946 Appl Microbiol Biotechnol (2012)93:941-963 appropriate concentration in the media is 100-150 mg/L. development.it is at 3.000 to 50.000 lx.At the condition Although some of the elements have existed in the raw mate- of light level about 3,000-10,000 Ix,the stip and cap (pileus) rials,the substrates are still added the Caso,KH2PO4,and shape are normal.The optimum light level is at 15,000 to MgSO4,especially the amount of usage CaSO4 is at most up 50,000 Ix.The optimum pH value for mycelial running is 5.0 to 1%of total substrate weight.It is a significant reason that to 5.5 (Zhou and Lin 1999;Hou and Liao 2009). calcium sulfate may adjust the pH value of substrate,change Artificial cultivation of Lingzhi has a history of more the substrate porosity,increase the air flow,fix the nitrogen, than 40 years.Many methods of cultivating Lingzhi derive and enhance the mounts of calcium and sulfur elements.In the from the name of cultivation raw materials (log cultivation, development process of Lingzhi,the growth factors are the sawdust cultivation,substituted cultivation,etc.),the pack- necessary components,including vitamin B,B6,and biotin, aging forms of raw materials(bag or bottle),or cultivation which are related to Lingzhi metabolism.Because of these location (outdoor cultivation,indoor cultivation.bionics growth factors existing in the natural substrates,they do not wild cultivation,etc.).For example,based on the cultivation necessarily need to be added into the substrates.In addition, raw materials,basswood cultivation technology is actually clean well water should be used in large-scale cultivation of designed to culture mushroom using basswood as raw mate- Lingzhi.It is necessary that the water is not contaminated with rials (Zhang et al.2004).The substituted cultivation tech- undesirable pollutants.The appropriate content of water nology was defined as using the sawdust of hardwood, should be 65-70%(Zhou and Lin 1999:Han et al.2003). cotton seed husk,or foot materials of farm crops to cultivate Lingzhi is found more frequently in subtropical regions mushroom(Zhang and Wang 2010).The word"cultivation than in the temperate zones.It is an annual mushroom, technology"may mean different things,in different people growing on a wide variety of dead and dying trees.The and countries.However,regarding the choice of what kind growth parameters (Lin 1996;Chang and Miles 2004)are of method or raw materials to use,the production of the summarized mainly to include four aspects:temperature, basic process is roughly the same.Similarly,as to other humidity,air,and light.Firstly,temperature for mycelia cultivated edible mushrooms,the process for producing growth ranges from 15 to 35 C and the optimum temper- Lingzhi fruiting bodies can be divided into two major ature is 25 to 30 C:for primordial initiation,it ranges from stages.The first stage is the preparation of various raw 18 to 25 C;and for fruiting body development,the range is materials,which involves the selection of mother spawn, from 24 to 28 C.Under the condition of 20 C or lower,the cultivation method,and cultivation relative to materials.The fruit body would become yellow and stops growing.In the second stage is the performance of cultivation,including the procedure of fruit body development,it is actually not planting spawn,stock culture,fruiting culture,and utiliza- necessary to be stimulated with temperature difference. tion of the growth substrates for mushroom production. The higher temperature differences easily cause macroce- Currently,the methods adopted for commercial production phalic Lingzhi.Secondly,humidity is a term for the amount shows a variety of forms,which mainly include the wood of water vapor in the air or in the substrates.During the log,short basswood segment,tree stump,sawdust bag,and process of Lingzhi cultivation,the water content in sub- bottle procedures (Mayzumi et al.1997:Lin and Zhou strates should be maintained at 60%to 65%level.Relative 1999a:Erkel 2009:Chen 1999:Han et al.2008). humidity for the mycelial running is within 60%to 70% Because some methods are not beneficial for the protec- level.the primordial initiation is within 85%to 90%level. tion of the ecological system,Lingzhi cultivation using and the fruit body development is with 85%to 95%humid- wood log is not adopted in production practice.Here,we ity level in the environmental air.Finally,in the different only restate the basic procedure related to substituted culti- development periods,Lingzhi have the different require- vation.In the research of substituted cultivation of Lingzhi, ments on the rate of oxygen and carbon dioxide.Lingzhi the research content often focuses on the following