Solid-state fermentation are there any biotechnological advantages?

Udo Holker1 and Jurgen Lenz2

Solid-state fermentation (SSF) has developed in eastern countries over many centuries, and has enjoyed broad application in these regions to date. By contrast, in western countries the technique had to compete with classical submerged fermentation and, because of the increasing pressure of rationalisation and standardisation, it has been widely superseded by classical submerged fermentation since the 1940s. This is mainly because of problems in engineering that appear when scaling up this technique. However, there are several advantages of SSF, for example high productivities, extended stability of products and low production costs, which say much about such an intensive biotechnological application. With increasing progress and application of rational methods in engineering, SSF will achieve higher levels in standardisation and reproducibility in the future. This can make SSF the preferred technique for special elds of application such as the production of enzymes and food.
Addresses 1 Institute of Cellular and Molecular Botany, University of Bonn, Kirschallee 1, 53115 Bonn, Germany 2 Bioreact GmbH, Mulheimer Str. 26, 53840 Troisdorf, Germany Corresponding author: Holker, Udo (u.hoelker@uni-bonn.de)

ing aspects, will rst be considered separately, and then compared from the economical viewpoint.

Historical aspects
When compared with the SSF processes that have long been established in Asian countries, SSF in western countries appears to still be in its infancy. SSF is the state-of-the-art technology that is used in many applications in the food industry in Asia. For example, SSF is used in the production of enzyme-rich Koji (which is made of rice and uses Aspergillus oryzae as an enzymatic starter for different hydrolytic processes), in the saccharication of rice used for the production of alcoholic beverages such as sake, in the production of cellulase (which uses Trichoderma strains), and in the production of angkak, which is rice that is coloured red by Monascus purpurea metabolites. In Asia, enzymes and metabolites are commonly produced on a large scale by SSF processes that may be thousands of years old. These processes vary widely depending on locality, and are transmitted as secret formulas from father to son. In western countries, SSF had to compete with the technically easier SmF, which was more strongly developed from the 1940s onwards because of the necessity to produce antibiotics on a large scale. Today, SSF has been nearly completely abandoned because of the pressure of increasing industrial rationalisation and standardisation. In the east, by contrast, SSF survived in many areas in the form of labour-convenient, hand-made processes; however, some of the traditional SSF processes have also been further developed to achieve higher industrial rationalisation through stronger automation.

Current Opinion in Microbiology 2005, 8:301306 This review comes from a themed issue on Ecology and industrial microbiology Edited by Sergio Sanchez and Betty Olson Available online 5th May 2005 1369-5274/$ see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2005.04.006

Biological features Introduction

The term solid-state fermentation (SSF) denotes cultivation of microorganisms on solid, moist substrates in the absence of a free aqueous phase; that is, at average water activities (dened as the relative humidity of the gaseous phase in equilibrium with the moist solid) signicantly below 1 [1]. In a broader denition, SSF can be seen as including processes during which microorganisms are cultivated in the presence of a liquid phase at maximal substrate concentrations [2] or on inert carriers [3]. To assess the possible advantages of the biotechnological uses of SSF relative to those of classical submerged fermentations (SmFs), different aspects of the two, including historical, biological, ecological and engineerwww.sciencedirect.com

