on various carbohydrates for the 
production of acetone and butanol. He reported that 
glucose, fructose, mannose, sucrose, lactose, dextrin, 
and starch were completely consumed, while galactose, 
xylose, and arabinose were partially utilised, and 
glycerol was not fermented. However, Nakhmanichov 
and Kochkina [ll] showed that xylose and arabinose 
could be fermented completely by most ABE bacteria. 
Ounine et at. [13] reported a relatively high yield of 
conversion into solvents of 28 % for xylose, close to the 
maximal value of 32 % for glucose. Wiley et al. [22] 
investigated sulphite waste liquids from the pulp and 
paper industry <;ontaining glucose, xyloset and arabinose 
as alternative substrates for ABE fermentation. They 
showed that good yields of solvents were obtained when 
sulphur dioxide, lignin, and excess calcium were 
removed from the sulphite liquid. Voget et al. [211 

*Corresponding author: Fax: (36)-1463-25-89. e-mail: Reczey@chem.bme.hu 



20 

investigated the use of apple pomace for butanol 
production and found that 80 % to 84 % of soluble 
sugars (about 10% of the dry weight) were consumed. 
The residue remaining after butanol separation could be 
used as animal feed. Lignocellulosics are, however, the 
most abundant renewable resources [6] and ~are 
recognised as having great potential as substrates for 
fermentation, provided that the hemicellulose and 
cellulose components can be degraded and utilised 
cheaply and efficiently. Fermentation can be performed 
by yeasts to ethanol [16] or to acetone-butanol by 
clostridia [7]. 

The ABE fermentation by clostridia holds further 
potential as the butyric acid bacteria are capable of 
utilising all of the prevalent pentose and hexose sugars 
present in hemicellulose and cellulose in contrast to 
conventional baker's yeast Saccharomyces cerevisiae 
used for ethanol fermentation. Lignocellulosic biomass 
contains hemicellulose in a range of about 20 to 40 % 
[19] 'Yhich means that the overall substrate utilisation is 
higher depending on the content of hemicellulose in the 
biomass, however, the (hemi)cellulose must be 
hydrolysed prior to fermentation. Substrates with high 
lignin content can usually be hydrolysed only after 
pretreatment under harsh conditions [16], often leading 
to high levels of substances inhibitory to the 
fermentation, and to loss of less thermally stable sugars 
like xylose. This combination of problems leads to poor 
economic prospects for the saccharification process 
starting from lignin-rich substrates like wood [5]. These 
problems have motivated this. survey of potentially 
"easier•• substrates which can be readily hydrolysed and 
are available locally in Hungary. Selected substrates 
should also be tested in practical hydrolysis and 
fermentation experiments. 

Sun·ey of a\·ailabfe by-products from agriculture and 
food industry in Hungary 

Hungarian agriculture produces large amount of plant 
biomass every year. A part of this biomass is a by-
product or waste, and its efficient utilisation is not yet 
solved. The aim of this survey in the agriculture and 
food industries was to identify potential substrates for 
ABE fermentation, which should be easy to hydrolyse 
and available in sufficiently large quantities. 

A.fJricultural b.\·~products 

Crop residues considered for collection are from corn. 
small grains. and grass. They are harvested by combines 
that leave stubble~ and the straw or stalks are returned to 
the field after harvest [23}. This is a rather large amount 

1.20E+07 com-StOw!lr 

..._ 
l,OOE+07 

.. 
~ 8,00E+06 

;Q 

= .. 6,00E+06 c. wneatstraw ..... .. 
';:; 4,00E+06 
~ 
~ 

R csidncs of fr~itjuicc 
processing 

2,00E+06 

O,OOE+OO mm • • t • 
!llllli::~al wheat barley com-stalk com-cob residues of extracted 

soil waste straw straw frul juke sugar-beet 
in processilg si:e 

Budapest 

Fig.l Amounts of various agricultural and food industry 
wastes in Hungary according to the survey performed in this 

study (in million tons per year) 

of biomass. The amount of corn stalk is tenfold more 
than the municipal solid waste of Budapest in a year 
(Fig. I). 

