Journal of Food Engineering 76 (2006) 291–302

www.elsevier.com/locate/jfoodeng

Application of solid-state fermentation to food industry—A review

a,b
Susana Rodrı́guez Couto , Ma Ángeles Sanromán 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 specific 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 flavours, enzymes (a-amylase, fructosyl transferase, lipase, pectinase),
organic acids (lactic acid, citric acid) and xanthan gum. In addition, different types of biorreactor for SSF processes have been
described.
Ó 2005 Elsevier Ltd. All rights reserved.

Keywords: Bioreactors; Enzyme production; Food processing industry; Solid-state fermentation

1. Introduction liquid (Pandey, 1992). The low moisture content means

Microorganisms have long played a major role in the
production of food (dairy, fish and meat products) and
alcoholic beverages. In addition, several products of
microbial fermentation are also incorporated into food
as additives and supplements (antioxidants, flavours,
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 defined 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 flowing
*
Corresponding author. Tel.: +34 986 812383; fax: +34 986 812380.
E-mail address: sanroman@uvigo.es (Ma. Á. Sanromán).
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 offers 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 different 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 Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jfoodeng.2005.05.022
292  Sanromán / Journal of Food Engineering 76 (2006) 291–302
S.R. Couto, Ma. A.

Table 1 & Nigam, 2003). Castilho, Alves, and Medronho (2000),

Main groups of microorganisms involved in SSF processes (extracted have performed a detail economic analysis of the pro-
from Raimbault, 1998)
duction of Penicillium restrictum lipase in both SmF
Microflora SSF process and SSF. They found that for a production scale of
Bacteria 100 m3 lipase concentrate per year, total capital invest-
Bacillus sp. Composting, natto, amylase ment needed for SmF was 78% higher than that needed
Pseudomonas sp. Composting
Serratia sp. Composting
for SSF. Also, SSF unitary product cost was 47% lower
Streptococcus sp. Composting than the selling price. These studies pointed out that the
Lactobacillus sp. Ensiling, food great advantage of SSF processes is the extremely cheap
Clostridium sp. Ensiling, food raw material used as main substrate. Therefore, SSF is
Yeast certainly a good way of utilising nutrient rich solid
Endomicopsis burtonii Tape cassava, rice wastes as a substrate. Both food and agricultural wastes
Saccharomyces cerevisiae Food, ethanol are produced in huge amounts and since they are rich in
Schwanniomyces castelli Ethanol, amylase
carbohydrates and other nutrients, they can serve as a
Fungi substrate for the production of bulk chemicals and en-
Altemaria sp. Composting zymes using SSF technique.
Aspergillus sp. Composting, industrial, food
The nature of the solid substrate employed is the
Fusarium sp. Composting, gibberellins
Monilia sp. Composting most important factor affecting SSF processes and its
Mucor sp. Composting, food, enzyme selection depends upon several factors mainly related
Rhizopus sp. Composting, food, enzymes, with cost and availability and, thus, may involve the
organic acids screening of several agro-industrial residues. In SSF pro-
Phanerochaete chrysosporium Composting, lignin degradation
cess the solid substrate not only supplies the nutrients to
Trichoderma sp. Composting, biological control,
bioinsecticide the culture but also serves as an anchorage for the
Beauveria sp., Metharizium sp. Biological control, bioinsecticide microbial cells. Among the several factors, which are
Amylomyces rouxii Tape cassava, rice important for microbial growth and activity in a partic-
Aspergillus oryzae Koji, food, citric acid ular substrate, particle size and moisture level/water
Rhizopus oligosporus Tempeh, soybean, amylase, lipase
activity are the most critical (Auria, Palacios, & Revah,
Aspergillus niger Feed, proteins, amylase,
citric acid 1992; Barrios-Gonzalez, Gonzalez, & Mejia, 1993;
Pleurotus oestreatus, sajor-caju Mushroom Echevarria, Leon, Espinosa, & Delgado, 1991; Liu
Lentinus edodes Shii-take mushroom & Tzeng, 1999; Pandey, Ashakumary, Selvakumar, &
Penicilium notatum, roquefortii Penicillin, cheese Vijayalakshmi, 1994; Pastrana, Gonzalez, Pintado, &
Murado, 1995; Roussos, Raimbault, Prebois, & Lon-
sane, 1993; Sarrette, Nout, Gervais, & Rombouts, 1992;
amounts of agricultural residues generated by this coun- Smail, Salhi, & Knapp, 1995; Zadrazil & Punia, 1995).
try (Soccol & Vandenberghe, 2003). Thus, the produc- Generally, smaller substrate particles provide a larger
tion of bulk chemicals and value-added fine products surface area for microbial attack but if they are too
such as ethanol, single-cell protein (SPC), mushrooms, small may result in substrate agglomeration as well as
enzymes, organic acids, amino acids, biologically active poor growth. In contrast, larger particles provide better
secondary metabolites, etc. (Hölker, Höfer, & Lenz, aeration but a limited surface for microbial attack.
2004; Pandey, 1992; Pandey, 1994; Pandey, Azmi, Therefore, a compromised particle size must be selected
Singh, & Banerjee, 1999b; Pandey et al., 1999c; Pandey, for each particular process (Pandey et al., 1999a).
Nigam, & Vogel, 1988; Pandey et al., 2000b; Pandey Research on the selection of suitable substrates for
et al., 1999d; Vandenberghe, Soccol, Pandey, & Lebea- SSF has mainly been centred around agro-industrial resi-
ult, 2000) has been produced from these raw materials dues due to their potential advantages for filamentous
by means of SSF technique. fungi, which are capable of penetrating into the hardest
In recent years, SSF has received more and more of these solid substrates, aided by the presence of turgor
interest from researchers, since several studies for en- pressure at the tip of the mycelium (Ramachandran
zymes (Pandey et al., 1999a), flavours (Ferron, Bonna- et al., 2004). In addition, the utilisation of these agro-
rame, & Durand, 1996), colourants (Johns & Stuart, industrial wastes, on the one hand, provides alternative
1991) and other substances of interest to the food indus- substrates and, on the other, helps in solving pollution
