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Appl Microbiol Biotechnol (2005) 68: 598–606 Complex media from processing of agricultural cropsfor microbial fermentation Received: 2 March 2005 / Revised: 3 June 2005 / Accepted: 3 June 2005 / Published online: 5 August 2005 Abstract This mini-review describes the concept of the more than the cost of transporting the material from their green biorefinery and lists a number of suitable agricultural point of origin to a processing plant. Increasing costs for by-products, which can be used for production of bioen- waste disposal and restrictions on land filling with certain ergy and/or biochemicals. A process, in which one possible kind of wastes allow some wastes to be acquired at neg- agricultural by-product from the green crop drying in- ative cost. Renewable resources can be converted into either dustry, brown juice, is converted to a basic, universal fer- mentation medium by lactic acid fermentation, is outlined.
The resulting all-round fermentation medium can be usedfor the production of many useful fermentation products when added a carbohydrate source, which could possiblybe another agricultural by-product. Two examples of such A wide variety of organic chemicals like amino acids, products—polylactic acid and L-lysine—are given. A cost enzymes and antibiotics can be produced by fermentation.
calculation shows that this fermentation medium can be Most microorganisms used in commercial fermentations produced at a very low cost ≈1.7 Euro cent/kg, when tak- require six carbon sugars or disaccharides as substrates, ing into account that the green crop industry has expenses although the microbial world contains organisms that can amounting to 270,000 Euro/year for disposal of the brown breakdown virtually any organic compound. This means juice. A newly built lysine factory in Esbjerg, Denmark, that in fermentation processes, complex raw materials can can benefit from this process by buying a low price medium be used, as opposed to chemical synthesis where fine chem- for the fermentation process instead of more expensive icals are required. Biotechnology offers several other ad- traditional fermentation liquids such as corn steep liquor.
vantages compared to chemical synthesis, e.g. high productspecificity, low production temperature and low energy con-sumption. As a result, fermentation is becoming increas- ingly important in the production of commodity chemicals.
In the green biorefinery, jointly described by the Uni- Renewable resources also known as biomass are organic versity of Southern Denmark and AgroFerm, different materials of biological origin and are, by definition, sus- renewable resources can be converted by means of me- tainable natural resources. Sustainable implies that the re- chanical and biotechnological methods (e.g. fermentation) source renews itself at such rate that it will be available for into useful materials such as food and feed products and use by future generations. Generally, renewable resources additives, as well as fermentation medium to be used in the are classified as either wastes or dedicated energy crops.
production of organic chemical compounds, materials and When a waste from one process can be utilized as feedstock bioenergy. Common for the production of green pellets, in another process, a more appropriate name is co-product/ potato starch, cornstarch and other agricultural products by-product. The major virtue of waste materials is their are that during processing of the crops, a liquid stream low cost. By definition, waste materials have little appar- containing part of the crop nutrients is produced.
ent economic value and often can be acquired for little These liquids (plant juices) are in most countries used as a fertiliser for crop plants, because they have a valuable content of nutrients such as potassium and nitrogen. How- Biosystems Department, Risoe National Laboratory, ever, due to environmental problems involved in applying excess nitrogen, which, if not taken up by plants in the autumn and winter period, will eventually end up in the ground water as nitrate, more and more restrictions have been introduced regarding the use of these residues as source in the green biorefinery (Bjerre et al. Schmidt The plant juices are regarded as “difficult” waste streams, Starch is another agricultural carbohydrate source. Wheat but in the concept of the green biorefinery, it is consid- has certain advantages over other carbohydrate sources.
ered a co-product from which useful products can be pro- Cereals, being low in moisture, are more energy inten- duced. The brown juice, green juice and corn steep liquor sive and have the advantage that they can be stored and are plant juices rich in protein and elements, and they are transported easily. The sugar is in the form of starch, and suitable as replacement for yeast extract in fermentation in addition, cereal grains contain nutrients that can be separated easily from the grain and sold as lucrative by- However, the plant juices often are low in carbohydrate, products: bran, gluten and A-starch. Gluten is the most and in order to obtain high yields in the green biorefinery, profitable of these by-products—a protein used in the bak- whether the production is bioenergy or biochemicals, a ing industry. Starch can be saccharified to fermentable sugars by either acid hydrolysis or by enzymatic hydrolysis.