By-products from the food industry 

• Cheese whey has attracted interest as an alternative 
substrate for ABE fermentation because of its 
disposal problem, lactose content, and availability in 
many countries. After the precipitation and removal 
of casein, whey filtrate contains a relatively low 
sugar content and is unsuitable for most fermentation 
processes without prior concentration steps, but it is 
suitable for ABE fermentation [7]. Maddox et al. [8] 
reported successful utilisation of whey permeate 
after removal of valuable proteins. In Hungary 
435*10

6
1itres of whey is produced annually and only 

10% of this is dried and utilised as animal feed. The 
ABE fermentation could be a good alternative for ~e 
remainder. 

• In Hungary, one of the largest companies in the fruit 
juice processing industry is Rauch Hungary Ltd., 
which produces annually 10,000 t apple residues 
(from juice and pulp processing) and apple pomace 
(from washing and sorting) and 1,500-2,000 t of 
other fruit waste. Expansion of the production 
facilities is anticipated. 

• At the canning factories there is no problem with 
wastes yet, because the entire quantity is used as 
animal feed or is composted. 

• According to information from breweries, their by-
products are sold as animal feed. However, with the 
concentration process going on in this industry as in 
many others, it might become difficult to distribute 
in large quantities. 



Experbnentalprocedure 

Substrates for hydrolysis experiments 

Industrial residues from the fruit processing industry for 
use in the comparison experiments were obtained from 
Rauch Hungary. The comparison experiments were 
performed with fresh residues in the factory without any 
treatment or preservation. Peach residue for the 
assessment effect of re-freezing was stored at -20 °C, 
thawed and hydrolysed. A portion was refrozen and 
hydrolysed after the second freeze-thaw cycle. 

Enzymatic hydrolysis 

Hydrolysis of fruit pulp for the substrate comparison 
was performed at 3 % dry material in a 0.05 M citrate 
buffer solution (pH 4.8) supplemented with Pulpzyme 
HC, 24.7 FPU (Filter Paper Unit) and Novozym 188, 
121.7 CbU (Cellobiase Unit) per g dried substrate. 
Hydrolysis was performed at 50 oc for 5 h in 1 OOml 
shake flasks. Samples were collected each hour, 
centrifuged at 10,000 rpm for 3 minutes and analysed 
for reducing sugar content by the DNS method. 

Enzyme preparations used 

Pulpzyme HC is an enzyme preparation with a cellulase 
and xylanase activity- produced in submerged 
fermentation with a genetically modified Bacillus strain 
(NOVO Nordisk, Denmark). Novozym 188 is a 
cellobiase enzyme preparation obtained by submerged 
fermentation with Aspergillus niger (NOVO Nordisk, 
Denmark). 

Organism, culture maintenance, and growth media 

The organism utilised was Clostridium acetobutylicum 
ATCC 824 maintained in spore stock suspension 
according to Gapes et al. [2]. The composition of the 
medium used for strain maintenance was 60 gil glucose, 
5 gil yeast extract, 1 g/1 K 2HPOiH20, 1 g/1 KHzP04, 1 
gil MgS047H20, 0.5 g/1 FeS047H20, 0.01 gil p-amino-
benzoic acid, 3 g/1 ammonium acetate. 

ABE fermentation 

Previously hydrolysed apple pulp was used for the ABE 
fermentation experiments, supplemented by nutrients of 
the standard semisynthetic medium for the maintenance 
of the C. acetobutylicum ATCC 824 strain according to 
Gapes et al. [2], or for the comparison experiment 
without supplementation. The sugar concentration was 
increased to 60 gil by adding glucose. The experiments 

21 

were performed in 300 ml sealable glass bottles filled 
with 150 m1 of medium. To ensure anaerobic conditions 
nitrogen was blown through the solutions for 10 min to 
remove oxygen from the headspace. Following 
sterilisation at 121 oc for 16 min the medium was 
inoculated with 10 ml of a culture of vegetative cells of 
C. acetobutylicum ATCC 824 grown in continuous 
cultivation in a wheat medium as described by Nimcevic 
et al. [12]. The fermentation experiments were 
performed at 35 °C. 

Ethanol fermentation 

The conditions of the experiment were identical to those 
of the ABE fermentation experiments except for the 
microorganism used. Commercially available baker's 
yeast (Saccharomyces cerevisiae) was used for the 
ethanol fermentation. An inoculum culture was grown 
on semisynthetic medium for 24 hours, and 6 m1 of this 
culture was used to inoculate the apple pulp medium. 