SSF processes simulate the living conditions of many higher lamentous fungi. Ascomycetes, basidiomycetes and deuteromycetes developed in terrestrial habitats on wet substrates. For this reason, SSF is the cultivation method of choice for instance in cases in which the biotechnological process is required to include morphological and metabolic differentiation into substrate-penetrating and aerial hyphae, such as in the production of conidiospores. Hence, SSF is used in the food industry in the production of conidiospores for the production of mould cheese (for which conidiospores of Penicillium roquefortii or P. camemberti are needed) or salami improvement (for which conidiospores of P. nalgiovensis can be used) and also in the eld of production of plant protection agents (which uses Coniothyrium minitans).
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Higher fungi and their enzymes, spores or metabolites are well adjusted to growth on solid wet substrates. For instance, fungal spores produced by SSF show higher stability, are more resistant to drying and exhibit higher germination rates for extended periods of time after freeze-drying than do spores produced by SmF. This can be attributed to the fact that the conidiospores produced in SSF have a higher hydrophobicity, as was demonstrated with Penicillium oxalicum [4], or also possess, apart from a higher hydrophobicity caused by a hydrophobin-like protein, a stronger cell wall and a smaller volume (this has been documented with Trichoderma harzianum [5]). A direct comparison between the SSF and SmF cultivation modes of fungi is difcult to make because the two processes differ. Studies of the production of fungal enzymes in SSF have repeatedly shown that SSF, in comparison with SmF, provides higher volumetric productivities, is less prone to problems with substrate inhibition and yields enzymes with a higher temperature or pH stability. Also, the fermentation time may be shorter and the degradation of the enzymes by undesirable proteases is minimised [6]. Interesting studies in this eld have explored the physiological differences that arise during the growth of microbial cells in the two types of processes. In the well-studied fungus Aspergillus oryzae, water activity seems to play a decisive role in this context [7]. This is apparent from the accumulation of glycerol, erythritol and arabinitol [8], and from the induction of glucoamylase genes, the promoters of which have been shown to contain motifs that suggest induction by temperature or water activity [9], under SSF conditions. Similar results were also obtained in studies of the production of secondary metabolites [10]. When grown on solid substrates, numerous fungi have shown a markedly altered spectrum of secondary metabolites. As described in streptomycetes, the production of secondary metabolites often seems to be associated with the formation of aerial hyphae and the onset of spore production in the stationary growth phase. Various secondary metabolites have even been shown to be produced solely during growth on solid substrates, although the fungi in question can be readily cultivated in submerged processes. For this reason, SSF could play an interesting role in the screening for new metabolites and also in the production of these metabolites by wild-type fungi. In the employment of microbial mixed cultures, SSF offers an option that cannot be achieved by SmF. This makes use of the fact that, during growth on natural substrates, fungal consortia secrete a broad spectrum of enzymes [1113]. During co-cultivation of different fungi, some of the individual enzyme activities show synergistic increase [14], whereas others remain unchanged [15]. Filamentous fungi and yeasts can be broadly applied in processes of simultaCurrent Opinion in Microbiology 2005, 8:301306

neous saccharication and fermentation that can be regulated by the joint action of aerobic and facultative anaerobic processes via the availability of oxygen. In addition, SSF offers the possibility to explore and to use interactions between fungi by properly regulating the water activity as a selection parameter depending on the different water demands of the individual co-cultivated fungi. Many SSF processes used in the food industry make use of mixed cultures that are important for the avour of the foods produced. For example, bamboo sprouts can be processed by undened mixed cultures that, during the fermentation process, produce a variety of metabolites including 29 volatile substances with aroma-active properties [16]. Despite the situation when using lamentous fungi, SSF has only found restricted applications in processes using unicellular organisms. SSF has, for instance, been used in the production of endospores (Bacillus thuringiensis) [17], secondary metabolites by means of aerial mycelium of streptomycetes [18] or alcohol by yeast on solid sugar-rich substrates, which can be conveniently combined with simultaneous or previous hydrolysis of the polysaccharides by extracellular fungal enzymes. Another example is the production of organic acids (such as vinegar production performed in Asia for thousands of years) or the traditional fermentation of foods [19] by microbial mixed cultures that involve yeasts and bacteria.

Ecological features
Ecological (environment-protective) advantages of SSF reect the fact that the processes are conducted in the absence of a free aqueous phase. This results in minimum water consumption and thus a low production of efuent water by the process, obviation of the necessity to use antifoam agents, and the possibility of conducting SSF processes in certain applications under semi-sterile conditions. Because SSF processes are performed at water activities below 1, the growth of contaminating bacteria and yeasts is minimised. This means that, in certain cases, energy- and instrumentation-demanding sterilisation processes can be eliminated. Another additional environmentally friendly feature of SSF is that, in many cases, it can use agricultural wastes as carbon and energy sources. The production of enzymes or organic acids makes frequent use of plant remnants as carbon sources and inducers or mediators.