aspects: mycelial running do not need oxygen.But during the fruc- selection of substrate and its formulation.cultivation models tification period,good ventilation is necessary.When the (bag or bottle,inside or outside),sterilization and inocula- concentration of COz is below 0.1%,the fruit body would tion,cultivation and administration of mycelia,and admin- be abnormally developed.When the concentration of CO2 is istration of the stage of fruit body development.Briefly, above 0.1%,the fruit body would be normally developed. spawn is transferred onto a sterile solid-state substrate, Similar to other mushrooms,Lingzhi do not require the which frequently consists of a mixture of hardwood saw- various types and amount of light to grow.Because of the dust,wheat bran,and other supplemental substances,and is light for Lingzhi mycelial running having an inhibiting incubated under dark conditions until colonization of sub- effect,colonizing substrate should be kept under dark con- strate is achieved.After this,incubation under the weak light ditions to make sure the substrate does not pin prematurely. condition and at increased oxygen level is carried out to During primordia initiation of Lingzhi growth,the light develop the primordial initiation.Once the desired primor- level is required at 500 to 1,000 Ix;for fruit body dial initiation is achieved,the conditions are again altered to ②Springer
appropriate concentration in the media is 100–150 mg/L. Although some of the elements have existed in the raw materials, the substrates are still added the CaSO4, KH2PO4, and MgSO4, especially the amount of usage CaSO4 is at most up to 1% of total substrate weight. It is a significant reason that calcium sulfate may adjust the pH value of substrate, change the substrate porosity, increase the air flow, fix the nitrogen, and enhance the mounts of calcium and sulfur elements. In the development process of Lingzhi, the growth factors are the necessary components, including vitamin B1, B6, and biotin, which are related to Lingzhi metabolism. Because of these growth factors existing in the natural substrates, they do not necessarily need to be added into the substrates. In addition, clean well water should be used in large-scale cultivation of Lingzhi. It is necessary that the water is not contaminated with undesirable pollutants. The appropriate content of water should be 65–70% (Zhou and Lin 1999; Han et al. 2003). Lingzhi is found more frequently in subtropical regions than in the temperate zones. It is an annual mushroom, growing on a wide variety of dead and dying trees. The growth parameters (Lin 1996; Chang and Miles 2004) are summarized mainly to include four aspects: temperature, humidity, air, and light. Firstly, temperature for mycelia growth ranges from 15 to 35 °C and the optimum temperature is 25 to 30 °C; for primordial initiation, it ranges from 18 to 25 °C; and for fruiting body development, the range is from 24 to 28 °C. Under the condition of 20 °C or lower, the fruit body would become yellow and stops growing. In the procedure of fruit body development, it is actually not necessary to be stimulated with temperature difference. The higher temperature differences easily cause macrocephalic Lingzhi. Secondly, humidity is a term for the amount of water vapor in the air or in the substrates. During the process of Lingzhi cultivation, the water content in substrates should be maintained at 60% to 65% level. Relative humidity for the mycelial running is within 60% to 70% level, the primordial initiation is within 85% to 90% level, and the fruit body development is with 85% to 95% humidity level in the environmental air. Finally, in the different development periods, Lingzhi have the different requirements on the rate of oxygen and carbon dioxide. Lingzhi mycelial running do not need oxygen. But during the fructification period, good ventilation is necessary. When the concentration of CO2 is below 0.1%, the fruit body would be abnormally developed. When the concentration of CO2 is above 0.1%, the fruit body would be normally developed. Similar to other mushrooms, Lingzhi do not require the various types and amount of light to grow. Because of the light for Lingzhi mycelial running having an inhibiting effect, colonizing substrate should be kept under dark conditions to make sure the substrate does not pin prematurely. During primordia initiation of Lingzhi growth, the light level is required at 500 to 1,000 lx; for fruit body development, it is at 3,000 to 50,000 lx. At the condition of light level about 3,000–10,000 lx, the stip and cap (pileus) shape are normal. The optimum light level is at 15,000 to 50,000 lx. The optimum pH value