Biochemical Engineering Journal 13 (2003) 197203

Bioreactor design for protein enrichment of agricultural residues by solid state fermentation
Tim Robinson, Poonam Nigam
School of Biomedical Sciences, University of Ulster, Coleraine BT52 1SA, Northern Ireland UK Received 14 November 2001; accepted after revision 24 July 2002

Abstract This paper reviews bioreactor designs and their use for protein production under solid state fermentation (SSF) conditions using various agricultural by-products. The advantages and disadvantages of various bioreactors and their potential for scale-up are described. SSF is proposed as a suitable low-tech strategy for protein enrichment for animal feed by converting a previously low value substance into a more nutritionally valuable one. The use of various substrates and microorganisms for protein enrichment are also listed. 2002 Elsevier Science B.V. All rights reserved.
Keywords: Agricultural residues; Bioconversion; Bioreactors; Protein; Solid state fermentation

1. Introduction Solid state fermentation (SSF) may be characterised by a fermentation process carried out on a solid medium with a low moisture content (Aw ), typically 0.400.90, which occurs in a non-septic and natural state [1,2]. SSF has been successfully exploited for food production [3,4], fuel [5,6], enzymes [7], animal feeds [8,9] and also for dye degradation [1012]. Many of the solids used for SSF are unrened and are of agricultural origin [13,14] making complete characterisation and exact reproducibility difcult [15]. In recent years, SSF has received more and more interest from researchers, as studies have demonstrated superior product yields and simplied downstream processing [16,17]. The use of solid matter, either as an inert support or substrate/support has, however, had serious implications on the engineering aspect of bioreactor design and operation. The low moisture content means that fermentation can only be carried out by a limited number of microorganisms, mainly yeasts and fungi, although some bacteria have been used [18,19]. This means that although SSF is a non-septic fermentation, spoilage or contamination by unwanted bacteria is reduced by the low Aw (<0.95), inhibits most bacterial growth [2022].

Corresponding author. Tel.: +44-28-7032-4053; fax: +44-28-7032-4906. E-mail address: p.nigam@ulst.ac.uk (P. Nigam).

Therefore, as a result of the low Aw in SSF bioreactors, smaller fermenters are required and a more concentrated product is produced, simultaneously reducing energy requirements for downstream processing [23]. Sterilisation costs are lower due to the limitation of free water, which in turn reduces operating costs needed for efuent treatment [2,15]. A large amount of metabolic heat is generated during SSF and its rate is directly proportional to the level of metabolic activity in the system [24,25]. Heat transfer in SSF reactors is not as efcient in comparison to submerged (liquid) fermentations (SmF). This is mainly due to the solidness of the substrate and the lack of free water available during fermentation [15,26]. In laboratory scale reactors, heat may be removed by keeping the culture vessel in a temperature-controlled environment, such as a water bath [27]. Problems in SSF occur when scale-up is considered; the problem of the lack of free water and generation of metabolic heat is greatly exaggerated as the system struggles to provide adequate agitation, aeration and cooling. Temperatures have been reported to be as high as 4750 C on the centre of the fermenting mass by Rathbun and Shuler [28] and temperatures as high as 6070 C by Hayes [29] in the innermost region. The use of SSF for protein enrichment of lignocellulose residues has received close attention due its low level technology, reduced reactor volume per unit weight of substrate converted and its direct applicability of the fermented product for feeding purposes [30]. Large quantities of brous crop residues are currently under utilised as potential

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animal feed sources, especially in developing countries. A major reason for this is the low protein content of the waste residues, which can be enriched utilising added urea as nitrogen source through fermentation by white-rot fungi, for example, as demonstrated by Zadrazil [31]. Three general reactor groups exist for SSF, and each in their own design, try to make conditions more favourable for fermentation. This paper will examine from the simple to the more complex in design for protein enrichment in order to discover how and why changes have been made in an attempt to control the major limiting factors in SSF.