Assays 

For analysis of the biomass, the first step was an acidic 
lignin extraction performed according to Hagglund ( 4 ]. 
The sugar content of this acidic supernatant was 
determined by the phenol-sulphuric acid method [1] and 
total available carbohydrate concentration was 
calculated from this. The reducing sugar concentration 
during enzymatic hydrolysis was estimated 
calorimetrically using a dinitrosalicylic acid reagent 
[10]. Glucose concentration was analysed by HPLC or 
estimated by reflex photometry with Reflolux 
(Boehringer Mannheim I Roche Diagnostics, Mannheim, 
Germany). 

Results 

Comparative hydrolysis experiments with various fruit 
juice processing by-products 

The purpose of these experiments was to identify those 
by-products of fruit juice processing, which are suitable 
for ABE fermentation with regard to their sugar and 
convertible carbohydrate content. The hydrolysis was 
performed with 12 types of fruit substrates (apple pulp, 
peach and apricot pulp, juice and pulp processing by-
products, black and red currant, raspberry, blackberry, 
and elder pomaces) in 100 m1 shake flasks, using the 
enzymes Pulpyzime and Novozym. Since a portion of 
the sugars in fruit processing residues is soluble, the 
water-soluble sugars were first removed from the 
samples by cold and hot water washing, followed by 
filtering. The amounts of dissolved sugars in the filtered 
Table 1 Soluble sugar and acid-soluble carbohydrate content 

of residues from fruit processing by-products 



22 

Samples 

Peach* 
Peach** 
Apricot* 
Apricot** 

Apple pomace 
Apple pulp 
Red currant 
Raspberry 

Blackcurrant 
Elder 

Tomato 
Blackberry 

Soluble sugar 
(g/100 g 

substrate dw) 
9.4 
0.9 
10 
2.6 
7.1 
12 
1.7 
2.1 
2.1 
1.4 
7.6 
0.5 

* residues from pulp production 
** residues from juice production 

Acid soluble carbohydrate 
(g/100 g 

washed substrate dw) 
39 
28 
38 
30 
40 
38 
35 
38 
31 
21 
28 
34 

water were determined by the phenol-sulphuric acid 
method.. The results, based on 100 g dry weight, are 
shown in Table 1. Together with the carbohydrate 

· contents the dry matter contents were also determined 
for each sample. The hydrolysis of the samples was 
performed in 5 h, in suspensions containing 3 % dry 
weight. Previous experiments [20] have shown that the 
amount of sugar obtained in 24 h enzyme conversions is 
approximately 10 % higher than when the hydrolysis is 
stopped after 5 h.. However, since our goal was the 
identification of easily convertible wastes, this time 
period was sufficient and informative enough for making 
the desired determinations. 

The conversion yields reached with enzymatic 
hydrolysis based on the samples' carbohydrate contents 
(Table 2) proved to be low (~20 %) in the cases of the 
so-called coloured fruits (red and black currant, 
blackberry, raspberry,. and elder). Among two types of 
peach and apricot processing wastes, the pressed waste 
(the remnant of fruit juice processing) is apparently 
largely composed of stone-seeds, which are difficult to 
hydrolyse, resulting in a rather low conversion yield. 
Data from the literature [9] show that the most efficient 
handling of stone-seeds is combustion. The conversion 
yields of the by-products of pulping are promising. and 
these wastes could be useful for fermentation, however, 
the amounts available are rather smaU (below 1,000 t/a). 
The conversion yield of tomato pulp is comparable to 
those of the apricot and peach and in this case the water-
soluble sugar content is rather important as its quantity 
is similar to that resulting from hydrolysis. 

The results are quite promising with the apple 
residues. the apple pulp~ as weU as the pulp preparation 
residues from peach and apricot. These by-products are 
also available in significant quantities above 10J)00 tons 
per year. Therefore1 further hydrolysis experiments were 
carried out with this group of fruit by-products. 