try have shown that SSF can give higher yields problems, which otherwise may cause their disposal
(Tsuchiya et al., 1994) or better product characteristics (Pandey et al., 1999a).
(Acuña-Arguelles, Gutierrez-Rojas, Viniegra-González, SSF offers numerous advantages over SmF such as
& Favela-Torres, 1995) than submerged fermentation simpler technique and lower cost (Table 2). However,
(SmF). In addition, costs are much lower due to the effi- there are few designs available in the literature for biore-
cient utilisation and value-addition of wastes (Robinson actors operating in solid-state conditions. This is princi-
  Sanromán / Journal of Food Engineering 76 (2006) 291–302
S.R. Couto, Ma. A.
Table 2
Advantages and disadvantages of SSF over SmF
Advantages Disadvantages
Higher productivity Difficulties on scale-up
Better oxygen circulation Low mix effectively
Low-cost media Difficult control of process
parameters (pH, heat, moisture,
nutrient conditions, . . .)
Less effort in downstream Problems with heat build-up
processing
Reduced energy and cost Higher impurity product, increasing
requirements recovery product costs
Simple technology
Scarce operational problems
It resembles the natural
habitat for several
microrganisms
pally due to several problems encountered in the control
of different 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 difficult 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, differ-
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 flavouring com-
pounds are produced via chemical synthesis or by
extraction from natural materials. However, recent mar-
ket surveys have shown that consumers prefer foodstuff
that can be labelled as natural. Plants have been major
sources of essential oils and flavours but their use de-
pends on natural factors difficult to control such as
weather conditions and plant diseases. An alternative
route for flavour 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 different 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 flavours
and the recommended SSF processes for their
production.
293
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 fibres, respectively.
Bramorski, Soccol, Christen, and Revah (1998) com-
pared fruity aroma production by Ceratocystis fimbriata
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 coffee 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 flavour, are
used as a food additive for flavouring (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, DalÕBoit,
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 specific mixtures of sugars
294  Sanromán / Journal of Food Engineering 76 (2006) 291–302
S.R. Couto, Ma. A.
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.
Different 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 influencing the organismÕs ability to syn-
thesise the enzyme. It is known that particle size (specific
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 affected
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), coffee-
and tea-processing by-products (coffee husk, coffee pulp,
spent coffee 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 coffee 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 efficient 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 different 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-Muñoz, 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
  Sanromán / Journal of Food Engineering 76 (2006) 291–302
S.R. Couto, Ma. A.
systems for lipase production. All they found that en-
zyme yields were higher and stable in the latter.
Several factors can affect 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 firmness
of several fruits and to degum fibres (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 different 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), coffee pulp and coffee husk (Antier, Minjares,
Roussos, & Viniegra-Gonzalez, 1993), husk (Antier,
Minjares, Roussos, Raimbault, & Viniegra-González,
1993; Boccas, Roussos, Gutierrez, Serrano, & Viniegra,
1994), cocoa (Schwan, Cooper, & Wheals, 1997), lemon
and orange peel (Garzón & 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 (Acuña-
Arguelles, Gutierrez-Rojas, Viniegra-González, &
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
295
showed more stable properties: they had a higher
stability to pH and temperature and they were less
affected by catabolic repression than pectinases pro-
duced by SmF (Acuña-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;
Lichtfield, 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 Träger (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 flavour-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 filamentous
fungus A. niger. Recently, in order to increase the
296  Sanromán / Journal of Food Engineering 76 (2006) 291–302
S.R. Couto, Ma. A.
efficiency 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 influence 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.
Different agro-industrial residues such as apple
pomace, coffee 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 Fe2+, Mn2+,
Zn2+, 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 1.4–1.9 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 emulsifiers, 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 efficient industrial-level reactor for
SSF is of significance 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 affect 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 effects 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 flat 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 humidified air is circulated
(Fig. 1). The main disadvantage of this configuration
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 humidified
air is continuously forced (Durand et al., 1993; Raimba-
ult, 1998; Rodrı́guez Couto, Rivela, Muñoz, &
Sanromán, 2000). It may be fitted with a jacket for
circulation of water to control the temperature during
fermentation (Fig. 2). This is the configuration usually
employed in commercial koji production. The main
drawbacks associated with this configuration are: diffi-
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 baffles (Domı́nguez,
Rivela, Rodrı́guez Couto, & Sanromán, 2001; Nagel,
Tramper, Bakker, & Rinzema, 2001a, Nagel, Tramper,
Bakker, & Rinzema, 2001b; Prado et al., 2004; Stuart,
  Sanromán / Journal of Food Engineering 76 (2006) 291–302
S.R. Couto, Ma. A. 297