for mycelial running is 5.0 to 5.5 (Zhou and Lin 1999; Hou and Liao 2009). Artificial cultivation of Lingzhi has a history of more than 40 years. Many methods of cultivating Lingzhi derive from the name of cultivation raw materials (log cultivation, sawdust cultivation, substituted cultivation, etc.), the packaging forms of raw materials (bag or bottle), or cultivation location (outdoor cultivation, indoor cultivation, bionics wild cultivation, etc.). For example, based on the cultivation raw materials, basswood cultivation technology is actually designed to culture mushroom using basswood as raw materials (Zhang et al. 2004). The substituted cultivation technology was defined as using the sawdust of hardwood, cotton seed husk, or foot materials of farm crops to cultivate mushroom (Zhang and Wang 2010). The word “cultivation technology” may mean different things, in different people and countries. However, regarding the choice of what kind of method or raw materials to use, the production of the basic process is roughly the same. Similarly, as to other cultivated edible mushrooms, the process for producing Lingzhi fruiting bodies can be divided into two major stages. The first stage is the preparation of various raw materials, which involves the selection of mother spawn, cultivation method, and cultivation relative to materials. The second stage is the performance of cultivation, including the planting spawn, stock culture, fruiting culture, and utilization of the growth substrates for mushroom production. Currently, the methods adopted for commercial production shows a variety of forms, which mainly include the wood log, short basswood segment, tree stump, sawdust bag, and bottle procedures (Mayzumi et al. 1997; Lin and Zhou 1999a; Erkel 2009; Chen 1999; Han et al. 2008). Because some methods are not beneficial for the protection of the ecological system, Lingzhi cultivation using wood log is not adopted in production practice. Here, we only restate the basic procedure related to substituted cultivation. In the research of substituted cultivation of Lingzhi, the research content often focuses on the following aspects: selection of substrate and its formulation, cultivation models (bag or bottle, inside or outside), sterilization and inoculation, cultivation and administration of mycelia, and administration of the stage of fruit body development. Briefly, spawn is transferred onto a sterile solid-state substrate, which frequently consists of a mixture of hardwood sawdust, wheat bran, and other supplemental substances, and is incubated under dark conditions until colonization of substrate is achieved. After this, incubation under the weak light condition and at increased oxygen level is carried out to develop the primordial initiation. Once the desired primordial initiation is achieved, the conditions are again altered to 946 Appl Microbiol Biotechnol (2012) 93:941–963
Appl Microbiol Biotechnol(2012)93:941-963 947 aid in the fruiting body development(shown in Fig.3).The Liquid-state (submerged)fermentation of Lingzhi mycelia entire growth of Lingzhi from spawn running to cropping in Liquid-state fermentation (LSF)of mushroom.also known artificial cultivation is different from the method of cultiva- as liquid culture or submerged fermentation,is a process of tion used.In general,it should take on average approximately culturing microorganism in liquid media,but not on the 90-150days. surface of liquid media.Preparation of mushroom mycelium It directly affects the Lingzhi product's quality to admin- in submerged culture was initially developed during the ister the development process of fruit body.In the overview 1970s.At that time,only lower fungi (fungi that are not of previous publications,the cultivation procedures were from basidiocarps)were successfully cultured in fermentors roughly the same;the difference was only diverse from for economical production of various natural products(Yang different cultivation methods or raw materials (Chen 1999: and Liau 1998).The main principle is that the liquid medi- Chi 2005:Erkel 2009:Hossain et al.2009:Xiao et al.2010). um is added to the fermentation tank or flask.When sterile Up to now,in the Lingzhi production practice in China,the air is led to reactor,it will increase the dissolved oxygen preparation of substrate,including substrate formulations, content in the medium to improve respiratory metabolism of making the synthetic logs in bag cultivation and substrate the mushroom mycelium.At the same time,the mushroom sterilization etc.,have appeared in the textbook of some mycelium is agitated or oscillated,and appropriate external vocational schools or popular science books (Zhang 