Lignocellulosic material such as agricultural waste, e.g.
wheat straw, represents an abundant renewable raw mate-rial source. In the lignocellulosic biomass, hemicellulose and cellulose are infiltrated with the stiffening bondingmaterial lignin. The two polysaccharides are not directly In this mini-review, a number of agricultural by-products available for bioconversion; a pretreatment is needed to suitable for use in the green biorefinery and other fer- overcome the physical barrier of lignin and make sugars mentation industry are described (Table ).
available for the microorganisms. A wet oxidation processhas been developed, using water, oxygen pressure andelevated temperature, for fractionation of wheat straw at alkaline conditions. By this treatment, most of the hemi-cellulose is solubilised mainly as oligomers and poly- The green crop drying industry produces fodder pellets by mers, and the cellulose is recovered in the solid fraction.
drying crops such as perennial rye grass (Lolium perenne), The fibre fraction as well as the solubilised hemicellulose Italian rye grass (Lolium multiflorum), clover grass and can be hydrolysed by enzymes and used as a carbohydrate alfalfa (Koegel and Bruhn ). In order to reduce the Table 1 Agricultural by-products, their main components and potential industrial use Lactic acid/L-lysine Andersen and Kiel Thomsen Lactic acid/L-lysine Andersen and Kiel Andersen and energy consumption in the drying process, part of the in order to remove water and to form sugar crystals. The water content in the grass is normally removed from the thick liquid with crystals is centrifuged, and sugar crystals green crops before drying in a drum dryer, resulting in and molasses (a thick brown liquid) are separated. The significant productions of green plant juice (green juice).
molasses is used as animal feed and in fermentation in- At some green pellets factories, the crops are steam-heat- dustry, and it generally consists of 50% of sucrose and ed in a cooker to a temperature of about 80°C before some amount of protein (6–9%) and elements (Wee et al.
pressing. This process results in coagulation of a large proportion of the protein, as well as the damage to theplant cells and the residue from these factories are knownas brown juice. Approximately 200,000 m3 of brown juice is produced in Denmark each year, and of this, approx-imately half is evaporated and used in the production of Wheat straw is an abundant by-product from farming, and pellets. Both green and brown juice has a dry matter (DM) large amounts are produced in many countries. In a small content of approximately 6%, and the green juice contains country as Denmark alone, approximately 6 million tons 13% carbohydrate (in DM) and 35% protein (in DM), and are produced. Of these 6 million tons, 35% is excess straw the brown juice contains 33% carbohydrate (in DM) and not used for burning, animal feed or bedding. Wheat straw 26% protein (in DM) (Andersen and Kiel ).
has a content of approximately 40% cellulose and 30%hemicellulose (Bjerre et al. ; Thygesen et al. ).
Corn steep liquor is a by-product from the corn wet millingindustry. The corn wet milling industry separates the corn Corn stover and cob are by-products from cultivation of into several fractions: starch, germ, fibres, and gluten.
corn. The stover is the stem and leaves, and the cob is the Steeping of the corn before the wet milling process is part of the plant, where the corns are attached. These essential for high yields and high starch quality. The materials are very abundant in large regions of the world cleaned corn is filled into a battery of large steeping tanks, and the production is increasing, e.g. in Spain, the pro- where the corn is soaked in hot water for up to 50 h. The duction shifted from 1,848 tons in 1970 to 3,898 tons in steeping is actually a controlled fermentation (with pri- 2000. Corn stover contains approximately 50% cellulose marily Lactobacillus), and the addition of 1,000–2,000 ppm and 28% hemicellulose (Varga et al. and the cob of sulphur dioxide helps to control that fermentation. Dur- contains 34% cellulose in DM and 39% hemicellulose in ing steeping, solubles are extracted and the kernel softens.
The steep water is drained from the kernels and evaporat-ed to approximately 50% DM. Corn steep liquor containsapproximately 47% protein and is a valuable nutrient source in the fermentation industry (Stock et al. ).