Table 2 Conversion of insoluble carbohydrate to soluble 
reducing sugar by enzymatic hydrolysis of washed fruit 

residues. given as the ratio (in percent) of reducing sugar after 
hydrolysis to total available carbohydrate content 

Examined residues Conversion (% l 

Peach* 47 
Peach** 21 
Apricot* 46 
Apricot** 23 

Apple pomace 63 
Apple pulp 73 
Red currant 16 
Raspberry 21 

Blackcurrant 17 
Elder 30 

Tomato 46 
Blackberry 28 

* residues from pulp production 
** residues·from juice production, containing the stones 

Hydrolysis experiments in a stirred system, with the 
most readily convertible fruit processing by-products 

These 250 m1 experiments were performed at 50 °C 
with 3 6 9 and 12 % dry weight content suspensions 

, ' ' • -1 • 
under mild stirring (100-150 mm ) for 24 hours m order 
to determine the maximum amount of sugar that can be 
reached, which is an important factor for further 
experiments. The more efficient stirring increased the 
effectiveness of the hydrolysis. By increasing the initial 
amount of substrate, the maximum sugar concentration 
reached also increased (Table 3). In the case of 
suspensions containing 12 % dry weight, the highest 
sugar concentration, 46 g/1, was obtained from apple 
residues, while the lowest, 9 g/1, from tomato juice 
processing by-products. At the same time, with the 
increase in the amounts of sugar obtained, the 
conversion values decreased. However, the decrease 
between the 3% and the 12% samples was less than 10 
% (Table 4). Among the conversion values obtained 
with the 5 h and the 24 h hydrolysis, even with the 
mixtures containing larger amounts of initial matter, the 
difference was smaller than 10 % (Fig.2), thus in future 
ABE fermentation experiments it would be practical to 
inoculate the hydrolysate after 5 hour hydrolysis. 
Statistical tests showed no significant difference at a 95 
% confidence level in the total amount of sugar 
extracted when the original, unwashed sample is directly 
hydrolysed, when the sample is first washed and only 
then hydrolysed. The amount of extractable sugar also 
does not change when using fresh, dried, or powdered 
samples. Freezing and thawing of the samples does not 
spoil the degradability, moreover it shows a positive 
effect on the enzymatic hydrolysis. (Table 5). 



[ 
5 
~ 
§! 
c 
0 
1.) 

Table 3 Reducing sugar concentrations of enzymatic 
hydrolysates starting from substrates at varied dry matter 

contents 

-Sugar concentration (g/1) 
Examined residues 3% 6% 9% 12% 

Apple pulp 12 24 36 46 
Apricot pulp 8.3 16 24 33 
Peach pulp 9.0 16 25 32 

Tomato 3.0 5.3 7.6 9.3 

Table 4 Conversions efficiency of enzymatic hydrolysis 
starting from substrates at varied dry matter contents 

Conversion of carbohydrate (%) 
Examined residues 3% 6% 9% 12% 

Apple pulp 98 96 94 91 
Apricot pulp 67 65 65 63 
Peach pulp 70 64 64 62 

Tomato 45 42 40 38 

95 

85 

75 

65 

I"" j 
"":' 

I"" 

] I. f- f- t-I- 1- ' f- t-
r- t- 1- 1- t-

55 t- t--- 1- 1- t-. 

45 h I 
0 2 3 4 5 24 

time[h] 

03 (m/m)% ~6 (m/m)% D9(m/m)% D12(m/m)% 

Fig.2 Conversion of insoluble carbohydrate to soluble 
reducing sugar in hydrolysis of apple pulp, in relation to initial 

dry matter content of the substrate (in mass percent). 
Conversion is calculated as in Table 2 

ABE fermentation on hydrolysed apple pulp 

ABE fermentation experiments were performed on 
enzymatically hydrolysed apple pulp. The apple pulp 
was supplemented with glucose to elevate its sugar 
concentration to 60 gil, which was necessary because the 
ABE bacteria used require a minimal sugar 
concentration to initiate solvent production. In addition 
to the glucose, the medium was also supplemented with 
nutrients as used in the semisynthetic glucose media [2]. 
A comparative experiment with an unsupplemented 
medium was done without adding the nutrients. 

