Application of solid-state fermentation to food industryA review

Susana Rodr guez Couto

a,b
, M
a
A

ngeles Sanroman
a,
*
a
Department of Chemical Engineering, Isaac Newton Building, University of Vigo, Lagoas Marcosende, 36310 Vigo, Spain
b
Department of Chemical Engineering, Chemical Engineering School, Rovira i Virgili University, 43007 Tarragona, Spain
Received 9 November 2004; accepted 19 May 2005
Available online 20 July 2005
Abstract
Solid state fermentation (SSF) has become a very attractive alternative to submerged fermentation (SmF) for specic applications
due to the recent improvements in reactor designs. This paper reviews the application of SSF to the production of several metabo-
lites relevant for the food processing industry, centred on avours, enzymes (a-amylase, fructosyl transferase, lipase, pectinase),
organic acids (lactic acid, citric acid) and xanthan gum. In addition, dierent types of biorreactor for SSF processes have been
described.
2005 Elsevier Ltd. All rights reserved.
Keywords: Bioreactors; Enzyme production; Food processing industry; Solid-state fermentation
1. Introduction
Microorganisms have long played a major role in the
production of food (dairy, sh and meat products) and
alcoholic beverages. In addition, several products of
microbial fermentation are also incorporated into food
as additives and supplements (antioxidants, avours,
colourants, preservatives, sweeteners, . . .). There is great
interest in the development and use of natural food and
additives derived from microorganisms, since they are
more desirable than the synthetic ones produced by
chemical processes.
Solid-state fermentation (SSF) reproduces the natural
microbiological processes like composting and ensiling.
In industrial applications this natural process can be uti-
lised in a controlled way to produce a desired product.
SSF is dened as any fermentation process performed
on a non-soluble material that acts both as physical sup-
port and source of nutrients in absence of free owing
liquid (Pandey, 1992). The low moisture content means
that fermentation can only be carried out by a limited
number of microorganisms, mainly yeasts and fungi,
although some bacteria have also been used (Pandey,
Soccol, & Mitchell, 2000a). Some examples of SSF pro-
cesses for each category of microorganisms are reported
in Table 1.
SSF oers numerous advantages for the production
of bulk chemicals and enzymes (Hesseltine, 1977;
Pandey, Selvakumar, Soccol, & Nigam, 1999a; Soccol,
Iloki, Marin, & Raimbault, 1994). This process is known
from ancient times and dierent fungi have been culti-
vated in SSF for the production of food. Typical exam-
ples of it are the fermentation of rice by Aspergillus
oryzae to initiate the koji process and Penicillium roque-
fortii for cheese production. Also, in China, SSF has
been used extensively to produce brewed foods (such as
Chinese wine, soy sauce and vinegar) since ancient time
(Chen, 1992). Also, in Japan SSF is used commercially
to produce industrial enzymes (Suryanarayan, 2003).
Since 1986 in Brazil a series of research projects for the
value-addition of tropical agricultural products and
sub-products by SSF has been developed due to the high
0260-8774/$ - see front matter 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jfoodeng.2005.05.022
*
Corresponding author. Tel.: +34 986 812383; fax: +34 986 812380.
E-mail address: sanroman@uvigo.es (M
a
. A

. Sanroman).
www.elsevier.com/locate/jfoodeng
Journal of Food Engineering 76 (2006) 291302
amounts of agricultural residues generated by this coun-
try (Soccol & Vandenberghe, 2003). Thus, the produc-
tion of bulk chemicals and value-added ne products
such as ethanol, single-cell protein (SPC), mushrooms,
enzymes, organic acids, amino acids, biologically active
secondary metabolites, etc. (Ho lker, Ho fer, & Lenz,
2004; Pandey, 1992; Pandey, 1994; Pandey, Azmi,
Singh, & Banerjee, 1999b; Pandey et al., 1999c; Pandey,
Nigam, & Vogel, 1988; Pandey et al., 2000b; Pandey
et al., 1999d; Vandenberghe, Soccol, Pandey, & Lebea-
ult, 2000) has been produced from these raw materials
by means of SSF technique.