Engineering features
The most serious reasons why SSF has not yet found a broad use in western countries are engineering problems, the low amenability of the processes to standardisation and the limited reproducibility of the results. Scale-up represents a particular bottleneck because several different gradients (temperature, humidity, substrate concentration, and so on), which can arise in the course of the process, can have an adverse effect especially in static
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solid bed processes but also in processes involving substrate agitation, such as those performed in rotating drums. Inter-relationships among environmental factors such as oxygen content, moisture level and temperature contribute to the difcult regulation of these parameters. The microbial growth under aerobic conditions in the bioreactor results in a considerable production of heat that causes fast increase in temperature. This effect, which is desirable in composting, is often fatal for biotechnological processes because a large part of the enzymes produced during the fermentation can be heat-denatured at the end of the process [20]. In the absence of a free aqueous phase, the produced heat is difcult to remove, for example, via the bioreactor double walls. Instead, the cooling of the process takes place through evaporation. However, this requires very high aeration rates because the rising metabolic activity and the associated increased heat production have to be overcompensated by high aeration intensity. The water escaping by evaporation has to be in many cases replenished, and this can lead to
Figure 1

undesirable local increase in water activity during static processing. In semi-sterile processes, this increased water activity can in turn have adverse effects by facilitating the growth of bacterial contaminants, whereas in sterile fermentations, it may bring about local insufcient oxygen supply to the microorganisms. Substrate mixing may help but it should be noted that many microorganisms respond very sensitively to the shear stress caused by it. Another factor that is difcult to account for is the production of metabolic water by aerobic microorganisms, which can cause problems especially in the formation of conidiospores. Despite all these problems, the development of rational, at least partly computer-controlled methods of process development within the past decade brought advances in the eld, leading to a better understanding of biological and physical processes that take place during the SSF (Figure 1), and paving the way to new starting points for further industrialisation of the process [21]. These

O2

Aerial hyphae

Mycelial mat l s NH3 pH Acids Enzymes Polymers Monomers

Current Opinion in Microbiology

s l

CO2

s H2O Q T

Substrate surface Substrate pores l Penetrative hyphae

Products

Schematic of some of the microscale processes that occur during solid-state fermentation (SSF). After sporulation, fungal hyphae develop into a mycelial mat (black), which spreads over the surface of the particles that comprise the solid substrate (brown). From the mycelial mat, aerial hyphae protrude into the gaseous space, whereas others penetrate the substrate by growing into liquid-filled pores. At normal moisture levels, the void spaces between the aerial hyphae are most likely to be filled with gas (g), whereas the void spaces within the mycelial mat and within the substrate are filled with liquid (l). The metabolic activities shown mainly occur near the substrate surface and within the pores; however, exposed regions of the mycelium (for instance the aerial hyphae) also show metabolism and there can be a transport of substances from the penetrative to the aerial hyphae. Hydrolytic enzymes (light blue), which are produced by the mycelium, diffuse to the solid matrix and catalyse the degradation of macromolecules into smaller units (green). The latter are taken up by the fungus to serve as nutrients. O2 is consumed and CO2, H20, heat and interesting biochemical products are produced during fermentation. Hence, gradients develop within the biofilm that, for instance, force O2 to diffuse from the gaseous phase into deeper regions of the biofilm (lilac) and CO2 to diffuse from these regions to the gaseous phase (red). Heat development (Q; orange) leads to a fast increase in temperature (T), which is a serious problem during SSF. Heat is therefore removed from the substrate not only via conduction but also by evaporation, which is part of the complex balance of water in the system (dark blue). Beside evaporation, water balance includes water uptake by the mycelium in the course of growth, water consumption during hydrolysis reactions and water production through respiration. As another important factor, local pH, might be changed owing to the release of carbon acids and the exchange of ammonia (grey). The biochemical products of interest (magenta) that are released into the solid matrix and the liquid-filled spaces during fermentation might absorb to the solid and might have to be extracted for further use at the end of the process. All these and many other phenomena can strongly influence process performance during SSF. www.sciencedirect.com Current Opinion in Microbiology 2005, 8:301306