The fermentation was inoculated with vegetative 
cells grown in continuous cultivation on a starch 
medium. The maximum average product concentrations 
during the fermentation are presented in Table 6. On the 
supplemented medium the sugar was almost completely 
consumed within 42 h, its concentration decreasing from 
60 gil to 1,2-3,7 gil. Coupled with the decrease in 

Table 5 Effect of refreezing of peach residue on conversion 
effectivity of enzymatic hydrolysis starting from varied dry 

matter contents in the substrate 

Sugar concentration [gil} 

23 

Time (h) S=3% 
S=3% 

S=12% 
S=12% 

refreezing refreezing 
0 1.8 3.0 3.5 18 
1 9.6 12 31 41 
2 10 14 32 42 
3 11 15 32 45 
4 13 17 37 50 
5 14 17 

5 14 17 39 54 

24 17 18 48 56 

Table 6 Results of ABE fermentation using enzymatically 
hydrolysed apple pulp and effect of medium supplementation 

on performance 

Maximum product Maximum product 

Product, Substrate 
concentration at concentration at not 

completed medium completed medium 
(gil) (gil) 

Butanol 11 9.6 

Ethanol 3.0 1.0 

Acetone 6.6 4.8 

Butyric acid 0.02 0.13 

Acetic acid 1.4 1.3 

Remaining sugar 2.3 20 

Start of solvent formation: - before 18 h for supplemented 
medium; - after 24 h for unsupplemented medium. Max. 
solvent concentration was reached- at 42 h for supplemented 
medium; - at 66 h for unsupplemented medium. 

the sugar, the products (acetone, butanol, ethanon 
appeared in the broth and reached their maximum 
concentrations after just 42 h when the sugar was almost 
completely consumed (95 % of the initial glucose). On 
the uncompleted medium, the maximum product 
concentrations were somewhat lower (Table 6). Glucose 
was not consumed completely - only about 70 % was 
consumed. The fermentation proceeded more slowly. 
solvent formation started later and maximum product 
concentrations were reached later. 

Ethanol fermentation experiments on apple pulp with 
Saccharomyces cerevisiae 

The fermentation experiments were also performed on 
hydrolysed apple pulp with commercially available 
baker's yeast. In order to run both fermentation 
experiments under comparable conditions~ the solutions 
were supplemented with enough glucose to reach the 
sugar concentration of 60 gil. As in the ABE 
experiments the medium contained aU the necessary 
nutrients. Following sterilisation, the media was 
inoculated for one night with a Saccharomyces 
cerevisiae inoculum and the fermentation was performed 
at 35 oc for 46 h. The sugar ~oncentration gradually 
decreased from the initial value of 60 gil and was 
completely consumed by the 46th hour. at which point 
the maximum ethanol concentration was reached as well 



24 

(24.4, 24.8 and 28.3 gil in three parallel experiments). 
This is close to the theoretical maximum yield (0.51 gig 
glucose). 

Conclusions 

There is large quantity of agricultural and food 
industrial by-product, which could be potential 
substrates for ABE fermentation. Among the 12 
different fruit processing residues there are 4 different 
by-products (apple, apricot, peach and tomato) which 
are worth further examination. The conversion yields of 
the apple processing by-products are the most 
promising, and the nearly 100 % conversion of the 
technological wastes is especially noteworthy. The 
experiments also show that in the case of apple residues 
the amount of sugar obtainable by washing is also 
significant. In these cases significantly better 
conversions are obtained, when the apple wastes are 
initially washed with water, which can be explained by 
product inhibition ({3-glucosidase). Apple processing by-
products are promising both in the ethanol and the ABE 
fermentation experiments. The results showed that the 
previously hydrolysed apple pulp contain no inhibitors 
that could interfere with or block the activities of the 
microorganisms. Supplementation of the hydrolysate 
with additional nutrients is not essential for growth and 
solvent production, however, it accelerates the 
fermentation process. The strain examined is able to 
utilise up to 96 % of the sugar extracted during the 
hydrolysis and during the anaerobic fermentation it is 
able to produce the desired products acetone, butanol, 
and ethanol in amounts close to the theoretical yields. 
Yeast ethanol fermentation on the hydrolysates is also 
possible. and the yields are reasonable (ca. 90 % based 
on glucose). 

... The pomaces of the berries, blackberry, red and 
black currant, raspberry. and elder were not easily 
accessible to enzymatic hydrolysis. Their conversion 
values are low~ therefore they cannot be recommended 
as ABE fermentation substrates based on these results 
despite their potentially high carbohydrate contents. 

Acknowledgement 

The Research Fund of the Ministry of Education 
(FKFP)~ Osterreichische Austauschdienst (OAD) 
National Research Fund of Hungary (OTKA) and Rauch 
Hungary Ltd. are gratefully acknowledged for their 
financial support~ and the NOVO Nordisk for the 
enzyme preparations. 

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