2. Bioreactors for SSF Three basic groups of reactor exist for SSF, and these may be distinguished by type of mixing and aeration used. In laboratory scale, SSF occurs mainly in asks and the following reactors used for larger scale product formation. 2.1. Tray bioreactors Tray bioreactors tend to be very simple in design, with no forced aeration or mixing for the solid substrate (Fig. 1). Such reactors are restrictive in the amount of substrate that can be fermented, as only thin layers can be used, in order to avoid overheating and to maintain aerobic conditions [32]. The undersides of the trays are perforated to allow

aeration of the solid substrate, with each tray arranged above each other. Temperature and relative humidity are the only controllable external parameters [14]. Wooden trays were initially used for soy sauce production in Koji fermentations by Aspergillus oryzae [33]. The use of tray reactors has remained largely unchanged, with the only engineering advance being the use of more modern materials such as, plastic and aluminium. This has been successfully used for lignocellulose fermentation [33]. The advantage of plastic or aluminium trays over wooden trays would make sterilization and cleaning easier and therefore reduce the possibility of contamination or spoilage. The use of tray fermenters in large-scale production is limited as they require a large operational area and tend to be labour intensive with mechanical handling also being difcult. It can be seen that the lack of adaptability of this type of fermenter makes it an unattractive design for any large-scale production [33]. 2.2. Drum bioreactors Drum bioreactors are designed to allow adequate aeration and mixing of the solid, whilst limiting the damage to the inoculum or product (sheer forces or heat build-up). Mixing and aeration of the medium has been explored in two ways: by rotating the entire vessel [34,35] or by various agitation devices, such as paddles and bafes [36,37]. Rotation or the use of agitation can be carried out on a continuous or periodic basis (Fig. 2), promoting surface

Fig. 1. A Koji-type tray bioreactor.

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Fig. 2. A rotating drum bioreactor.

mass heat transfer and a more uniform distribution of nutrients [27]. Growth of the inoculum in drum bioreactors is considered to be better and more uniform than that in tray fermenters [33]. Fungal cultures are prone to damage in both rotating drum bioreactors (RDBs) and mixed reactors, as the increased sheer forces through mixing, affecting the ultimate product yield being compromised [33]. Air may be supplied into the vessel via an inlet, with excess gas escaping through an outlet valve. Temperature control in drum bioreactors is quite difcult with mixing along with the growth of the microorganism producing high temperatures [36]. Any rise in temperature above the optimal range may have a detrimental effect on product formation. Although the mass heat transfer, aeration and mixing of the substrate is increased, in drum reactors, damage to inoculum and heat build-up through sheer forces may affect the nal product yield. Application of drum reactors for large-scale fermentations also poses handling difculties [33]. 2.3. Packed bed bioreactors Packed bed reactors, usually in the form of a column, have emerged over the past 20 years as a potential alternative to the previously mentioned reactors (Fig. 3). Columns are usually constructed from glass or plastic with the solid substrate supported on a perforated base through which forced aeration is applied [15]. Packed bed column bioreactors have been reported to be useful for product developments with efcient process controls, particularly for heat removal. They have been used for the production of enzymes, organic acids and secondary metabolites [23,33].

Forced aeration is generally applied at the bottom of the column, with the humidity of the air kept high to avoid desiccation of the substrate. Columns, at lab-scale, are typically 30 cm in height and may be places in water baths in order to maintain a constant fermentation temperature [38,39]. In larger-scale fermentations the column temperature can be controlled by the use of a water jacket [14,15,33]. The use of thin columns have been reported as helping to facilitate heat transfer in the solid mass due to the increase in surface area and therefore avoiding the need for any cooling devices [15]. Disadvantages associated with packed bed column bioreactors for SSF include difculties in obtaining the product, non-uniform growth, poor heat removal and scale-up problems [15,40].

Fig. 3. A packed column bioreactor.

