Aplicaciones de fermentación en sólido- Industria alimentaria

8/13/2019 Aplicaciones de fermentacin en slido- Industria alimentaria

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Application of solid-state fermentation to food industryA review

Susana Rodrguez Couto a,b, Ma Angeles Sanroman a,*

a Department of Chemical Engineering, Isaac Newton Building, University of Vigo, Lagoas Marcosende, 36310 Vigo, Spainb Department of Chemical Engineering, Chemical Engineering School, Rovira i Virgili University, 43007 Tarragona, Spain

Received 9 November 2004; accepted 19 May 2005Available online 20 July 2005

Abstract

Solid state fermentation (SSF) has become a very attractive alternative to submerged fermentation (SmF) for specific applicationsdue 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 beendescribed. 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 theproduction of food (dairy, fish and meat products) andalcoholic beverages. In addition, several products ofmicrobial fermentation are also incorporated into foodas additives and supplements (antioxidants, flavours,colourants, preservatives, sweeteners, . . .). There is greatinterest in the development and use of natural food andadditives derived from microorganisms, since they aremore desirable than the synthetic ones produced bychemical 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 performedon a non-soluble material that acts both as physical sup-port and source of nutrients in absence of free flowing

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

that fermentation can only be carried out by a limitednumber 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 reportedinTable 1.

SSF offers numerous advantages for the productionof bulk chemicals and enzymes (Hesseltine, 1977;Pandey, Selvakumar, Soccol, & Nigam, 1999a; Soccol,Iloki, Marin, & Raimbault, 1994). This process is knownfrom 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 Aspergillusoryzaeto initiate the koji process and Penicillium roque-fortii for cheese production. Also, in China, SSF hasbeen used extensively to produce brewed foods (such asChinese wine, soy sauce and vinegar) since ancient time(Chen, 1992). Also, in Japan SSF is used commerciallyto produce industrial enzymes (Suryanarayan, 2003).Since 1986 in Brazil a series of research projects for thevalue-addition of tropical agricultural products andsub-products by SSF has been developed due to the high

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* Corresponding author. Tel.: +34 986 812383; fax: +34 986 812380.E-mail address:[email protected](Ma. A. Sanroman).

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amounts of agricultural residues generated by this coun-try (Soccol & Vandenberghe, 2003). Thus, the produc-tion of bulk chemicals and value-added fine productssuch as ethanol, single-cell protein (SPC), mushrooms,enzymes, organic acids, amino acids, biologically activesecondary metabolites, etc. (Holker, Hofer, & Lenz,2004; Pandey, 1992; Pandey, 1994; Pandey, Azmi,Singh, & Banerjee, 1999b; Pandey et al., 1999c; Pandey,Nigam, & Vogel, 1988; Pandey et al., 2000b; Pandeyet al., 1999d; Vandenberghe, Soccol, Pandey, & Lebea-ult, 2000) has been produced from these raw materialsby means of SSF technique.

In recent years, SSF has received more and moreinterest from researchers, since several studies for en-zymes (Pandey et al., 1999a), flavours (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(Acuna-Arguelles, Gutierrez-Rojas, Viniegra-Gonzalez,& Favela-Torres, 1995) than submerged fermentation(SmF). In addition, costs are much lower due to the effi-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 SmFand SSF. They found that for a production scale of100 m3 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% lowerthan the selling price. These studies pointed out that thegreat advantage of SSF processes is the extremely cheapraw material used as main substrate. Therefore, SSF iscertainly a good way of utilising nutrient rich solidwastes as a substrate. Both food and agricultural wastesare produced in huge amounts and since they are rich incarbohydrates and other nutrients, they can serve as asubstrate for the production of bulk chemicals and en-zymes using SSF technique.