1998; conditions are controlled so that mushroom cells grow in the Liu and Jiang 2007).Meanwhile,the cultivation processes liquid depths of breeding,producing a lot of the mycelium of Lingzhi have also been described in these books.Such or metabolites.Another review indicated that there were few questions regarding the crucial stages in Lingzhi cultivation, investigations into the development of high fungi(basidio- how to speed up the spawn running,how to control the mycetes)bioprocesses (Fang and Zhong 2002a,b). environmental factors,and how to produce Lingzhi with Compared with the artificial cultivation of Lingzhi fruit caps have been introduced in various media,including mag- bodies,LSF holds obvious advantages.Firstly,the advan- azines,popular science books,DVD video,TVetc.Growers tage of LSF over the traditional,multinational cultivation of will benefit from concentrating on the step-by-step instruc- fruit body is the reduction in the time spent to obtain the tion on how to cultivate it. product of interest (Mshandete and Mgonja 2009).The production of fruit bodies takes at least 3-5 months,while Artificial cultivation of Lingzhi mycelia Both the liquid- reasonable amounts of ganoderic acids(GAs)and polysac- state fermentation and solid-state fermentation are popular charides can be obtained by submerged fermentation after for the production of Lingzhi mycelia. 3-5 weeks only (Wagner et al.2003).Secondly,the myce- lium grown in LSF holds high stability and standardization in which the environmental conditions (temperature,dis- solved oxygen,pH,etc.)are easier to control.This is im- portant not only for producing the desired product but also might be beneficial for producing mushroom-based medi- cines and nutraceuticals.The products obtained by this method are easy to achieve,with higher quality standards and safety (Wasser and Weis 1999).Thirdly,advantage of mycelia culturing in LSF is to provide a new method for scalable production and increased yields of biologically active compounds.By LSF method,the producer can in- Development stage crease the yield of protein rich in essential amino acids and vitamins serving as functional foods compared with the yield of these components in carpophores of the standard Bud-developing stage fruiting basidiomycetes(Friel and McLoughlin 2000).Thus, Growth stage production of fungus metabolites through LSF method has received a lot of attention because of short time cultivation, high productivity,fewer chances of contamination,and easy recovery of producing metabolites (Huang and Liu 2008; Kim et al.2007). In general,the methods of artificial cultivation typically Bud-breaking stage in liquid media involve five stages:(1)selection of Lingzhi Maturity stage strain;(2)preparation of culture maintenance medium for Fig.3 The developmental process of Lingzhi fruit body different culture phases;(3)inoculation;(4)cultivation of ②Springer
aid in the fruiting body development (shown in Fig. 3). The entire growth of Lingzhi from spawn running to cropping in artificial cultivation is different from the method of cultivation used. In general, it should take on average approximately 90–150 days. It directly affects the Lingzhi product’s quality to administer the development process of fruit body. In the overview of previous publications, the cultivation procedures were roughly the same; the difference was only diverse from different cultivation methods or raw materials (Chen 1999; Chi 2005; Erkel 2009; Hossain et al. 2009; Xiao et al. 2010). Up to now, in the Lingzhi production practice in China, the preparation of substrate, including substrate formulations, making the synthetic logs in bag cultivation and substrate sterilization etc., have appeared in the textbook of some vocational schools or popular science books (Zhang 1998; Liu and Jiang 2007). Meanwhile, the cultivation processes of Lingzhi have also been described in these books. Such questions regarding the crucial stages in Lingzhi cultivation, how to speed up the spawn running, how to control the environmental factors, and how to produce Lingzhi with caps have been introduced in various media, including magazines, popular science books, DVD video, TV etc. Growers will benefit from concentrating on the step-by-step instruction on how to cultivate it. Artificial cultivation of Lingzhi mycelia Both the liquidstate fermentation and solid-state fermentation are popular for the production of Lingzhi mycelia. Liquid-state (submerged) fermentation of Lingzhi mycelia Liquid-state fermentation (LSF) of mushroom, also known as liquid culture or submerged fermentation, is a process of culturing microorganism in liquid media, but not on the surface of