Biotechnol Lett (2014) 36:2283–2289 DOI 10.1007/s10529-014-1605-3
ORIGINAL RESEARCH PAPER
Increased mycelial biomass production by Lentinula edodes intermittently illuminated by green light emitting diodes Lubov B. Glukhova • Ludmila O. Sokolyanskaya • Evgeny V. Plotnikov • Anna L. Gerasimchuk • Olga V. Karnachuk • Marc Solioz • Raisa A. Karnachuk
Received: 29 May 2014 / Accepted: 24 June 2014 / Published online: 22 July 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Fungi possess a range of light receptors to regulate metabolism and differentiation. To study the effect of light on Lentinula edodes (the shiitake mushroom), mycelial cultures were exposed to blue, green, and red fluorescent lights and light-emitting diodes, as well as green laser light. Biomass production, morphology, and pigment production were evaluated. Exposure to green light at intervals of 1 min/d at 0.4 W/m2 stimulated biomass production by 50–100 %, depending on the light source. Light intensities in excess of 1.8 W/m2 or illumination longer than 30 min/d did not affect biomass
production. Carotenoid production and morphology remained unaltered during increased biomass production. These observations provide a cornerstone to the study of photoreception by this important fungus.
L. B. Glukhova L. O. Sokolyanskaya E. V. Plotnikov A. L. Gerasimchuk O. V. Karnachuk M. Solioz (&) R. A. Karnachuk Dept. of Plant Physiology and Biotechnology, Tomsk State University, Lenin Prospect 36, 634050 Toms, Russian Federation e-mail: [email protected]
Filamentous fungi can sense light and use it as a signal to physiological and morphological responses and it has been known for many decades that processes, such as sporulation, morphogenesis, phototropism, circadian clock regulation or carotenoid biosynthesis, are influenced by light (for review see Rodriguez-Romero et al. 2010). However, the light-sensor proteins involved in these regulatory processes have only recently been characterized. The first red-light receptor, phytochrome, was characterized in Aspergillus nidulans by Blumenstein et al. (2005). In addition to phytochrome, blue-light and green-light sensing systems have been found in fungi (Bahn et al. 2007; Crosthwaite et al. 1997; Linden et al. 1997). With the advent of genome sequencing, it has become clear that most, if not all, fungi possess multiple light sensors and light-regulated systems which regulate not only enzymes, but also the expression of genes [see Tisch
L. B. Glukhova e-mail: [email protected] L. O. Sokolyanskaya e-mail: [email protected] E. V. Plotnikov e-mail: [email protected] A. L. Gerasimchuk e-mail: [email protected] O. V. Karnachuk e-mail: [email protected] R. A. Karnachuk e-mail: [email protected]
Keywords Fungal biomass Green light Hyphal morphology Intermittent illumination Lentinula edodes Phototropism Shiitake mushroom
Introduction
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and Schmoll (2010) and Rodriguez-Romero et al. (2010) for recent review]. Nevertheless, the study of these complex systems is still in its infancy in most fungi. Light of different wavelengths can potentially be applied to improve the growth yield of biotechnologically important basidiomycetes. Lentinula edodes, the shiitake mushroom, is one of the most cultivated mushrooms in the world (Bruhn et al. 2009). It is known not only for its culinary properties but also for its antitumor and antimicrobial activities (Poyedinok et al. 2005; Bisen et al. 2010; Falandysz and Borovicka 2013; Rao et al. 2009; Xu et al. 2012). However, as in many fungi, the role of light perception is largely unknown. In the present study, we exposed growing mycelia of L. edodes to a range of illumination regimes, from continuous light to short daily exposures of 1 min, using a range of light sources. Short illumination of 1 min/d with green light afforded a marked stimulation of biomass production. This effect is not only biotechnologically of interest but lays a cornerstone for the study of the mechanisms of photoreception by L. edodes.