Among the easily available agricultural by-products, ricestraw is the most abundant, with 82 million metric tons annually available in India, and the estimated amount ofrice straw in Asia in 1993 was 483 million tons. Rice straw The potato processing industry uses large volumes of wa- contains approximately 40% cellulose and 18% hemicel- ter during operations such as washing, peeling and blanch- lulose (Kaur et al. Liu et al. The rice hull is ing during production of potato chips, slices and shredded another waste from rice production. Rice hull is also a potatoes. The effluent generated in these processes are lignocellulosic material and contains 26% cellulose and characterised by high content of carbohydrates (starch) 13.5% hemicellulose (Schultz et al. ).
(19.5 g/l) and also some protein (2.9 g/l) (Mishra et al.
These starch-rich wastes can cause environmentalproblems if discharged in nature.
Pruning/trimming of vine stocks produces large amounts of lignocellulosic residues. These wastes are usually burnedin the field, causing environmental problems as well as Molasses is a by-product of sugar manufacturing. The risk to human health owing to the formation of toxic com- sugar is extracted from the beets (in the case of beet sug- pounds from lignin combustion. Wine-trimming wastes ar production) with hot water (70°C), and the pulp and contain approximately 34% cellulose and 19% hemicellu- liquid are separated. This liquid is purified with calcium carbonate, and the purified liquid is cooked in two steps is starch and approximately 24% is cellulose (Krishna andChandrasekaran ; Krishna Shah et al. ).
Sugar cane bagasse is the fibrous residue obtained afterextraction of sugar from sugar cane. Sugar cane bagassecontains significant amounts of cellulose (40% of DM) and hemicellulose (21%) (Gong et al. ; Schultz et al. Residue from peanut (groundnut) is abundant in Nigeria,where the annual production of peanut in 1996 was 7,608 tons. The peanut shell has a carbohydrate content ofapproximately 17%, a protein content of 7% and a fibre Sweet sorghum is a crop in the grass family that is con- sidered to be one of the most drought-resistant agriculturalcrops. Sweet sorghum is mostly used in the production ofsyrups, brown sugar and forage. The residual plant matter from syrup and brown sugar production the sorghum ba-gasse contains significant amounts of cellulose (37% of From a theoretical point of view, brown juice and other DM) and hemicellulose (26% of DM) (Gnansounou et al.
plant juices are well suited as fermentation media, as they contain all the necessary growth factors such as aminoacids, vitamins and minerals (Thomsen et al. submitted forpublication). But in practice. it is difficult to handle due to the transportation and storage difficulties of these sub-strates. If the substrates have to be stored or transported Olive cake is a by-product generated by olive oil extrac- before use in an industrial process, it is necessary to pre- tion. Olive oil cake can be used as fuel, but is mostly treat them in some way. Traditional heat sterilisation at discarded as waste in the environment. Olive cake contains 121°C for about 30 min reduces the quality of the juice as a approximately 10% cellulose in DM, 16% hemicellulose fermentation medium due to formation of inhibitors from, in DM and 5% protein in DM (Cordova et al. e.g. Maillard reactions (Andersen and Kiel How-ever, a process has been developed in which plant juicescan be converted to an all-round, storable and stable fer- mentation medium by means of lactic acid fermentation.
The fresh non-sterilised juice is inoculated with a suit- In Italy alone, 1,376,600 tons of citrus fruits were used in able lactic acid bacteria, Lactobacillus salivarius subsp.
the production of juices, essential oils and other products salivarius DSM 20555, which has been shown to be the during 2001–2002. As a result of the processing of citrus best strain (Thomsen et al. thereby converting the fruits, a large amount of residue is produced consisting of crop sugars into lactic acid. It has been shown that in fresh washing waters, peel, membranes, seeds and pulp. The brown juice, all sugars, even fructans, are converted to pulp is rich in sugar and fibres, and contains some protein lactic acid (by the help of plant and microbial enzymes) (Tripodo et al. ). The peel (orange bagasse) contains lowering pH and giving a good preservation of the juice fibres (11.8% of DM), reducing sugars (9% of DM) and (Andersen and Kiel In order to effectively preserve the juice, fermentation is continued until the pH drops tobelow 4.5, and preferably to below 4.0 (Andersen and Kiel). The resulting lactic acid fermented juice is a stable product that can be easily stored under anaerobic condi-tions (Andersen and Kiel for further use as a fer- Banana is one of the most consumed fruits in the world.
mentation medium either as it is or in concentrated form.