liquid media. Preparation of mushroom mycelium in submerged culture was initially developed during the 1970s. At that time, only lower fungi (fungi that are not from basidiocarps) were successfully cultured in fermentors for economical production of various natural products (Yang and Liau 1998). The main principle is that the liquid medium is added to the fermentation tank or flask. When sterile air is led to reactor, it will increase the dissolved oxygen content in the medium to improve respiratory metabolism of the mushroom mycelium. At the same time, the mushroom mycelium is agitated or oscillated, and appropriate external conditions are controlled so that mushroom cells grow in the liquid depths of breeding, producing a lot of the mycelium or metabolites. Another review indicated that there were few investigations into the development of high fungi (basidiomycetes) bioprocesses (Fang and Zhong 2002a, b). Compared with the artificial cultivation of Lingzhi fruit bodies, LSF holds obvious advantages. Firstly, the advantage of LSF over the traditional, multinational cultivation of fruit body is the reduction in the time spent to obtain the product of interest (Mshandete and Mgonja 2009). The production of fruit bodies takes at least 3–5 months, while reasonable amounts of ganoderic acids (GAs) and polysaccharides can be obtained by submerged fermentation after 3–5 weeks only (Wagner et al. 2003). Secondly, the mycelium grown in LSF holds high stability and standardization in which the environmental conditions (temperature, dissolved oxygen, pH, etc.) are easier to control. This is important not only for producing the desired product but also might be beneficial for producing mushroom-based medicines and nutraceuticals. The products obtained by this method are easy to achieve, with higher quality standards and safety (Wasser and Weis 1999). Thirdly, advantage of mycelia culturing in LSF is to provide a new method for scalable production and increased yields of biologically active compounds. By LSF method, the producer can increase the yield of protein rich in essential amino acids and vitamins serving as functional foods compared with the yield of these components in carpophores of the standard fruiting basidiomycetes (Friel and McLoughlin 2000). Thus, production of fungus metabolites through LSF method has received a lot of attention because of short time cultivation, high productivity, fewer chances of contamination, and easy recovery of producing metabolites (Huang and Liu 2008; Kim et al. 2007). In general, the methods of artificial cultivation typically in liquid media involve five stages: (1) selection of Lingzhi strain; (2) preparation of culture maintenance medium for Fig. 3 The developmental process of Lingzhi fruit body different culture phases; (3) inoculation; (4) cultivation of Appl Microbiol Biotechnol (2012) 93:941–963 947
948 Appl Microbiol Biotechnol (2012)93:941-963 strain in Erlenmeyer flasks,seeding tank,and fermentor. (IPS),and 582 mg/L for GA.In recent years,Chinese respectively;and(5)harvest of Lingzhi production.In the scientists have reported that they produced G.lucidum process of Lingzhi mycelia LSF,it is very important to 730 mycelia in a 500-L automatic stainless steel fer- select and control the appropriate fermentation conditions, mentor.The results showed that after a fermentation such as strain,amount of inoculation,temperature,pH,air time of 70 h,there was no obvious germination and flow,stirring rate,etc.The determination of the indices of lock-like concentration on the mycelia wall.There was the filamentous morphology,concentration,nutrients con- a little mycelium dissolved with no other bacteria.The sumption,and the outward appearance and viscosity of consistence of G.lucidum clump is about 30%.EPS broth could be used as the final quality standard of control and IPS is 3.5 g/L and 4.8 g/L,respectively (Wei et al. fermentation(Zhao 2002;Sanchez 2004;Wang et al.2007a, 2007a,b).Other reported studies of fermentation with b,c). G.lucidum were carried out in volumes of 10 m'with However,a multi-objective analysis and research was the fermentation liquid 7.5 m,the average production presented in the previous study of the Lingzhi LSF. cycle 150 h,and pH reduced from 6.5 to 3.5.The Some aimed simply to produce biomass,with no con- results showed that after the mycelia fermented,the cern for its composition.Others aimed to maximize the average weight of dry powders in every fermentor is production of either GAs or polysaccharides,and to 66.1 kg by spray drying,the average recovery 8.76 kg/ understand how different variables affect their produc- m',the content of pure polysaccharide 68.5 g/kg in the dry