Materials and methods
Biotechnol Lett (2014) 36:2283–2289
12 day old mycelial cultures and incubated at 22–24 °C and 70–80 % relative humidity. Cultures were grown either in the dark or under the illumination regimes described under Results and discussion. To obtain different illumination levels, cultures were placed at different distances to the light sources. The following illumination schemes were used: blue, green, and red fluorescent lights at illumination intensities at the culture surfaces of 0.44, 0.88, 1.77, 3.1 and 6.66 W/m2. Blue, green, and red LED with maxima at 465, 515 and 645 nm, respectively, were used at 0.4 W/m2. Green laser light of 532 nm was used at 0.4 W/m2. Exposure times were as detailed under Results. Light intensities at the culture surfaces and spectral characteristics of the light sources were determined using an AvaSpec-2048-2 optical fiber spectrophotometer (Avantes, Netherlands), using the instrument software. Biomass determination For biomass measurement, the cultures were grown for 12 days. Mycelium was collected from the surface of solid medium, air dried and the dried mycelium was weighted. The radius of each colony was measured at four fixed points. For statistical analysis, 20–40 measurements were taken for each type of treatment.
Strain characterization A commercial strain of L. edodes, W4, was purchased from Fungi Perfecti, Olympia, WA. The 18S rRNA sequence was determined by PCR amplification with the primers 50 -AACCTGGTTGATCCTGCCAGT and 50 -TGATCCTTCTGCAGGTTCACCTAC (Medlin et al. 1988), followed by DNA sequencing of the product of 1,727 base pairs. The sequence was deposited in GenBank under accession number of KM015456. Phylogenetic analysis was performed with the program MEGA5, using the maximum likelihood method, using the default parameters (Tamura et al. 2011).
Quantification of mycelial carotenoid accumulation Intracellular pigments were determined essentially as described (Yoshida and Hasunuma 2004). Briefly, 40 mg fresh mycelium was extracted with 3 ml methanol for 30 min at 60 °C. After centrifugation for 10 min at 20,0009g, the pellet was re-extracted with 3 ml acetone for 30 min at 50 °C. The combined methanol and acetone fractions were re-centrifuged as before and spectral absorption was determined from 300 to 500 nm. Carotenoid content was calculated using an extinction coefficient E1 % of 2,500 cm-1 (Pocock et al. 2004).
Strains and growth conditions Hyphal morphology Mycelia were grown in Petri dishes 9 cm diam. containing 20 ml solid Czapec medium: 4 % (w/v) yeast extract, 3 % (w/v) glucose, 0.2 % NaNO3, 0.1 % KH2PO4, 0.05 % KCl, 0.05 % MgSO47H2O, and 10 mg FeSO4 l-1. Petri dishes were inoculated with
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Hyphal diameters were assessed from growth plates by transferring 0.5 mm of the top agar layer to a microscope slide and photographing it with a Carl Zeiss AxioStar microscope, fitted with a digital
Biotechnol Lett (2014) 36:2283–2289
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Fig. 1 Light emission spectra of light sources used in this study. a blue fluorescent lamp (kmax = 460 nm), b green fluorescent lamp (kmax = 545 nm), c red fluorescent lamp
(kmax = 660 nm), d blue LED (kmax = 465 nm), e green LED (kmax = 515 nm), f red LED (kmax = 645 nm), g green laser (kmax = 532 nm)
camera. Plates were inspected for complete mycelia removal. From each of five independent experiments, 30–50 images representing different hyphen densities were taken and on each image, 30–80 random measurements of hyphen diameters were performed. The measurements were calibrated with a calibration slide with 10 lm divisions, photographed at the same magnification.