Each hectare of banana crop generates nearly 220 tons of This substrate can be used as fermentation broth (by ad- plant residue that consists mainly of lignocellulose mate- dition of a carbon source) in a number of different fer- rial. In, e.g. India, where 4.796·105 ha of banana is cul- mentation processes producing valuable products such as tivated, farmers discard banana waste into rivers, lake and organic acids, amino acids, feed additives, enzymes, pro- on roads, causing serious environmental problems. The teins, peptides or a fungus or bacteria (Andersen and Kiel main residuals of banana crops are leaves and pseudostem, ). In the following, a couple of examples of products both containing high levels of lignocellulose. Banana fruit based on acidified brown juice medium will be given, and stalks contain approximately 57% total sugar of which 27% finally, the price of this acidified brown juice medium willbe evaluated.
and purification, which could amount to almost 50% of thefinal product cost (Evangelista et al. For the prep- aration of lactic acid polymers, it is advantageous to useaqueous solutions of lactic acid at about 90% by weight Polylactic acid (PLA) is a polymer made by polymerisation (Mantovani et al. Several methods for purification of lactic acid. PLA shows great potential as material for of lactic acid from fermentation broth have been described, food packaging due to its mechanical properties. Due to the such as gypsum precipitation (Datta ), extraction moisture and gas barrier and the ability to produce flexible using a trialkyl amine in an organic solvent as employed water-resistant films, PLA is suitable for packaging of by Cargill (Bizzari et al. ), ion-exchange (Mantovani respiring fruit and vegetables and for liquid food applica- tions, e.g. juice. PLA can be used as a pure product or it can be used in combination with other polymers (Shogren It may contain natural extracts/components, e.g.
Kascak et al. and membrane processes such as lignin and waxes, acting as preservatives or antioxidants electrodialysis (Czytko et al. Hongo et al. ; preventing oxidation-sensitive products from deteriorating Madzingaidzo et al. A process has been developed (Petersen et al. Other uses for PLA are surgical whereby lactic acid is neutralised with piperazine; an amine sutures, implantable medical devices, pharmaceutical con- that combined with two molecules of lactic acid makes trolled drug delivery systems, fibres and yarns for manu- piperazinium dilactate. The piperazinium dilactate can be facturing of clothing, etc. In order for PLA to be able to converted into dilactid (a building block in production of compete with conventional packaging materials, it has to polylactate) without the production of undesired by-prod- be produced from cheap raw materials and feasible pro- ucts. Ultrafiltration, nanofiltration and electrodialysis are cesses. The typical fermentative route to lactic acid starts used for purification of the lactide from the fermentation with some sort of substrate pretreatment, either just addi- broth. In this process, no problems with fouling of the ul- tion of essential growth components, e.g. suitable mineral trafiltration membrane was experienced, but still, some and proteinaceous nutrients, or in addition, mechanical optimising is needed on the nanofiltration and electrodi- treatment combined with chemical/enzymatic hydrolysis.
alysis processes (Kamm et al. ). At the Technical Nutritionally lactic acid bacteria are extremely fastidious University of Denmark, a process has been developed, in due to their limited ability to synthesize B vitamins and which lactic acid is continuously removed and purified amino acids (Chopin a medium that will support from the fermentation broth using various membrane pro- their growth must contain a fermentable carbohydrate and cesses (Donnan dialysis, electrodialysis with bipolar mem- many growth factors such as amino acids, peptides, nuclei branes and electrodialysis). In this process, problems with acid derivatives and vitamins. Brown juice is a cheap me- fouling of the membranes when using a complex medium dium, and it contains all nutrients necessary for lactic acid such as brown juice was minimised or avoided (Garde et al.
fermentation (Andersen and Kiel Thomsen et al.
If the acidified brown juice medium is to be used for actuallactic acid fermentation, the fresh brown juice is typical- L-Lysine is an essential amino acid in animal nutrition.
ly supplemented with additional carbohydrate, so that the Many plant products used for livestock feed, e.g. wheat initial acidification step for preservation of the juice is and corn, are deficient in L-lysine, and thus, it must be followed by additional lactic acid fermentation. That is, in added as a supplement. The bulk of L-lysine production order to profitably utilize brown juice in the production of throughout the world depends on direct fermentation of PLA, a carbohydrate source must be added with the pur- carbohydrates by auxotrophic and regulatory mutants of pose of increasing the lactic acid yield (Andersen and Kiel Corynebacteria and Brevibacteria spp. in batch culture The carbohydrate source could be another waste product from the agricultural industry such as molasses L-Lysine is added to feed in amounts of 0.2 to 1% and from beet sugar production, a lignocellulosic by-product as reduces the livestock’s consumption of nitrogen-rich feed the ones described in Table , or wheat starch.
such as soybean, fish protein, and flesh and bone meal.