Gas exit
Gas exit
3 rpm
Support susbstrate

Culture medium level

Sampling port Air diffuser
Air inlet

Fig. 3. Scheme of a horizontal drum bioreactor (humidified air;

Trays mechanical agitation).

Solid substrate

Air inlet

Fig. 1. Scheme of a tray bioreactor (passive aeration; static).

Mitchell, Johns, & Litster, 1999) (Fig. 3). Its main disad-
vantage is that the drum is filled to only 30% capacity,
otherwise mixing is inefficient.
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 affect the final
product yield.
The different disadvantages detected in the above-
mentioned bioreactor designs to perform SSF processes
have promoted the necessity of developing new bioreac-
tor configurations or modifying the already existing de-
signs. These bioreactor configurations 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 field, resulting
Fig. 4. Scheme of a fluidised-bed bioreactor (humidified air; pneu-
matic agitation).

in the design of a new bioreactor, called immersion bio-

Fig. 2. Scheme of a packed-bed bioreactor (humidified air; static).
reactor. This bioreactor consists of a jacketed cylindrical
glass vessel with a round bottom, inside which several
wire mesh baskets filled 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, Rodrı́guez
Couto, & Sanromán, 2000) (Fig. 5). It is noteworthy
that this bioreactor configuration was also able to run
in continuous mode without operational problems,
attaining high ligninolytic enzyme activities (Rodriguez
Couto, Barreiro, Rivela, Longo, & Sanroman, 2002).
Different studies were carried out for the production
of natural food and additives derived from micro-
organisms in different bioreactor configurations. For
298  Sanromán / Journal of Food Engineering 76 (2006) 291–302
S.R. Couto, Ma. A.
Compressor
Compressor Pneumatic system
Gas exit
Culture medium level
Water cooling
Mesh with substrate
Sampling port
Air inlet
Fig. 5. Scheme of an immersion bioreactor (humidified air; mechanical
agitation).
example, the production of aroma compounds by K.
marxianus grown on cassava bagasse in solid state fer-
mentation using packed bed reactors, testing two differ-
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-Bolaños, Jiménez-Islas, Botello-
Alvarez, Rico-Martı́nez, and Paredes-López (2004) have
employed a modular rotating drum bioreactor
(equipped with inlet air injection, variable speed pumps,
humidifier, and gas analyser) for xanthophylls extrac-
tion from marigold flowers. 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 flowers 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 airflow rate had a sig-
nificant effect 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: airflow 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 offers 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 financial support of the re-
search post of Susana Rodrı́guez Couto under the pro-
gramme Isidro Parga Pondal.
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