In recent years, SSF has received more and more
interest from researchers, since several studies for en-
zymes (Pandey et al., 1999a), avours (Ferron, Bonna-
rame, & Durand, 1996), colourants (Johns & Stuart,
1991) and other substances of interest to the food indus-
try have shown that SSF can give higher yields
(Tsuchiya et al., 1994) or better product characteristics
(Acun a-Arguelles, Gutierrez-Rojas, Viniegra-Gonzalez,
& Favela-Torres, 1995) than submerged fermentation
(SmF). In addition, costs are much lower due to the e-
cient utilisation and value-addition of wastes (Robinson
& Nigam, 2003). Castilho, Alves, and Medronho (2000),
have performed a detail economic analysis of the pro-
duction of Penicillium restrictum lipase in both SmF
and SSF. They found that for a production scale of
100 m
3
lipase concentrate per year, total capital invest-
ment needed for SmF was 78% higher than that needed
for SSF. Also, SSF unitary product cost was 47% lower
than the selling price. These studies pointed out that the
great advantage of SSF processes is the extremely cheap
raw material used as main substrate. Therefore, SSF is
certainly a good way of utilising nutrient rich solid
wastes as a substrate. Both food and agricultural wastes
are produced in huge amounts and since they are rich in
carbohydrates and other nutrients, they can serve as a
substrate for the production of bulk chemicals and en-
zymes using SSF technique.
The nature of the solid substrate employed is the
most important factor aecting SSF processes and its
selection depends upon several factors mainly related
with cost and availability and, thus, may involve the
screening of several agro-industrial residues. In SSF pro-
cess the solid substrate not only supplies the nutrients to
the culture but also serves as an anchorage for the
microbial cells. Among the several factors, which are
important for microbial growth and activity in a partic-
ular substrate, particle size and moisture level/water
activity are the most critical (Auria, Palacios, & Revah,
1992; Barrios-Gonzalez, Gonzalez, & Mejia, 1993;
Echevarria, Leon, Espinosa, & Delgado, 1991; Liu
& Tzeng, 1999; Pandey, Ashakumary, Selvakumar, &
Vijayalakshmi, 1994; Pastrana, Gonzalez, Pintado, &
Murado, 1995; Roussos, Raimbault, Prebois, & Lon-
sane, 1993; Sarrette, Nout, Gervais, & Rombouts, 1992;
Smail, Salhi, & Knapp, 1995; Zadrazil & Punia, 1995).
Generally, smaller substrate particles provide a larger
surface area for microbial attack but if they are too
small may result in substrate agglomeration as well as
poor growth. In contrast, larger particles provide better
aeration but a limited surface for microbial attack.
Therefore, a compromised particle size must be selected
for each particular process (Pandey et al., 1999a).
Research on the selection of suitable substrates for
SSF has mainly been centred around agro-industrial resi-
dues due to their potential advantages for lamentous
fungi, which are capable of penetrating into the hardest
of these solid substrates, aided by the presence of turgor
pressure at the tip of the mycelium (Ramachandran
et al., 2004). In addition, the utilisation of these agro-
industrial wastes, on the one hand, provides alternative
substrates and, on the other, helps in solving pollution
problems, which otherwise may cause their disposal
(Pandey et al., 1999a).
SSF oers numerous advantages over SmF such as
simpler technique and lower cost (Table 2). However,
there are few designs available in the literature for biore-
actors operating in solid-state conditions. This is princi-
Table 1
Main groups of microorganisms involved in SSF processes (extracted
from Raimbault, 1998)
Microora SSF process
Bacteria
Bacillus sp. Composting, natto, amylase
Pseudomonas sp. Composting
Serratia sp. Composting
Streptococcus sp. Composting
Lactobacillus sp. Ensiling, food
Clostridium sp. Ensiling, food
Yeast
Endomicopsis burtonii Tape cassava, rice
Saccharomyces cerevisiae Food, ethanol
Schwanniomyces castelli Ethanol, amylase
Fungi
Altemaria sp. Composting
Aspergillus sp. Composting, industrial, food
Fusarium sp. Composting, gibberellins
Monilia sp. Composting
Mucor sp. Composting, food, enzyme
Rhizopus sp. Composting, food, enzymes,
organic acids
Phanerochaete chrysosporium Composting, lignin degradation
Trichoderma sp. Composting, biological control,
bioinsecticide
Beauveria sp., Metharizium sp. Biological control, bioinsecticide
Amylomyces rouxii Tape cassava, rice
Aspergillus oryzae Koji, food, citric acid
Rhizopus oligosporus Tempeh, soybean, amylase, lipase
Aspergillus niger Feed, proteins, amylase,
citric acid
Pleurotus oestreatus, sajor-caju Mushroom
Lentinus edodes Shii-take mushroom
Penicilium notatum, roquefortii Penicillin, cheese
292 S.R. Couto, M
a
.