The nature of the solid substrate employed is themost important factor affecting SSF processes and itsselection depends upon several factors mainly related

with cost and availability and, thus, may involve thescreening of several agro-industrial residues. In SSF pro-cess the solid substrate not only supplies the nutrients tothe culture but also serves as an anchorage for themicrobial cells. Among the several factors, which areimportant for microbial growth and activity in a partic-ular substrate, particle size and moisture level/wateractivity 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 largersurface area for microbial attack but if they are toosmall may result in substrate agglomeration as well aspoor growth. In contrast, larger particles provide betteraeration but a limited surface for microbial attack.Therefore, a compromised particle size must be selectedfor each particular process (Pandey et al., 1999a).

Research on the selection of suitable substrates forSSF has mainly been centred around agro-industrial resi-dues due to their potential advantages for filamentousfungi, which are capable of penetrating into the hardestof these solid substrates, aided by the presence of turgorpressure at the tip of the mycelium (Ramachandranet al., 2004). In addition, the utilisation of these agro-industrial wastes, on the one hand, provides alternativesubstrates and, on the other, helps in solving pollutionproblems, which otherwise may cause their disposal(Pandey et al., 1999a).

SSF offers numerous advantages over SmF such assimpler 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 1Main groups of microorganisms involved in SSF processes (extractedfrom Raimbault, 1998)

Microflora SSF process

Bacteria

Bacillus sp. Composting, natto, amylasePseudomonas sp. Composting

Serratia sp. CompostingStreptococcus sp. CompostingLactobacillus sp. Ensiling, food

Clostridium sp. Ensiling, food

YeastEndomicopsis burtonii Tape cassava, riceSaccharomyces cerevisiae Food, ethanolSchwanniomyces castelli Ethanol, amylase

Fungi

Altemaria sp. CompostingAspergillus sp. Composting, industrial, foodFusarium sp. Composting, gibberellinsMonilia sp. Composting

Mucorsp. Composting, food, enzymeRhizopus sp. Composting, food, enzymes,

organic acidsPhanerochaete chrysosporium Composting, lignin degradation

Trichoderma sp. Composting, biological control,bioinsecticide

Beauveria sp., Metharizium sp. Biological control, bioinsecticideAmylomyces rouxii Tape cassava, rice

Aspergillus oryzae Koji, food, citric acidRhizopus oligosporus Tempeh, soybean, amylase, lipase

Aspergillus niger Feed, proteins, amylase,citric acid

Pleurotus oestreatus, sajor-caju MushroomLentinus edodes Shii-take mushroomPenicilium notatum, roquefortii Penicillin, cheese

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pally due to several problems encountered in the controlof different parameters such as pH, temperature, aera-

tion and oxygen transfer and moisture. SSF lacks thesophisticated control mechanisms that are usually asso-ciated with SmF. Control of the environment within thebioreactors 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 metabolitesof 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 byextraction from natural materials. However, recent mar-ket surveys have shown that consumers prefer foodstuffthat can be labelled as natural. Plants have been majorsources of essential oils and flavours but their use de-pends on natural factors difficult to control such asweather conditions and plant diseases. An alternativeroute 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 fortheir ability to synthesise different aroma compounds.Attempts to use these microorganisms in SmF resultedin low productivity of aroma compounds (Yamguchiet 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 theprospects of microbial production of food flavoursand the recommended SSF processes for theirproduction.

Several researchers have studied the production of ar-oma compounds by SSF from several microorganismssuch 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 byCeratocystis fimbriatain solid-state cultures using several agro-industrialwastes (cassava bagasse, apple pomace, amaranth andsoybean), 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 aromawhen SSF was carried out using coffee husk as a sub-strate by this strain.Bramorski, Christen, Ramirez, Soc-col, and Revah (1998)andChristen, Bramorski, Revah,and Soccol (2000) described the production of volatile

compounds such as acetaldehyde and 3-methylbutanolby the edible fungus Rhizopus oryzae during SSF ontropical agro-industrial substrates.

Kluyveromyces marxianus produced aroma com-pounds, such as monoterpene alcohols and isoamyl ace-tate (responsible for fruity aromas), in SSF usingcassava bagasse or giant palm bran as a substrate(Medeiros et al., 2001).