304 Ecology and industrial microbiology

developments include progress in the modelling of microbial growth on solid substrates and energy and mass transfer in different types of bioreactors [2226,27, 28,29,30], formulation of scale-up rules and recommendations for optimal design and scale-up of industrial bioreactors [3133], new methods for measuring the spatial distribution of process parameters, evaluation of parameters that are difcult to measure on-line such as humidity and biomass [3436], the use of statistical methods for parameter optimisation [3742], and the development of new strategies for process control in SSF [4345]. Interestingly, the practical development of commercially employed bioreactors proceeded largely independently of the results of the above studies. This was in part because of the difculties in converting the general statements of rational studies pertaining to bioreactor and process design into concrete guidelines for specic applications. The reason again is the lack of sufcient bodies of process data needed for validating the mathematical models. Hence, the commercially employed industrial bioreactors often still represent empirical developments of traditional processes, or they are large but technically relatively lowlevel facilities restricted correspondingly to non-sterile and robust processes suitable for manufacture of bulk products (e.g. bioreactors for Koji fermentation). The engineering shortcomings are then compensated by high space and time gains and low production costs so that these processes can be operated with satisfactory economy. Although a variety of different SSF bioreactors are available on the laboratory and pilot scale [46], only some of these variants have the potential for scale-up to the industrial level because large-volume operations may give rise to effects, such as the inuence of compaction, which are not perceptible on a smaller scale.

sured in solid substrates can reach levels common in commercial enzyme preparation [48].

Conclusion
Yet, despite the multitude of the above advantages, a broad application of SSF is, at least at present, hard to envisage. The main obstacles are the low amenability of the process to regulation, the strongly heterogeneous fermentation conditions and the ensuing frequently unsatisfactory reproducibility of the results, difcult scale-up, the often unfeasible biomass determination and complicated product purication by downstream processes resulting from the use of heterogeneous organic growth substrates. For these reasons, SSF will never replace or supersede the established processes for the production of enzymes and metabolites by bacteria or yeasts that have for decades been successfully cultivated in submerged bioreactors and also optimised as heterologous expression systems. Yet only in special cases, SSF can be applicable also with unicellular organisms. A different situation exists in the cultivation of differentiating microorganisms, in which SSF simulates the natural growth conditions and its biological and environmental advantages can outweigh the engineering problems. In principle, SSF processes can be economically operated on a large scale despite their engineering difculties when the technical demands (for example, in nonsterile processes or in those that do not require further expensive product processing) and hence also the production costs are low. In these cases, SSF can provide substantial advantages over SmF. At any rate, even though further progress in the eld of SSF can be expected to bring improved biomonitoring and thereby optimisation and higher standardisation of industrial processes, the future applications of SSF for the above purposes can be expected to be mainly limited to those that even today exhibit substantial advantages above all the production of spores, screening for secondary metabolites and enzymes, protein enrichment of bre-rich natural materials, degradation and bioconversion of plant material [49], and co-cultivation of several fungi for the production of enzymes [50] and foods.