Furthermore, L-lysine improves nitrogen’s biological avail-ability in the animal, thereby diminishing the agricultural nitrogen emission to the environment (Ruklisha et al.
).
The commercial success of PLA hinges on the purity (heat The world marked for L-lysine is about 600,000 tons stable grade) and the cost of the fermentation-produced a year. In Europe, approximately 95,000 tons a year is lactic acid. Therefore, one of the major challenges in lactic consumed, and the market is up going. The L-lysine prod- acid production is to reduce the cost of the acid recovery ucts produced to day are based on industrial fermentation of sugar and hydrolysed starch and are marketed in the tation as described above is a perfect substrate for amino form of a lysine–HCl purified by ion exchange. A few big acid producing microorganisms, which are able to utilize manufacturers dominate the world market, and about 35 to the available organic acids and amino acids as building 40% of the L-lysine consumed in Europe today is imported blocks and as energy source for production of both cell mass and the desired amino acids such as L-lysine. Ithas been shown that the use of the acidified brown juicemakes possible a very high microorganism growth rate as A Danish L-lysine production—from brown juice well as a very high yield and productivity.
In particular, it has been found that the biomass yield is The Danish marked for L-lysine is approximately 14,000 increased in the lactic acid fermented juice, as the lactic tons a year. Up till now, there have been no Danish pro- acid bacteria are able to utilize a higher proportion of the ducers of L-lysine. The company AgroFerm now initiates organic acids and sugar present in the juice than is the case a Danish production of L-lysine; the new L-lysine factory for the Corynebacteria alone. Also, the lactic acid bacteria is planned to produce approximately 12,500 tons of L- hydrolyse some of the proteins in the juice making them lysine a year based on the acidified brown juice medium.
available for the Corynebacteria in the subsequent amino In the lysine factory, the acidified brown juice is sterilised acid fermentation. The net result is that the lactic acid in a continuous steriliser and led to a sterile, aerated stirred fermentation converts compounds that are otherwise non- tank reactor (STR) reactor (sterilisation of the brown juice fermentable for the Corynebacterium to compounds, which is now possible because the sugars are converted to lactic the Corynebacteria are able to ferment. The overall yield acid, so no damage by Maillard reactions between carbo- is therefore greatly increased. The acidified brown juice hydrates and amino acids can take place). The fermentors medium is a complete medium with a high content of free are inoculated with a culture of Corynebacterium glutami- amino acids, which is highly useful for microorganisms cum and carried out as fed batch fermentation. A sterile able to utilize lactic acid as a carbon source such as carbohydrate solution is added continuously to the fermen- Corynebacteria, which are able to utilize the produced tation tank, after the sugar in the initial medium has been lactic acid for both cell mass and amino acid production utilized. pH of the medium is controlled by addition of am- monia. The fermentation continues until a certain L-lysineconcentration is reached. pH in the media is dropped to 4.0by adding sulphuric acid, and the final liquid product with 25% L-lysine is achieved after vacuum evaporation of thewhole media containing all remaining nutrients and bio- Based on chemical analyses of the juice, laboratory as well mass. The result is production of a new valuable product as large pilot scale fermentation experiments, and simula- without formation of new waste streams.
tion in a design and simulation program called Super Pro Andersen and Kiel (has shown that the acidified Designer, a plant for production of 10,000 tons of acidified, brown juice resulting from the initial lactic acid fermen- concentrated (25% DM) brown juice a year has been Fig. 1 The lactic acid fermentation of brown juice in the green crop drying plant as it was simulated in Super Pro Designer (Thomsen ) designed (Thomsen et al. The price of the acidified ing to approximately 20 DDK/m3 fresh brown juice for brown juice medium was determined from investment disposal. About 100,000 tons will be spread on the fields costs, interests and the total operating cost, which was every year, giving an annual expense of 2,000,000 DDK determined by simulation of the process in Super Pro a year. The remaining part of the brown juice will be re- cycled and evaporated in the production of green pellets.