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3. Current SSF strategies Many of the current research papers concerning SSF have tended to focus on reporting lab-scale ndings. This is understandable, as parameters tend to be more controllable, with data collection simplied and running costs low. There are therefore few papers describing current state of the art reactors, giving design and yield details, as this is industrially sensitive material. Fermentation strategies have scaled-up and adopted to suit various situations and needs. Singh and Gupta [41], describe the SSF of cereal straw under a polythene cover, known as the Karnal process. This method is carried out in two stages, and uses thinly layered substrate as in a tray fermenter, but without the use of a perforated tray support. In the rst stage, the cereal straw is treated with 4% urea at a 40% moisture level and is ensiled under the polythene cover for 30 days. The second stage involves the use of a rectangular brick structure (200 cm 150 cm) which acts as the bioreactor. The loosely stacked bricks help provide aeration from all sides, with a thin layer of urea-treated straw spread thinly inside the brick enclosure as a nitrogen source for the fungi. Coprinus metarius was added and its subsequent growth enriched the protein content of the straw. Although external parameters could not be nely controlled, the brick reactor and the covering of polythene helped to maintain an adequate balance between heat generation and loss, and also allow a reasonable level of aeration. Tripathi and Yadav [42], also employed a similar strategy to ferment wheat straw using polypropylene bags inoculated with Pleurotus ostreatus. Their method employed the use of perforated bags to allow aeration and cooling whilst ultimately enriching the wheat straw for animal feed. 3.1. Parameter controls in drum reactors 3.1.1. Mixed reactors A major problem with scale-up is the removal of heat generated by metabolic activity of the microorganism [43]. Heat removal is limited in a solid mass, with a lack of heat exchange surface on a large-scale. Current strategies have concentrated on improving cooling through evaporation of moisture from the substrate, subsequently reducing the reactor temperature. This has however has its disadvantages with great loss of water [44] and so research has focused on maintaining a balance between temperature and moisture. Evaporative cooling may be combined with spraying of water onto the substrate to prevent drying [45]. This evaporation and moisture replenishment needs to occur in uniform over the substrate and mixed bioreactors are required. Nagel et al. [36], attempted to ensure uniform mixing of the solid mass allowing adequate and uniform mixing of the substrate whilst simultaneously maintaining uniform evaporation and moisture replenishment. This was carried out in a 35 l bioreactor with a paddle mixer. Six V-shaped paddles were mounted along a central axis, equidistant and

at 90 to each other. This achieved both radial and axial mixing. Axial mixing was important for homogenous water distribution in the bed as individual spraying nozzles cannot spray evenly over the total surface of the bed. Adequate radial mixing was achieved after six revolutions. Various studies have also demonstrated that the monitoring of moisture content of the solid substrate is a vital and necessary aspect of SSF [36,38,46]. Moisture in the SSF system is required for cooling, as mentioned previously, and also for the incorporation of water into new microbial cells [37]. This theory is supported by Oriol et al. [38], who found that fungal growth could be hampered by limited water availability. Nagel et al. [37] estimated that the amount of water needed for growth of new cells and evaporative cooling was a moisture content of 45%. A model was developed which allowed the overall water content as well as the extracellular water content to be incorporated in an on-line control system. Experiments in a 1.5 and 35 l mixed reactor were carried out to validate the model that was able to predict experimental moisture control levels to high degree. 3.1.2. Rotating drum bioreactors Heat removal and mixing in RDBs are carried out through the rotation of the vessel. The speed of rotation, however, causes sheer forces to act on the microorganism affecting growth and product formation. The effect of rotational speed on SSF performance has received much attention with some studies reporting reduced SSF productivity with a high rpm [34,47,48]. Other studies by Lindenfelser and Ciegler [49], indicated that high rotational speeds actually favoured SSF, possibly due to better aeration and substrate mixing. RDBs are designed to aid aeration and heat removal through the rotational movement of the vessel. This improved mixing and aeration leads to an increase in microbial growth and a subsequent increase in metabolic heat. Stuart et al. [35] showed that in a 18.7 l reactor, at 50 rpm, 40% less protein was produced in comparison to static experiments. Poor fungal growth due to sheer forces was a possible explanation for the decline in protein production. Fermentation on wheat bran at 5 rpm improved heat transfer by making the substrate more homogenous in temperature, increasing the surface area and also increasing evaporative cooling. For any process using RDBs rotational speed, the loading of solids and aeration characteristics have to be taken into account.