Economic viewpoint
The described biological and environmental benets of SSF can bring direct economical advantages that have much to say for an intensive biotechnological application of SSF. For instance, calculation of the costs of production of crude enzymes by SSF showed that, for example in the case of cellulase production, the economical efciency is higher by a factor of 100 than in the case of SmF [47]. This striking difference has several reasons. In addition to the much cheaper growth substrates, the minimised requirements for sterility and low requirements for instrumentation and equipment, this approach can make downstream processes in the production of enzymes unnecessary. In general, this holds for those cases when the fermented substrate can be used directly in a follow-up process such as the saccarication of renewable raw materials employed in ensiling, production of bio-ethanol or biogas, in the pulpand-paper industry, textiles industry or in the production of animal fodder supplements. This is usually made possible thanks to the high concentrations of enzymes in the solid substrates, which makes further concentrating processes unnecessary. The enzyme activities that have been meaCurrent Opinion in Microbiology 2005, 8:301306

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:  of special interest  of outstanding interest 1. Pandey A: Solid-state fermentation. Biochem eng 2003,  13:81-84. The author gives a compact, yet very informative, overview of the present status and applications of SSF. He envisages SSF to become on a par with SmF technology, with respect to industrial application in the future, if the current trends in rational engineering are continued. 2. Mitchell DA, Berovic M, Krieger N: Biochemical engineering of solid state bioprocessing. Adv Biochem Eng Biotechnol 2000, 68:61-138. www.sciencedirect.com

 Solid-state fermentation Holker and Lenz 305

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This study investigates critically the effect of heat production on productivity during SSF. Overheating, which results from difculties in heat removal, is a severe problem when trying to scale up a process. 21. Lenz J, Hofer M, Krasenbrink JB, Holker U: Computational and physical methods in solid-state fermentation. Appl Microbiol Biotechnol 2004, 65:9-17. 22. Ashley VM, Mitchell DA, Howes T: Evaluating strategies for overcoming overheating problems during solid-state fermentation in packed bed bioreactors. Biochem Eng J 1999, 3:141-150. 23. Gervais P, Molin P: The role of water in solid-state fermentation. Biochem Eng J 2003, 13:85-101. 24. Ikasari L, Mitchell DA: Two-phase model of the kinetics of growth of Rhizopus oligosporus in membrane culture. Biotechnol Bioeng 2000, 68:619-627. 25. Mitchell DA, Tongta A, Stuart DM, Krieger N: The potential for establishment of axial temperature proles during solid-state fermentation in rotating drum bioreactors. Biotechnol Bioeng 2002, 80:114-122. 26. Mitchell DA, von Meien OF, Krieger N: Recent developments in modelling of solid-state fermentation: heat and mass transfer in bioreactors. Biochem Eng J 2003, 13:137-147. 27. Mitchell DA, von Meien OF, Krieger N, Dalsenter FDH: A review of  recent developments in modelling of microbial growth kinetics and intraparticle phenomena in solid-state fermentation. Biochem Eng J 2004, 17:15-26. In an excellent review, the authors describe various approaches to the modelling of those processes that occur on the particle scale during SSF. Mechanistic modelling of, for instance, intra-particle diffusion of enzymes, O2 and hydrolysis products helps us to understand how these effects inuence fungal growth and could potentially limit bioreactor performance. 28. Oostra J, le Comte EP, van der Heuvel JC, Tramper J, Rinzema A: Intra-particle oxygen diffusion limitation in solid-state fermentation. Biotechnol Bioeng 2001, 75:13-24. 29. Schutyser MAI, de Pagter P, Weber FJ, Briels WJ, Boom RM,  Rinzema A: Substrate aggregation due to aerial hyphae during discontinuously mixed solid-state fermentation with Aspergillus oryzae: experiments and modelling. Biotechnol Bioeng 2003, 83:503-513. This study is the rst attempt to quantify and to predict the effect of mycelial bonds on particle mixing and vice versa during SSF. The tensile strength of mycelial bonds was measured using a new device on the basis of two wheat-dough-plates and texture analysis. Using these measurements, mixing experiments performed with Aspergillus oryzae that grows on wheat grain in a rotating drum bioreactor were compared with results obtained from computer simulations. 30. Schutyser MAI, Weber FJ, Briels WJ, Rinzema A, Boom RM: Heat and water transfer in a rotating drum containing solid substrate particles. Biotechnol Bioeng 2003, 82:552-563. 31. Hardin MT, Mitchell DA, Howes T: Approach to designing rotating drum bioreactors for solid-state fermentation on the basis of dimensionless design factors. Biotechnol Bioeng 2000, 67:274-282. 32. Mitchell DA, Pandey A, Sangsurasak P, Krieger N: Scale-up strategies for packed-bed bioreactors for solid-state fermentation. Process Biochem 1999, 35:167-178. 33. Weber FJ, Tramper J, Rinzema A: A simplied material and energy balance approach for process development and scaleup of Coniothyrium minitans conidia production by solid-state cultivation in a packed-bed reactor. Biotechnol Bioeng 1999, 65:447-458. 34. Aldarf M, Amrane A, Prigent Y: Reconstruction of the biomass history from carbon and nitrogen substrate consumption, ammonia release and proton transfer during solid cultures of Geotrichum candidum and Penicillium camembertii. Appl Microbiol Biotechnol 2002, 58:823-829. 35. Bellon-Maurel V, Orliac O, Christen P: Sensors and measurements in solid state fermentation: a review. Process Biochem 2003, 38:881-896. Current Opinion in Microbiology 2005, 8:301306