A. Sanroma n / Journal of Food Engineering 76 (2006) 291302
pally due to several problems encountered in the control
of dierent parameters such as pH, temperature, aera-
tion and oxygen transfer and moisture. SSF lacks the
sophisticated control mechanisms that are usually asso-
ciated with SmF. Control of the environment within the
bioreactors is also dicult to achieve, particularly tem-
perature and moisture.
The aim of this paper is to review the potential appli-
cation of SSF for the production of several metabolites
of great interest to the food industry. In addition, dier-
ent types of biorreactor for SSF processes are described.
2. Some examples of applications of SSF to food industry
2.1. Flavours
Flavours comprise over a quarter of the world mar-
ket for food additives. Most of the avouring com-
pounds are produced via chemical synthesis or by
extraction from natural materials. However, recent mar-
ket surveys have shown that consumers prefer foodstu
that can be labelled as natural. Plants have been major
sources of essential oils and avours but their use de-
pends on natural factors dicult to control such as
weather conditions and plant diseases. An alternative
route for avour synthesis is based on microbial biosyn-
thesis or bioconversion (Janssens, de Pooter, Van-
damme, & Schamp, 1992). Several microorganisms,
including bacteria and fungi, are currently known for
their ability to synthesise dierent aroma compounds.
Attempts to use these microorganisms in SmF resulted
in low productivity of aroma compounds (Yamguchi
et al., 1993), which hampered their industrial applica-
tion. SSF could be of high potential for this purpose
(Berger, 1995). Thus, Ferron et al. (1996) reviewed the
prospects of microbial production of food avours
and the recommended SSF processes for their
production.
Several researchers have studied the production of ar-
oma compounds by SSF from several microorganisms
such as Neurospora sp. (Pastore, Park, & Min, 1994),
Zygosaccharomyces rouxii (Sugawara, Hashimoto,
Sakurai, & Kobayashi, 1994), Aspergillus sp. (Ito,
Yoshida, Ishikawa, & Kobayashi, 1990), using pre-
gelatinised rice, miso and cellulose bres, respectively.
Bramorski, Soccol, Christen, and Revah (1998) com-
pared fruity aroma production by Ceratocystis mbriata
in solid-state cultures using several agro-industrial
wastes (cassava bagasse, apple pomace, amaranth and
soybean), determining that the media with cassava ba-
gasse, apple pomace or soybean produced a strong fru-
ity aroma. Soares, Christen, Pandey, and Soccol (2000)
also reported the production of strong pineapple aroma
when SSF was carried out using coee husk as a sub-
strate by this strain. Bramorski, Christen, Ramirez, Soc-
col, and Revah (1998) and Christen, Bramorski, Revah,
and Soccol (2000) described the production of volatile
compounds such as acetaldehyde and 3-methylbutanol
by the edible fungus Rhizopus oryzae during SSF on
tropical agro-industrial substrates.
Kluyveromyces marxianus produced aroma com-
pounds, such as monoterpene alcohols and isoamyl ace-
tate (responsible for fruity aromas), in SSF using
cassava bagasse or giant palm bran as a substrate
(Medeiros et al., 2001).
Esters are the source of the aromas and among them
pyrazines, which possess a nutty and roasty avour, are
used as a food additive for avouring (Seitz, 1994). Bes-
son, Creuly, Gros, and Larroche (1997) and Larroche,
Besson, and Gros (1999) studied the production of
2,5-dimethylpyrazine (2,5-DMP) and tetramethyl-
prazine (TTMP) using B. natto and B. subtilis, respec-
tively, on soybeans in SSF. They found that SSF
was very suitable for the production of these
compounds.
2.2. Enzyme production
Recently, dos Santos, Souza da Rosa, DalBoit,
Mitchell, and Krieger (2004) evaluated whether SSF is
the best system for producing enzymes. They found that
SSF is appropriate for the production of enzymes and
other thermolabile products, especially when higher
yields can be obtained than in SmF.
2.2.1. a-Amylase
a-Amylases (endo-1,4-a-D-glucan glucanohydrolase
EC 3.2.1.1) are extra-cellular endo enzymes that ran-
domly cleave the 1,4-a linkages between adjacent glu-
cose units in the linear amylose chain and ultimately
generates glucose, maltose and maltotriose units. Since
the 1950s, fungal amylases have been used to manufac-
ture sugar syrups containing specic mixtures of sugars
Table 2
Advantages and disadvantages of SSF over SmF
Advantages Disadvantages
Higher productivity Diculties on scale-up
Better oxygen circulation Low mix eectively
Low-cost media Dicult control of process
parameters (pH, heat, moisture,
nutrient conditions, . . .)