4. Substrates for protein enrichment by SSF As previously mentioned crop residues represent a potential source of dietary energy to ruminants if the protein content of can be enriched. As these residues are renewable and in an abundant supply (3.5 billion tonnes of agricultural by-products per year) they represent a potential

T. Robinson, P. Nigam / Biochemical Engineering Journal 13 (2003) 197203 Table 1 Various substrates and microorganisms used for protein enrichment of agricultural waste residues (some examples) Substrate Apple pomace Canola meal Carob pods Grape stalks, orange peels Sugarcane baggase Microorganism C. utilis, Kloeckera apiculata, Saccharomyces cerevisae, C. utilis, C. tropicalis, Trichoderma viriae, A. niger Aspergillus carbonarius A. niger Agrocybe aegirata, Amillarialla mellea, P. ostreatus Chaetomium celluloticum

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Reference [50,51] [52] [53] [54] [55]

solution to feeding animals in developing countries [33]. Table 1 shows a selection of substrates and microorganisms used for the protein enrichment of agricultural waste residues. 4.1. SSF of starchy material Cassava is an important food for millions of people in Africa, Asia and South America. It is, however, low in protein, vitamins and minerals, as well as sulphur containing amino acids [19,33]. Sterz et al. [56] found that Rhizopus formusa was a suitable fungus for improving the nutritional quality of raw cassava our. Other studies by Soccol et al. [57] looked at the biotransformation of cassava [57,58] and cassava wastes [42,5961] for nutritional improvement, using various strains of Rhizopus sp. Raimbault and Germon [62] increased protein enrichment of cassava to the extent of 20%. This was achieved in a 30 h fermentation period with Aspergillus niger. Other microorganisms used for cassava enrichment include R. olegosporus and A. oryzae [63]. Research has also been carried out in a farm setting [64] and on a pilot plant scale [65]. 4.2. SSF of lignocellulosic material Lignocellulosic crop residues may be characterised by being high in cellulose, hemicellulose and lignin, but low in protein. They tend to be difcult to digest by ruminants and so are limited as potential animal feed [19,33]. The SSF of these residues has been explored using fungi for protein enrichment [30,31,6668]. The use of temperate lignocellulosic wastes, such as wheat straw, has been examined by many researchers [41,42,69]. The use of rice and maize straw in developing countries may also be exploited [33,41]. 4.3. SSF of citrus wastes Citrus wastes are wastes remaining after juicing on an industrial scale. These dried peels contain simple sugars, pectin and cellulose but are low in protein [30,70,71]. A. niger has been reported to utilise the dried citrus peels under SSF conditions, by fermenting the simple sugars present [30].

4.4. SSF of apple pomace After pulping, apple pomace, which consists of crushed skins, pips and stalks may be fermented for protein enrichment. It has a high sugar content, as well as a high moisture content (80%) which poses disposal problems for the pulping industry. Unfermented apple pomace had been previously fed directly to pigs, but was mostly dumped in landll sites [30]. The use of co-cultures of yeasts and fungi to enrich the protein content of the pomace has been explored by Bhalla and Joshi [72]. Protein and pectin increased by 20 and 17%, respectively, when apple pomace was fermented by Candida utilis and A. niger under SSF conditions. 4.5. SSF of carob pobs Carob or locust tree (Ceratunia siliqua) is found extensively in Mediterranean countries, with the pods containing large amounts of sugars, making them an attractive substrate for protein enrichment by SSF. Carob pods also contain high amounts of tannin, which has an adverse effect on animal growth [30]. Tannins may be degraded by certain fungi, including A. niger [72] whilst also being simultaneously enriching the protein content of the carob pods [73]. Protein enrichment of 20% was achieved in 4 days of SSF with a 83% tannin decrease by Smail et al. [53].

5. Conclusions The potential advantages of SSF for protein enrichment can be seen if scale-up parameters, such as cooling and heat transfer can be more easily controlled. The low Aw , although beneting the inoculum in terms of competition, has a detrimental effect on the basis of heat transfer. Metabolic heat generated by rapidly growing microorganisms due to better mixing and aeration of reactors, also poses problems for the SSF system affecting product formation. Additional heat through the friction of mixing supplements to the problems of heat exchange. SSF can produce a more concentrated protein product that may be used as an animal feed in both developing and developed countries. As SSF bioreactors have increased in size in order to try and increase the product concentration so too have the problems

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