tion.Among these studies,many researchers studied powders,and the weight of the pure polysaccharide 4.225 kg Lingzhi LSF to obtain substances and special metabolic in every fermentor(Hu 2006). products (food,medicine or industrial enzyme,etc.) In addition,the cost-benefit ratio of liquid culture (Fang and Zhong 2002a;Yang 2005;Chen et al. and solid culture technologies for spawn production 2008;Songulashvili et al.2007).Also,other researchers were investigated by some researchers.The results dem- focused on the parameters in Lingzhi LSF including onstrated that liquid culture technology for spawn pro- culture media (Sun et al.2000),initial pH (Fang and duction is obviously more advantageous,which is Zhong 2002b),culture times and temperature,etc.(Wei reflected in better spawn quality,reduction of contami- et al.2007a,b;Xia et al.2007;Zhang et al.2008a,b; nation,increased efficiency,reduction of cost,and in- Liang 2011).In summary,in the process of culturing creased production stability.For this technology to be Lingzhi by LSF method,the suitable carbon and nitro- broadly used,it is key to improve the incubator shakers, gen resources are corn flour and soybean meal with fermentation tanks,and inoculation equipment.Efforts concentrations of 3%and 2.5%,respectively.The opti- to facilitate researches according to the demand of a mum cultivation conditions were as follows:initial pH 4.5- dynamic market would promote the application of this 5.0,rotation speeds 120-150 rpm,100-120 mL medium/ technology in the fast-growing mushroom spawn pro- 500 mL Erlenmeyer flask at 25 C.When the diameter of duction industry (Guo and Liu 2011).Based on the mycelium pellet was 0.85-0.9 mm in the process of culturing character of Lingzhi mycelium having bio-enrichment with Erlenmeyer flask,the biomass of Lingzhi mycelia is the of mineral nutrition,some researchers also focused their highest(Dong et al.2004;Zhu et al.2009). interest on the accumulation of some minerals by sub In spite of having achieved significant progress in merged fermentation,which include the selenium- Lingzhi LSF,the applications of LSF techniques have enriched Lingzhi (Xie et al.1996;Shen and Yu 2008; an interval to industrialization production.In other Ling et al.2008),the calcium-enriched Lingzhi (Gao et words,the successful example in large-scale culture al.2007).the zinc-enriched Lingzhi (Liu et al.2005: Lingzhi by LSF is extraordinary.In the previous review Wei et al.2010),the iron-enriched Lingzhi (Miao and articles,the researchers greatly summarized the pub- Lv 2007),and so on.The abovementioned studies pro- lished data on submerged fermentation with Lingzhi vide a foundation of development of functional food (Wagner et al.2003;Sanodiya et al.2009).In this using mineral nutrition enrichment Lingzhi as raw mate- review article,only one report of a large-scale fermen- rials (Wang et al.2001;Mao and Ma 2009). tation was described in which Ganoderma tsugae was cultivated in tanks with a volume of 20 m'.All other Solid-state fermentation of Lingzhi mycelia In contrast to reported studies of LSF with Lingzhi were done only in LSF,solid-state fermentation(SSF),also known as SSC,is volumes of 10 L or less.By estimating results general- the cultivation of microorganisms under controlled condi- ly,half the studies were undertaken in Erlenmeyer tions in the absence of free water.The production examples flasks and others were in fermentors.However,the best of SSF include the industrial enzymes (Wu et al.2000), yields reported to date are 22.1 g/L for biomass,1.71 g/ biofuels,biopesticide and nutrient-enriched animal feeds. L for EPS,2.49 g/L for intercellular polysaccharides etc.(Habijanic and Berovic 2000;Sun et al.2007;Chen et ②Springer
strain in Erlenmeyer flasks, seeding tank, and fermentor, respectively; and (5) harvest of Lingzhi production. In the process of Lingzhi mycelia LSF, it is very important to select and control the appropriate fermentation conditions, such as strain, amount of inoculation, temperature, pH, air flow, stirring rate, etc. The determination of the indices of the filamentous morphology, concentration, nutrients consumption, and the outward appearance and viscosity of broth could be used as the final quality standard of control fermentation (Zhao 2002; Sánchez 2004; Wang et al. 2007a, b, c). However, a multi-objective analysis and research was presented in the previous study of the Lingzhi LSF. Some aimed simply to produce biomass, with no concern for its composition. Others aimed to maximize the production of either GAs or polysaccharides, and to understand how different variables affect their production. Among these studies, many researchers studied Lingzhi