Less eort in downstream
processing
Problems with heat build-up
Reduced energy and cost
requirements
Higher impurity product, increasing
recovery product costs
Simple technology
Scarce operational problems
It resembles the natural
habitat for several
microrganisms
S.R. Couto, M
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A. Sanroma n / Journal of Food Engineering 76 (2006) 291302 293
that could not be produced by conventional acid hydro-
lysis of starch. Amylases are extensively employed in
processed-food industry such as baking, brewing, prepa-
ration of digestive aids, production of cakes, fruit juices,
starch syrups, etc.
The production of a-amylases has generally been car-
ried out using SmF; however, SSF systems appear as a
promising technology. Recently, Francis et al. (2003)
used spent brewing grains in SSF for the production
of a-amylase and determined that the supplement of fer-
mentation media with Tween-80 or calcium ions en-
hanced a-amylase activity.
Krishna and Chandrasekaran (1996) used banana
fruit stalk as a substrate in SSF with Bacillus subtilis.
Dierent factors such as initial moisture content, parti-
cle size, thermal treatment time and temperature, pH,
incubation temperature, additional nutrients, inoculum
size and incubation period on the production of a-amy-
lase were characterised. Results obtained for the optimi-
sation of process parameters clearly shown their impact
on the gross yield of enzymes as well as their indepen-
dent nature in inuencing the organisms ability to syn-
thesise the enzyme. It is known that particle size (specic
surface area) is a critical factor in SSF. Banana fruit
stalk particles of 400 lm favoured maximal a-amylase
production compared to larger particles. A similar
trend was reported for the production of glucoamylases
with wheat bran (Pandey, 1991) and cellulases with coir
pith of small particle size (Muniswaran & Charyulu,
1994).
Nowadays, gelatinisation is coupled with liquefac-
tion, which is possible by the action of thermostable
amylases, which have been reported in both SmF (Stam-
ford, Stamford, Coelho, & Araujo, 2001) and SSF
(Babu & Satyanarayana, 1995). Sodhi, Sharma, Gupta,
and Soni (2005) determined that the productivity of
thermostable amylases from Bacillus sp. was aected
by the nature of the solid substrate (wheat bran, rice
bran, corn bran and combination of two brans), nature
of the moistening agent, level of moisture content,
incubation temperature, presence or absence of sur-
factant, carbon, nitrogen, mineral, amino acid and
vitamin supplements. Maximum enzyme production
was obtained on wheat bran supplemented with glycerol
(1.0%, w/w), soyabean meal (1.0%, w/w), L-proline
(0.1%, w/w), vitamin B-complex (0.01%) and moistened
with tap water containing 1% Tween-40.
Recently, Ramachandran et al. (2004) reported the
use of coconut oil cake (COC) as a substrate for the pro-
duction of a-amylase by A. oryzae under SSF condi-
tions. Raw COC supported the growth of the culture,
resulting in the production of 1372 U/gds a-amylase in
24 h. Supplementation with 0.5% starch and 1% peptone
to the substrate positively enhanced the enzyme synthe-
sis producing 3388 U/gds, proving COC a promising
substrate for a-amylase production.
2.2.2. Fructosyl transferase
Fructosyl transferase (EC 2.4.1.10) catalyses the
formation of fructo-oligosaccharides from sucrose.
Fructo-oligosaccharides are present in various com-
monly consumed foods like fruits, vegetables, cereals
and honey in trace amounts. The production of fructosyl
transferase derived from microorganisms has attracted
attention in recent years by SmF using Aspergillus spp,
Penicillium spp and Aureobasidium spp (Prapulla, Sub-
haprada, &Karanth, 2000) and SSF using both Aspergil-
lus foetidus and A. oryzae (Hang, Woodams, & Jang,
1995; Sangeetha, Ramesh, & Prapulla, 2004).
Recently, Sangeetha et al. (2004) have studied the
production of fructosyl transferase by A. oryzae employ-
ing a great variety of agricultural by-products as sub-
strates: Cereal brans (wheat bran, rice bran and oat
bran), corn products (corn cob, corn bran, corn germ,
corn meal, corn grits and whole corn powder), coee-
and tea-processing by-products (coee husk, coee pulp,
spent coee and spent tea), sugarcane bagasse and cas-
sava bagasse. They found that, among them, the best re-
sults were obtained when rice bran, wheat bran, corn
germ, spent coee and tea were used supplemented with
yeast extract and complete synthetic media.