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Ruijter GJ, Visser J, Rinzema A: Polyol accumulation by Aspergillus oryzae at low water activity in solid-state fermentation. Microbiology 2004, 150:1095-1101. In this interesting paper, it was shown that Aspergillus oryzae accumulates high concentrations of polyols at water activities between 0.96 and 0.97 during solid-state processing, which seems to be typical for this type of processing. In addition, four different polyol dehydrogenases were detected, which might be induced by osmotic stress, as concluded by the authors. 9. Ishida H, Hata Y, Kawato A, Abe Y, Suginami K, Imayasu S: Identication of functional elements that regulate the glucoamylase-encoding gene (glab) expressed in solid-state culture of Aspergillus oryzae. Curr Genet 2000, 37:373-379.

10. Robinson T, Singh D, Nigam P: Solid-state fermentation: a promising microbial technology for secondary metabolite production. Appl Microbiol Biotechnol 2001, 55:284-289. 11. Koroleva OV, Gavrilova VP, Stepanova EV, Lebedeva VI, Sverdlova NI, Landesman EO, Yavmetdinov IS, Yaropolov AI: Production of lignin modifying enzymes by co-cultivated white-rot fungi Cerrena maxima and Coriolus hirsutus and characterization of laccase from Cerrena maxima. Enz Microbial Technol 2002, 30:573-580. 12. Stepanova EV, Koroleva OV, Vasilchenko LG, Karapetyan KN, Landesman EO, Yavmetdinov IS, Kozlov YP, Ranbinovich ML: Fungal decomposition of oat straw during liquid and solidstate fermentation. Prikl Biokhim Mikrobiol 2003, 39:65-74. 13. Yang YH, Wang BC, Wang QH, Xiang LJ, Duan CR: Research on solid-state fermentation on rice chaff with a microbial consortium. Colloids Surf B Biointerfaces 2004, 34:1-6. 14. Gutierrez-Correa M, Portal L, Moreno P, Tengerdy RP: Mixed culture solid substrate fermentation of Trichoderma reesei with Aspergillus niger on sugar cane bagasse. Bioresour Technol 1999, 68:173-178. 15. Massadeh MI, Yusoff WMW, Omar O, Kader J: Synergism of cellulase enzymes in mixed culture solid substrate fermentation. Biotechnol Lett 2001, 23:1771-1774. 16. Fu SG, Yoon Y, Bazemore R: Aroma-active compounds in fermented bamboo shoots. J Agric Food Chem 2002, 50:549-554. 17. Hongzhang C, Fujian X, Zhonghou T, Zuohu L: A novel industriallevel reactor with two dynamic changes of air for solid-state fermentation. J Biosci Bioeng 2002, 93:211-214. 18. Ellaiah P, Srinivasulu B, Adinarayana K: Optimisation studies on neomycin production by a mutant strain of Streptomyces marinensis in solid state fermentation. Process Biochem 2003, 39:529-534. 19. Han B-Z, Rombouts FM, Nout MJR: A Chinese fermented soybean food. Int J Food Microbiol 2001, 65:1-10. 20. Muller dos Santos M, Souza das Rosa A, DalBoit S, Mitchell DA,  Krieger N: Thermal denaturation: is solid-state fermentation really a good technology for the production of enzymes? Bioresour Technol 2004, 93:261-268. www.sciencedirect.com