The price of power and utilities was determined by simulation in Super Pro Designer and maintenance set as 4% of the fixed capital investment, operating supplies as15% of maintenance and insurance as 1% of the fixed The acidification process should be simple, robust, cost capital investment. Transportation costs of the brown juice efficient, have low energy consumption and be run in the of course depend on how far away from the green crop green crop factory by personnel not educated in microbi- drying plant the fermentation factory is situated, but in the ology. The hot (60–70°C) brown juice from the green crop case of the L-lysine factory AgroFerm in Esbjerg, Den- drying plant is at the first step cooled to fermentation mark, transportation of the brown juice amounts to a cost temperature. Only the best quality of the juice is used in of 50 DDK/m3 brown juice, that is, 500,000 DDK or the acidification process, that is, juice with a pH higher approximately 70,000 Euro a year (the distance is approx- that 5.5, whereas juice with a pH below 5.5 is returned to the pellet factory in a concentrated form. Fermentation Summing it up, the price the L-lysine factory will have is performed in a 100 m2 continuous stirred tank reactor to pay for the acidified brown juice medium (25% DM) (CSTR) inoculated with a strain of L. salivarius and run will be approximately 1,150,000 DDK or 156,000 Euro a at high dilution rate (0.5–1.0) (Thomsen et al. The year (10,000 m3) or 1.7 Euro cent/kg (Thomsen acidified brown juice with pH between 4.0 and 4.5 is ledto a sedimentation tank, from where the supernatant is ledto a storage/buffer tank before evaporation to 25% DM.
The sediment is led to a sediment buffer tank. The con-centrated acidified brown juice is stored until transport to The actual price of the acidified brown juice medium the lysine factory. Using this concept, it is possible to sup- might be even lower because of the protein (value) added to ply the lysine factory with stabilized high quality brown the green pellet production. It is assumed that the flow of juice all year round and produce about 50,000 tons of liq- brown juice is halved after sedimentation. In reality, it will uid lysine feed concentrate a year (Fig. ) (Thomsen et al.
be more because brown juice with a pH below 5.5 will be led directly to a sediment buffer tank, where also the sed-iment from the sedimentation tank is led. After evapora-tion, the concentrated brown juice is used in the production of fodder pellets. The estimated amount (by simulation) ofsediment led back to the production of fodder pellets is Following unit operations is used in the production of the approximately 10,000 tons DM/year. The sediment is rich acidified brown juice medium: cooling tower, fermenta- in precipitated protein and will increase the protein content tion tank, sedimentation tank, buffer tank, evaporator and a storage tank (Fig. The total capital investment of The price of the acidified brown juice medium can be this simple process has been found to be approximately compared to corn steep liquor, which is also a waste prod- 10,000,000 DDK or 1,300,000 Euro (Thomsen Price of cooling tower and evaporator was neglected be- as fermentation medium. The price of corn steep liquor cause these unit operations are already present at the green (25% DM) in Denmark is 1.5 DDK/kg or 20 Euro cent/kg.
crop drying plant (and the capacity of this equipment is From this comparison, it is evident that converting the sufficient to also be used in the acidification process).
fresh brown juice to a universal stable fermentation me- It was assumed that the capital investment comes from dium by lactic acid fermentation is a feasible process. The loan (annuity loan), and that the investment is written off lysine factory benefits from this process by buying a low in a period of 10 years at an interest rate of 5% per year.
price medium for the fermentation process instead of more The annual costs of such a loan will be approximately expensive traditionally fermentation liquids such as corn 1,255,000 DDK or 170,000 Euro. The annual operating steep liquor, and at the same time, pollution of the ground costs is determined as cost of raw materials, power and water by this nutrient-rich liquid in the autumn period is utilities, maintenance, operating supplies, insurance and transportation of the brown juice to the fermentation fac-tory. The cost of the brown juice was set to a negative value of 2,000,000 DDK or approximately 270,000 Euro, be- Ferm) are acknowledged for their extensive research leading to the cause the green crop drying industry has expenses amount- development of this process and for input to this mini-review.
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