2.2.3. Lipase
Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3)
are well known as ecient biocatalysts for the hydroly-
sis of water-insoluble fatty-acid esters, being triacylgly-
cerols of long chain fatty acids their natural substrates.
Lipases are nowadays widely used at industrial scale
with applications in food, detergent, cosmetic and phar-
maceutical industries (Jaeger & Reetz, 1998).
Most studies on lipolytic enzymes production by bac-
teria, fungi and yeasts have been performed in sub-
merged cultures; however, there are few reports on
lipase synthesis in solid state cultures. In recent years,
increasing attention has been paid to the conversion of
processing industry wastes in lipase by solid state
cultures.
There are several reports dealing with extracellular li-
pase production by fungus such as Rizhopus sp., Asper-
gillus sp., Penicillium sp. on dierent solid substrates
(Christen, Angeles, Corzo, Farres, & Revah, 1995; Cor-
dova et al., 1998; Gombert, Pinto, Castilho, & Freire,
1999; Kamini, Mala, & Puvanakrishnan, 1998; Miranda
et al., 1999) under submerged conditions. However, few
researchers have investigated the synthesis of lipase by
yeasts using SSF technique. Among them, Rao,
Jayaraman, and Lakshmanan (1993) determined that
the C/N ratio of the medium is an important parameter
for lipase production by the yeast Candida rugosa.
Rivera-Mun oz, Tinoco-Valencia, Sanchez, and
Farres (1991), Ohnishi, Yoshida, and Sekiguchi (1994),
Christen et al. (1995) and Benjamin and Pandey
(1996a, 1996b, 1997a, 1997b), compared SmF and SSF
294 S.R. Couto, M
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A. Sanroma n / Journal of Food Engineering 76 (2006) 291302
systems for lipase production. All they found that en-
zyme yields were higher and stable in the latter.
Several factors can aect extracellular lipase produc-
tion such as pH, temperature, aeration and medium
composition. Furthermore, the presence of triglycerides
or fatty acids has been reported to increase lipolytic en-
zyme secretion by a certain number of microorganisms
(Marek & Bednarski, 1996). Therefore, in SSF the type
of substrate could be used to enhance the production of
enzymes, as several food and agroindustrial wastes are
rich in fatty acids, triglycerides and/or sugars.
Dominguez, Costas, Longo, and Sanroman (2003)
have reported the great potential of food-agroindustrial
wastes (ground nut and barley bran) as support-sub-
strates for lipase production in solid state cultures of
the yeast Y. lipolytica, since they led to much higher
activities than those found using an inert support.
2.2.4. Pectinases
They constitute a heterogeneous group of enzymes
that catalyse the degradation of pectins, which are the
structural polysaccharides present in vegetable cells
and are responsible for maintaining the plant tissues
integrity (Alkorta, Garbisu, Llama, & Serra, 1998). Pec-
tinases are widely used in the food industry to clarify
fruit juices and wine, to improve oil extraction, to re-
move the peel from citrus fruit, to increase the rmness
of several fruits and to degum bres (Baker & Wicker,
1996; Chang, Siddiq, Sinha, & Cash, 1994).
Commercial pectinase preparations are produced
from fungal microorganisms, mainly by Aspergillus
niger strains. The use of SSF for pectinase production
has been proposed using dierent solid agricultural
and agro-industrial residues as substrates such as wheat
bran (Castilho, Alves, & Medronho, 1999; Singh, Platt-
ner, & Diekmann, 1999), soy bran (Castilho et al., 2000),
cranberry and strawberry pomace (Zheng & Shetty,
2000), coee pulp and coee husk (Antier, Minjares,
Roussos, & Viniegra-Gonzalez, 1993), husk (Antier,
Minjares, Roussos, Raimbault, & Viniegra-Gonzalez,
1993; Boccas, Roussos, Gutierrez, Serrano, & Viniegra,
1994), cocoa (Schwan, Cooper, & Wheals, 1997), lemon
and orange peel (Garzon & Hours, 1991; Ismail, 1996;
Maldonado, Navarro, & Callieri, 1986), orange bagasse,
sugar cane bagasse and wheat bran (Martins, Silva, Da
Silva, & Gomes, 2002), sugar cane bagasse (Acun a-
Arguelles, Gutierrez-Rojas, Viniegra-Gonzalez, &
Favela-Torres, 1994) and apple pomace (Hours, Voget,
& Ertola, 1988a, 1988b). Also, Bai, Zhang, Qi, Peng,
and Li (2004) produced pectinase from A. niger by SSF
using sugar beet pulp as a carbon source and wastewater
from monosodium glutamate production as nitrogen
and water source. This allowed not only reducing pro-
duction costs but also decreasing the pollution source.