Esters are the source of the aromas and among thempyrazines, which possess a nutty and roasty flavour, areused as a food additive for flavouring (Seitz, 1994).Bes-son, Creuly, Gros, and Larroche (1997) andLarroche,

Besson, and Gros (1999) studied the production of2,5-dimethylpyrazine (2,5-DMP) and tetramethyl-prazine (TTMP) using B. natto and B. subtilis, respec-tively, on soybeans in SSF. They found that SSFwas very suitable for the production of thesecompounds.

2.2. Enzyme production

Recently, dos Santos, Souza da Rosa, DalBoit,Mitchell, and Krieger (2004) evaluated whether SSF isthe best system for producing enzymes. They found that

SSF is appropriate for the production of enzymes andother thermolabile products, especially when higheryields can be obtained than in SmF.

2.2.1. a-Amylase

a-Amylases (endo-1,4-a-D-glucan glucanohydrolaseEC 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 ultimatelygenerates glucose, maltose and maltotriose units. Sincethe 1950s, fungal amylases have been used to manufac-ture sugar syrups containing specific mixtures of sugars

Table 2Advantages and disadvantages of SSF over SmF

Advantages Disadvantages

Higher productivity Difficulties on scale-upBetter oxygen circulation Low mix effectivelyLow-cost media Difficult control of process

parameters (pH, heat, moisture,

nutrient conditions, . . .)Less effort in downstream

processingProblems with heat build-up

Reduced energy and costrequirements

Higher impurity product, increasingrecovery product costs

Simple technologyScarce operational problemsIt resembles the natural

habitat for severalmicrorganisms

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that could not be produced by conventional acid hydro-lysis of starch. Amylases are extensively employed inprocessed-food industry such as baking, brewing, prepa-ration of digestive aids, production of cakes, fruit juices,starch syrups, etc.

The production ofa-amylases has generally been car-

ried out using SmF; however, SSF systems appear as apromising technology. Recently, Francis et al. (2003)used spent brewing grains in SSF for the productionofa-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 bananafruit 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, inoculumsize and incubation period on the production ofa-amy-lase were characterised. Results obtained for the optimi-

sation of process parameters clearly shown their impacton the gross yield of enzymes as well as their indepen-dent nature in influencing the organisms ability to syn-thesise the enzyme. It is known that particle size (specificsurface area) is a critical factor in SSF. Banana fruitstalk particles of 400 lm favoured maximal a-amylaseproduction compared to larger particles. A similartrend was reported for the production of glucoamylaseswith wheat bran (Pandey, 1991) and cellulases with coirpith of small particle size (Muniswaran & Charyulu,1994).

Nowadays, gelatinisation is coupled with liquefac-

tion, which is possible by the action of thermostableamylases, 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 ofthermostable amylases from Bacillus sp. was affectedby the nature of the solid substrate (wheat bran, ricebran, corn bran and combination of two brans), natureof the moistening agent, level of moisture content,incubation temperature, presence or absence of sur-factant, carbon, nitrogen, mineral, amino acid andvitamin supplements. Maximum enzyme productionwas 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 moistenedwith tap water containing 1% Tween-40.

Recently, Ramachandran et al. (2004) reported theuse 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 in24 h. Supplementation with 0.5% starch and 1% peptoneto the substrate positively enhanced the enzyme synthe-sis producing 3388 U/gds, proving COC a promisingsubstrate for a-amylase production.

2.2.2. Fructosyl transferase

Fructosyl transferase (EC 2.4.1.10) catalyses theformation of fructo-oligosaccharides from sucrose.Fructo-oligosaccharides are present in various com-monly consumed foods like fruits, vegetables, cerealsand honey in trace amounts. The production of fructosyl

transferase derived from microorganisms has attractedattention 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 theproduction of fructosyl transferase byA. oryzaeemploy-ing a great variety of agricultural by-products as sub-strates: Cereal brans (wheat bran, rice bran and oatbran), 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, corngerm, spent coffee and tea were used supplemented withyeast 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 onlipase synthesis in solid state cultures. In recent years,increasing attention has been paid to the conversion ofprocessing industry wastes in lipase by solid statecultures.