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36. Pena y Lillo M, Perez-Correa R, Agosin E, Latrille E: Indirect measurement of water content in an aseptic solid substrate cultivation pilot-scale bioreactor. Biotechnol Bioeng 2001, 76:44-51. 37. Abdel-Fattah YR, Olama ZA: l-Asparaginase production by Pseudomonas aeruginosa in solid-state culture: evaluation and optimization of culture conditions using factorial designs. Process Biochem 2002, 38:115-122. 38. Banerjee R, Bhattacharyya BC: Evolutionary operation as a tool of optimization for solid-state fermentation. Biochem Eng J 2003, 13:149-155. 39. Francis F, Sabu A, Nampoothiri KM, Ramachandran S, Ghosh S, Szakacs G, Pandey A: Use of response surface methodology for optimizing process parameters for the production of a-amylase by Aspergillus oryzae. Biochem Eng J 2004, 15:107-115. 40. Hamidi-Esfahani Z, Shojaosadati SA, Rinzema A: Modelling of simultaneous effect of moisture and temperature on A. niger growth in solid-state fermentation. Biochem Eng J 2004, 21:265-272. 41. Kar B, Banerjee R, Bhattacharyya BC: Optimization of physicochemical parameters for gallic acid production by evolutionary operation-factorial design technique. Process Biochem 2002, 37:1395-1401. 42. Park YS, Kang SW, Lee JS, Hong SI, Kim SW: Xylanase production in solid state fermentation by Aspergillus niger mutant using statistical experimental designs. Appl Microbiol Biotechnol 2002, 58:761-766. 43. Nagel FJI, Tramper J, Bakker MSN, Rinzema A: Temperature control in a continuously mixed bioreactor for solid-state fermentation. Biotechnol Bioeng 2001, 72:219-230.

44. Nagel FJI, Tramper J, Marjolein SN, Bakker MSN, Rinzema A: Model for on-line moisture-content control during solid-state fermentation. Biotechnol Bioeng 2001, 72:231-243. 45. Thibault J, Acuna G, Perez-Corea R, Jorquera H, Molin P, Agosin E: A hybrid representation approach for modelling complex dynamic bioprocesses. Bioprocess Eng 2000, 22:547-556. 46. Durand A: Bioreactor designs for solid state fermentation. Biochem Eng J 2003, 13:113-125. 47. Tengerdy RP: Cellulase production by solid substrate fermentation. J Sci Ind Res 1996, 55:313-316. 48. Holker U, Janssen M, Lenz J: Directed selective solid-phase culturing of stable microbial mixed populations for the continuous preparation of dened enzyme and metabolite mixtures. PCT / EP2004 / 051234. 49. Tengerdy RP, Szakacs G: Bioconversion of lignocellulose  in solid substrate fermentation. Biochem Eng J 2003, 13:169-179. This review presents the state-of-the-art of lignocellulose bioconversion by solid-substrate fermentation. It also contains descriptions of novel solid-state bioreactors with on-line process control. 50. Viniegra-Gonzalez G, Favela-Torres E, Aguilar CN,  Romero-Gomez S, de J, Diaz-Godinez G, Augar C: Advantages of fungal enzyme production in solid state over liquid fermentation systems. Biochem Eng J 2003, 13:157-167. This paper attempts to explain why enzyme production in solid-state fermentation is higher than in SmF. The authors showed that solid-state culture seems to be working as a natural fed-batch culture with fast oxygenation but slow sugar supply.

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