It was found that SSF was more productive than
SmF and, in addition, the pectinases produced by SSF
showed more stable properties: they had a higher
stability to pH and temperature and they were less
aected by catabolic repression than pectinases pro-
duced by SmF (Acun a-Arguelles et al., 1995).
2.3. Organic acids
Organic acids have been utilised for long time by the
food industry as food additives and preservatives for
prevent deterioration and extending the shelf life of
perishable food ingredients. Here, two common organic
acids widely used on the food industry have been
considered.
2.3.1. Lactic acid
Lactic acid fermentation has received extensive atten-
tion since long time (Benninga, 1990; Vickroy, 1985). It
has wide applications in food, pharmaceutical, leather
and textile industries and as a chemical feed stock. It
has two enantiomers L(+) and D() of which L(+) is
used by human metabolism due to the presence of L lac-
tate dehydrogenase and is preferred for food. Nowa-
days, lactic acid is in great demand due to its use as
starting material to produce biodegradable polymers
used in medical, industrial and consumer products
(Bohlmann & Yoshida, 2000; Gross & Kalra, 2002;
Lichteld, 1996; Malhotra, Raina, & Sanjay, 2000).
Soccol, Marin, Rimbault, and Labeault (1994) stud-
ied the production of L(+)-lactic acid by Rhizopous ory-
zae in solid-state conditions operating with sugarcane
bagasse as a support. They obtained a slightly higher
productivity than in submerged cultivation. Also, Rich-
ter and Trager (1994) investigated the L(+)-lactic acid
production by Lactobacillus paracasei in solid-state con-
ditions using sweet sorghum as a support. More re-
cently, Naveena, Altaf, Bhadrayya, Madhavendra, and
Reddy (2005) and Naveena, Altaf, Bhadriah, and Reddy
(2005) have reported the production of L(+) lactic acid
by Lactobacillus amylophilus GV6 under SSF conditions
using wheat bran as both support and substrate.
2.4. Citric acid
Citric acid is one of the most commonly used organic
acids in food and pharmaceutical industries. The food
industry is the largest consumer of citric acid, using al-
most 70% of the total production, followed by about
12% for the pharmaceutical industry and 18% for other
applications (Shah, Chattoo, Baroda, & Patiala, 1993).
Its pleasant taste, high solubility and avour-enhancing
properties have ensured its dominant position in the
market. Although citric acid can be obtained by chemi-
cal synthesis, the cost is much higher than using fermen-
tation. It is mainly produced by SmF, by the lamentous
fungus A. niger. Recently, in order to increase the
S.R. Couto, M
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A. Sanroma n / Journal of Food Engineering 76 (2006) 291302 295
eciency of citric acid production using A. niger, SSF
has been studied as a potential alternative to SmF.
The production of citric acid depends strongly on an
appropriate strain and on operational conditions. Oxy-
gen level is an important parameter for citric acid fer-
mentation. Several researchers (Pintado, Lonsane,
Gaime-Perraud, & Roussos, 1998; Prado et al., 2004)
have studied the inuence of forced aeration on citric
acid production and the metabolic activity of A. niger
in SSF by respirometric analysis. They showed that
citric acid production was favoured by a limited biomass
production, which occurred with low aeration rates.
Both works showed the feasibility of using the strain
A. niger for citric acid production by SSF.
Dierent agro-industrial residues such as apple
pomace, coee husk, wheat straw, pineapple waste,
mixed fruit, maosmi waste, cassava bagasse, banana,
sugar beet cosset and kiwi fruit peel have been investi-
gated for their potential to be used as substrates (Hang
& Woodams, 1985; Khare, Krishana, & Gandhi, 1995;
Kumar, Jain, Shanker, & Srivastava, 2003a, 2003b;
Shojaosadati & Babaripour, 2002). In addition, SSF
gave high citric acid yield without inhibition related to
presence of certain metal ions such as Fe
2+
, Mn
2+
,
Zn
2+
, etc. (Gutierrez-Rozas, Cordova, Auria, Revah,
& Favela-Torres, 1995), although Shankaranand and
Lonsane (1994) reported that addition of these minerals
into the production media to a certain level enhanced
citric acid production by 1.41.9 fold with respect to
SmF. Therefore, SSF is a good way of using nutrient
rich solid waste as a substrate.