LSF to obtain substances and special metabolic products (food, medicine or industrial enzyme, etc.) (Fang and Zhong 2002a; Yang 2005; Chen et al. 2008; Songulashvili et al. 2007). Also, other researchers focused on the parameters in Lingzhi LSF including culture media (Sun et al. 2000), initial pH (Fang and Zhong 2002b), culture times and temperature, etc. (Wei et al. 2007a, b; Xia et al. 2007; Zhang et al. 2008a, b; Liang 2011). In summary, in the process of culturing Lingzhi by LSF method, the suitable carbon and nitrogen resources are corn flour and soybean meal with concentrations of 3% and 2.5%, respectively. The optimum cultivation conditions were as follows: initial pH 4.5– 5.0, rotation speeds 120–150 rpm, 100–120 mL medium/ 500 mL Erlenmeyer flask at 25 °C. When the diameter of mycelium pellet was 0.85–0.9 mm in the process of culturing with Erlenmeyer flask, the biomass of Lingzhi mycelia is the highest (Dong et al. 2004; Zhu et al. 2009). In spite of having achieved significant progress in Lingzhi LSF, the applications of LSF techniques have an interval to industrialization production. In other words, the successful example in large-scale culture Lingzhi by LSF is extraordinary. In the previous review articles, the researchers greatly summarized the published data on submerged fermentation with Lingzhi (Wagner et al. 2003; Sanodiya et al. 2009). In this review article, only one report of a large-scale fermentation was described in which Ganoderma tsugae was cultivated in tanks with a volume of 20 m3 . All other reported studies of LSF with Lingzhi were done only in volumes of 10 L or less. By estimating results generally, half the studies were undertaken in Erlenmeyer flasks and others were in fermentors. However, the best yields reported to date are 22.1 g/L for biomass, 1.71 g/ L for EPS, 2.49 g/L for intercellular polysaccharides (IPS), and 582 mg/L for GA. In recent years, Chinese scientists have reported that they produced G. lucidum 730 mycelia in a 500-L automatic stainless steel fermentor. The results showed that after a fermentation time of 70 h, there was no obvious germination and lock-like concentration on the mycelia wall. There was a little mycelium dissolved with no other bacteria. The consistence of G. lucidum clump is about 30%. EPS and IPS is 3.5 g/L and 4.8 g/L, respectively (Wei et al. 2007a, b). Other reported studies of fermentation with G. lucidum were carried out in volumes of 10 m3 with the fermentation liquid 7.5 m3 , the average production cycle 150 h, and pH reduced from 6.5 to 3.5. The results showed that after the mycelia fermented, the average weight of dry powders in every fermentor is 66.1 kg by spray drying, the average recovery 8.76 kg/ m3 , the content of pure polysaccharide 68.5 g/kg in the dry powders, and the weight of the pure polysaccharide 4.225 kg in every fermentor (Hu 2006). In addition, the cost–benefit ratio of liquid culture and solid culture technologies for spawn production were investigated by some researchers. The results demonstrated that liquid culture technology for spawn production is obviously more advantageous, which is reflected in better spawn quality, reduction of contamination, increased efficiency, reduction of cost, and increased production stability. For this technology to be broadly used, it is key to improve the incubator shakers, fermentation tanks, and inoculation equipment. Efforts to facilitate researches according to the demand of a dynamic market would promote the application of this technology in the fast-growing mushroom spawn production industry (Guo and Liu 2011). Based on the character of Lingzhi mycelium having bio-enrichment of mineral nutrition, some researchers also focused their interest on the accumulation of some minerals by submerged fermentation, which include the seleniumenriched Lingzhi (Xie et al. 1996; Shen and Yu 2008; Ling et al. 2008), the calcium-enriched Lingzhi (Gao et al. 2007), the zinc-enriched Lingzhi (Liu et al. 2005; Wei et al. 2010), the iron-enriched Lingzhi (Miao and Lv 2007), and so on. The abovementioned studies provide a foundation of development of functional food using mineral nutrition enrichment Lingzhi as raw materials (Wang et al. 2001; Mao and Ma 2009). Solid-state fermentation of Lingzhi mycelia In contrast to LSF, solid-state fermentation (SSF), also known as SSC, is the cultivation of microorganisms under controlled conditions in the absence of free water. The production examples of SSF include the industrial enzymes (Wu et al. 2000), biofuels, biopesticide and nutrient-enriched animal feeds, etc. (Habijanic and Berovic 2000; Sun et al. 2007; Chen et 948 Appl Microbiol Biotechnol (2012) 93:941–963