There are several reports dealing with extracellular li-pase production by fungus such as Rizhopussp., 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; Mirandaet al., 1999) under submerged conditions. However, fewresearchers have investigated the synthesis of lipase byyeasts using SSF technique. Among them, Rao,Jayaraman, and Lakshmanan (1993) determined thatthe C/N ratio of the medium is an important parameterfor lipase production by the yeast Candida rugosa.

Rivera-Munoz, Tinoco-Valencia, Sanchez, andFarres (1991), Ohnishi, Yoshida, and Sekiguchi (1994),Christen et al. (1995) and Benjamin and Pandey(1996a, 1996b, 1997a, 1997b), compared SmF and SSF



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efficiency of citric acid production using A. niger, SSFhas been studied as a potential alternative to SmF.

The production of citric acid depends strongly on anappropriate 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 citricacid production and the metabolic activity of A. nigerin SSF by respirometric analysis. They showed thatcitric acid production was favoured by a limited biomassproduction, which occurred with low aeration rates.Both works showed the feasibility of using the strainA. niger for citric acid production by SSF.

Different agro-industrial residues such as applepomace, 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, SSFgave high citric acid yield without inhibition related topresence of certain metal ions such as Fe2+, Mn2+,Zn2+, etc. (Gutierrez-Rozas, Cordova, Auria, Revah,& Favela-Torres, 1995), although Shankaranand andLonsane (1994)reported that addition of these mineralsinto the production media to a certain level enhancedcitric acid production by 1.41.9 fold with respect toSmF. Therefore, SSF is a good way of using nutrientrich solid waste as a substrate.

2.5. Xanthan gum

Xanthan gum is a hetero-polysaccharide producedindustrially by the bacterium Xanthomonas campestris,fermenting commonly glucose or sucrose. It is the mostimportant microbial polysaccharide from the commer-cial point of view, with a worldwide production of about30,000 tons per year, corresponding to a market of $408million (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 totheir rheological properties. Because of these properties,they have been used as emulsifiers, as stabilisers and astexture enhancers in the food industry.

Recently, the feasibility of this exopolysaccharideproduction using SSF has been reported by the groupof Stredansky and Conti (1999), Stredansky, Conti,Navarini, and Bertocchi (1999). X. campestris strainswere cultivated on a great variety of solid substrates orby-products such as spent malt grains, apple pomace,grape pomace and citrus peels, easily available andlow cost substrates, in order to evaluate their ability toproduce the exopolysaccharide xanthan. With most of

the substrates, xanthan yields were comparable to thoseobtained from conventional submerged cultivation. Inaddition, the products were analysed by NMR spectros-copy, revealing a composition consistent with that ofcommercial xanthan.

2.6. SSF bioreactors

The design of an efficient industrial-level reactor forSSF is of significance because SSF is more environmen-tally friendly than SmF. However, it shows considerabledrawbacks such as transfer resistance, steep gaseousconcentration and heat gradients that develop withinthe medium bed, which may adversely affect solid-statefermentor performances (Ghildyal, Ramalaishna, Lon-sane, & Karantb, 1992; Lonsane, Saucedo-Castuneda,& Raimbault, 1992; Sargantanis, Karim, Murphy,Ryoo, & Tengerdy, 1992). Agitation and rotation inSSF were often carried out to improve mass and heat

transfers, but the shearing force caused by agitationand rotation has adverse effects on medium porosityand 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 stateconditions. The bioreactors commonly used, which canbe distinguished by the type of aeration or the mixedsystem employed, include the following:

Tray: It consists of flat trays. The substrate is spreadonto 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 configurationis 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 ofglass or plastic with the solid substrate retained on a per-forated base. Through the bed of substrate humidifiedair is continuously forced (Durand et al., 1993; Raimba-ult, 1998; Rodrguez Couto, Rivela, Munoz, &Sanroman, 2000). It may be fitted with a jacket forcirculation of water to control the temperature duringfermentation (Fig. 2). This is the configuration usuallyemployed in commercial koji production. The maindrawbacks 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 thedamage 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 (Domnguez,Rivela, Rodrguez Couto, & Sanroman, 2001; Nagel,Tramper, Bakker, & Rinzema, 2001a, Nagel, Tramper,Bakker, & Rinzema, 2001b; Prado et al., 2004; Stuart,



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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 andaggregation of substrate particles, this design suppliesa continue agitation with forced air (Fig. 4). Althoughthe mass heat transfer, aeration and mixing of the sub-strate is increased, damage to inoculum and heatbuild-up through sheer forces may affect the finalproduct yield.

The different disadvantages detected in the above-mentioned bioreactor designs to perform SSF processeshave promoted the necessity of developing new bioreac-

tor configurations or modifying the already existing de-signs. These bioreactor configurations should be able tooperate in continuous mode with high productivity forprolonged periods of time without operational problemsas well as permit the scale-up of the process. Our re-search group has been working in this field, resulting

in the design of a new bioreactor, called immersion bio-reactor. This bioreactor consists of a jacketed cylindricalglass vessel with a round bottom, inside which severalwire mesh baskets filled with support colonised by thefungus are placed. They moved upwards and down-wards by means of a pneumatic system, remaining moretime outside than inside the medium (Rivela, RodrguezCouto, & Sanroman, 2000) (Fig. 5). It is noteworthythat this bioreactor configuration was also able to runin continuous mode without operational problems,attaining high ligninolytic enzyme activities (RodriguezCouto, Barreiro, Rivela, Longo, & Sanroman, 2002).

Different studies were carried out for the productionof natural food and additives derived from micro-organisms in different bioreactor configurations. 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 (humidified 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 (humidified air;mechanical agitation).

Fig. 4. Scheme of a fluidised-bed bioreactor (humidified air; pneu-matic agitation).



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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 byMedeiros 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 andacetaldehyde. The fruity aroma was attributed to theproductions of esters.

Recently,Navarrete-Bolanos, Jimenez-Islas, Botello-Alvarez, Rico-Martnez, and Paredes-Lopez (2004)haveemployed 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 beencommercialised internationally and are used as additivesfor poultry feed, as they provide bright colours in eggyolks, skin, and fatty tissues. For this reason, they areused as an additive in several food and pharmaceuticalindustries. Based on experimental design strategies, opti-mum operation values were determined for aeration,moisture, agitation and marigold-to-inoculum ratio inSSF of marigold flowers by mixed culture of threemicroorganisms (Flavobacterium IIb,Acinetobacter anit-ratus, andRhizopus nigricans), leading to a xanthophyllsyield of 17.8-g/kg dry weight. This value represented a65% increase in relation to the control.

Milagres, Santos, Piovan, and Roberto (2004) haveshown that Thermoascus aurantiacus was able to pro-duce a high level of thermostable xylanase when sugarcane bagasse was used as a substrate in a glass-columnreactor with forced aeration. The airflow rate had a sig-nificant effect on enzyme activity, whereas initial mass ofbagasse had none. The highest yield of xylanase(1597 U/g) was obtained operating in the bioreactor atthe 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 citricacid by A. niger using apple pomace as a substrate.Under the optimised conditions, 124 g citric acid wasproduced from 1 kg dry apple pomace with yield of80% based on total sugar (Shojaosadati & Babaripour,

2002).

3. Conclusion

Critical analysis of the literature shows that produc-tion of relevant compounds for the food processingindustry by SSF offers several advantages. It has beenwell 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 iskept in mind while developing novel bioreactors forSSF processes.

Acknowledgments

The authors are grateful to Xunta de Galicia (localgovernment of Spain) for the financial support of the re-search post of Susana Rodrguez Couto under the pro-gramme Isidro Parga Pondal.

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