2.5. Xanthan gum
Xanthan gum is a hetero-polysaccharide produced
industrially by the bacterium Xanthomonas campestris,
fermenting commonly glucose or sucrose. It is the most
important microbial polysaccharide from the commer-
cial point of view, with a worldwide production of about
30,000 tons per year, corresponding to a market of $408
million (Demain, 2000; Sutherland, 1998). This water-
soluble microbial polysaccharide gives aqueous solu-
tions with several industrial applications in the food,
cosmetic, textile and pharmaceutical industries due to
their rheological properties. Because of these properties,
they have been used as emulsiers, as stabilisers and as
texture enhancers in the food industry.
Recently, the feasibility of this exopolysaccharide
production using SSF has been reported by the group
of Stredansky and Conti (1999), Stredansky, Conti,
Navarini, and Bertocchi (1999). X. campestris strains
were cultivated on a great variety of solid substrates or
by-products such as spent malt grains, apple pomace,
grape pomace and citrus peels, easily available and
low cost substrates, in order to evaluate their ability to
produce the exopolysaccharide xanthan. With most of
the substrates, xanthan yields were comparable to those
obtained from conventional submerged cultivation. In
addition, the products were analysed by NMR spectros-
copy, revealing a composition consistent with that of
commercial xanthan.
2.6. SSF bioreactors
The design of an ecient industrial-level reactor for
SSF is of signicance because SSF is more environmen-
tally friendly than SmF. However, it shows considerable
drawbacks such as transfer resistance, steep gaseous
concentration and heat gradients that develop within
the medium bed, which may adversely aect solid-state
fermentor performances (Ghildyal, Ramalaishna, Lon-
sane, & Karantb, 1992; Lonsane, Saucedo-Castuneda,
& Raimbault, 1992; Sargantanis, Karim, Murphy,
Ryoo, & Tengerdy, 1992). Agitation and rotation in
SSF were often carried out to improve mass and heat
transfers, but the shearing force caused by agitation
and rotation has adverse eects on medium porosity
and disrupts fungal mycelia.
There are four types of reactors to perform SSF pro-
cesses and each in their own design tries to make condi-
tions more favourable for fermentation under solid state
conditions. The bioreactors commonly used, which can
be distinguished by the type of aeration or the mixed
system employed, include the following:
Tray: It consists of at trays. The substrate is spread
onto each tray forming a thin layer, only a few centime-
tres deep. The reactor is kept in a chamber at constant
temperature through which humidied air is circulated
(Fig. 1). The main disadvantage of this conguration
is that numerous trays and large volume are required,
making it an unattractive design for large-scale produc-
tion (Pandey, Soccol, Rodriguez-Leon, & Nigam, 2001).
Packed-bed: It is usually composed of a column of
glass or plastic with the solid substrate retained on a per-
forated base. Through the bed of substrate humidied
air is continuously forced (Durand et al., 1993; Raimba-
ult, 1998; Rodr guez Couto, Rivela, Mun oz, &
Sanroman, 2000). It may be tted with a jacket for
circulation of water to control the temperature during
fermentation (Fig. 2). This is the conguration usually
employed in commercial koji production. The main
drawbacks associated with this conguration are: di-
culties in obtaining the product, non-uniform growth,
poor heat removal and scale-up problems.
Horizontal drum: This design allows adequate aera-
tion and mixing of the substrate, whilst limiting the
damage to the inoculum or product. Mixing is per-
formed by rotating the entire vessel or by various agita-
tion devices such as paddles and baes (Dom nguez,
Rivela, Rodr guez Couto, & Sanroman, 2001; Nagel,
Tramper, Bakker, & Rinzema, 2001a, Nagel, Tramper,
Bakker, & Rinzema, 2001b; Prado et al., 2004; Stuart,
296 S.R. Couto, M
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.

A. Sanroma n / Journal of Food Engineering 76 (2006) 291302
Mitchell, Johns, & Litster, 1999) (Fig. 3). Its main disad-
vantage is that the drum is lled to only 30% capacity,
otherwise mixing is inecient.
Fluidised bed: In order to avoid the adhesion and
aggregation of substrate particles, this design supplies
a continue agitation with forced air (Fig. 4). Although
the mass heat transfer, aeration and mixing of the sub-
strate is increased, damage to inoculum and heat
build-up through sheer forces may aect the nal
product yield.
The dierent disadvantages detected in the above-
mentioned bioreactor designs to perform SSF processes
have promoted the necessity of developing new bioreac-
tor congurations or modifying the already existing de-
signs. These bioreactor congurations should be able to
operate in continuous mode with high productivity for
prolonged periods of time without operational problems
as well as permit the scale-up of the process. Our re-
search group has been working in this eld, resulting
in the design of a new bioreactor, called immersion bio-
reactor. This bioreactor consists of a jacketed cylindrical
glass vessel with a round bottom, inside which several
wire mesh baskets lled with support colonised by the
fungus are placed. They moved upwards and down-
wards by means of a pneumatic system, remaining more
time outside than inside the medium (Rivela, Rodr guez
Couto, & Sanroman, 2000) (Fig. 5). It is noteworthy
that this bioreactor conguration was also able to run
in continuous mode without operational problems,
attaining high ligninolytic enzyme activities (Rodriguez
Couto, Barreiro, Rivela, Longo, & Sanroman, 2002).
Dierent studies were carried out for the production
of natural food and additives derived from micro-
organisms in dierent bioreactor congurations. For
Trays
Gas exit
Air inlet
Solid substrate
Fig. 1. Scheme of a tray bioreactor (passive aeration; static).
Fig. 2. Scheme of a packed-bed bioreactor (humidied air; static).
Air inlet
Gas exit
Sampling port
Culture medium level
Support susbstrate
Air diffuser
3 rpm
Fig. 3. Scheme of a horizontal drum bioreactor (humidied air;
mechanical agitation).
Fig. 4. Scheme of a uidised-bed bioreactor (humidied air; pneu-
matic agitation).
S.R. Couto, M
a
.

A. Sanroma n / Journal of Food Engineering 76 (2006) 291302 297
example, the production of aroma compounds by K.
marxianus grown on cassava bagasse in solid state fer-
mentation using packed bed reactors, testing two dier-
ent aeration rates was studied by Medeiros et al. (2001).
Headspace analysis of the culture by gas chromatogra-
phy showed the production of 11 compounds. The pre-
dominant compounds were ethyl acetate, ethanol and
acetaldehyde. The fruity aroma was attributed to the
productions of esters.
Recently, Navarrete-Bolanos, Jimenez-Islas, Botello-
Alvarez, Rico-Mart nez, and Paredes-Lo pez (2004) have
employed a modular rotating drum bioreactor
(equipped with inlet air injection, variable speed pumps,
humidier, and gas analyser) for xanthophylls extrac-
tion from marigold owers. Marigold extracts have been
commercialised internationally and are used as additives
for poultry feed, as they provide bright colours in egg
yolks, skin, and fatty tissues. For this reason, they are
used as an additive in several food and pharmaceutical
industries. Based on experimental design strategies, opti-
mum operation values were determined for aeration,
moisture, agitation and marigold-to-inoculum ratio in
SSF of marigold owers by mixed culture of three
microorganisms (Flavobacterium IIb, Acinetobacter anit-
ratus, and Rhizopus nigricans), leading to a xanthophylls
yield of 17.8-g/kg dry weight. This value represented a
65% increase in relation to the control.
Milagres, Santos, Piovan, and Roberto (2004) have
shown that Thermoascus aurantiacus was able to pro-
duce a high level of thermostable xylanase when sugar
cane bagasse was used as a substrate in a glass-column
reactor with forced aeration. The airow rate had a sig-
nicant eect on enzyme activity, whereas initial mass of
bagasse had none. The highest yield of xylanase
(1597 U/g) was obtained operating in the bioreactor at
the optimal conditions: airow rate (6 l/h g) and sub-
strate (8 g).
A packed-bed bioreactor with four stages was con-
structed and operated for microbial production of citric
acid by A. niger using apple pomace as a substrate.
Under the optimised conditions, 124 g citric acid was
produced from 1 kg dry apple pomace with yield of
80% based on total sugar (Shojaosadati & Babaripour,
2002).
3. Conclusion
Critical analysis of the literature shows that produc-
tion of relevant compounds for the food processing
industry by SSF oers several advantages. It has been
well established that enzyme titres produced in SSF sys-
tems are much higher than the achieved in SmF ones.
Although the reasons for this are not clear, this fact is
kept in mind while developing novel bioreactors for
SSF processes.
Acknowledgments
The authors are grateful to Xunta de Galicia (local
government of Spain) for the nancial support of the re-
search post of Susana Rodr guez Couto under the pro-
gramme Isidro Parga Pondal.
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