and edible. Genome sequences are, unfortunately, not available for any of these strains/species but it is clear that the cultivar of L. edodes used in this study is a typical representative of the shiitake family (Fig. 1). To identify phototropic responses of L. edodes mycelia, a range of different light sources was employed to test light effects on biomass formation, morphology and carotenoid content. Fig. 1 shows the spectral characteristics of the light sources used. Blue, green, and red fluorescent lights provided relatively broad-range illumination with maxima at 460, 545, and 660 nm, respectively. While fluorescent lights are very cost-effective, they have the disadvantage of containing intense bands at other wavelength (cf. Fig. 1a–c). These bands are due to emission lines of the mercury vapor, used to produce light in these lamps. Blue, green, and red LED, on the other hand, provide narrower-band spectra with maxima at 465, 515, and 645, respectively, and are free of intense
Results A commercially-cultivated shiitake strain, L. edodes W4, was used to investigate the effect of light on this fungus. Phylogenetic analysis of the 18S rRNA sequence revealed that L. edodes W4 was closely related to strains FJ379277, FJ379282, and FJ379282 with sequence similarities of 99.4, 99.9, and 99.9 %, respectively. All these strains are commercially used
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Fig. 2 Dry biomass of L. edodes mycelia grown under exposure to different fluorescent light sources at varying exposure times and intensities. a blue fluorescent light, b green fluorescent light, c red florescent light. Light intensities were as follows: 0.44 W/m2 (black bars), 0.88 W/m2 (grey bars), 1.77 W/m2 (hatched bars), 3.1 W/m2 (stippled bars), 6.66 W/m2 (open bars). Other details of the experiment are described under Materials and methods. The error bars indicate the standard error of three independent experiments
wavelength bands (Fig. 1d–f). Laser light, finally, can provide essentially monochromatic illumination, (Fig. 1g).
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Biomass production of L. edodes W4 under illumination with blue, green, and red fluorescent lights for 1–60 min per day and at intensities ranging from 0.44 to 6.66 W/m2 was determined. Exposure of L. edodes culture to blue fluorescent light at intensities of 0.44–1.77 W/m2 for 1–30 min/d stimulated biomass production by approximately 100 % (Fig. 2a; P \ 0.01), compared to dark controls. This effect declined at higher light intensities or longer exposure times. Essentially the same observation was made under illumination with green fluorescent light (Fig. 2b). However, blue fluorescent light has a strong emission band in the green at 546 nm due to a mercury emission line (cf. Fig. 1a). Illumination with red fluorescent light also produced significant growth stimulation, but in a much narrower range of exposure times and light intensities (Fig. 2c). Overall, a roughly two-fold stimulation of biomass production by L. edodes could be observed by relatively short, lowlevel intermittent illumination with fluorescent light. (Fig. 2). To obtain more precise information on the wavelength characteristics of the light-stimulation of biomass production by L. edodes, LED with narrower and cleaner emission spectra were employed. Under these conditions, it became apparent that the best stimulatory effect was produced by green LED light of 0.35 W/m2 for 1 min/d, which led to 1.6 times the biomass compared to dark control cultures (Fig. 3).
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Fig. 4 Hyphen diameters under different growth conditions. Hyphen measurements of L. edodes grown in the dark (solid bars) and with intermittent green LED illumination at 0.35 W/ m2 (open bars). The error bars show the standard error of five independent experiments
The over 50 % increase in growth yield observed under these conditions was statistically highly significant (P \ 0.01). In contrast, the effects of illumination with red or blue LED light were not or only marginally significantly. The positive effect on growth by green light was corroborated by exposing cultures to green laser light of wavelength 532 nm under comparable conditions. Biomass accumulation was similarly stimulated by nearly 50 %, namely from (44.3 ± 4.2) mg to (62.1 ± 3.1) mg under illumination by green laser light for 1 min/d (P \ 0.01). (Fig. 3). To obtain information about the possible reasons for the increase in biomass when L. edodes was grown with green light illumination, the hyphal morphology was assessed. There was a large variation in hyphae diameters, as apparent from microscopic examination. However, quantitative morphometric analysis revealed that hyphae diameters did not differ significantly between cultures grown in the dark and those exposed to green light (Fig. 4). Hyphae diameters differed widely, from \1 to [7 lm; approx. 50 % of the hyphae displayed diameters from 3 to 4.9 lm. There were also no other apparent morphological differences between light and dark grown hyphae. (Fig. 4). Light induction of carotenoid biosynthesis has been described in many fungi and has received extensive attention in Neurospora crassa (Rodriguez-Romero et al. 2010). It was thus of interest to look at the influence of illumination on carotenoid content. In carotenoid extracts of hyphae, there was a single peak
at (440 ± 2) nm, corresponding to 41 ± 2 ng of carotenoid per mg of fresh mycelium; this value did not significantly vary between extracts from hyphae grown either in the dark or exposed to green light. Thus, there was no detectable light effect on carotenoid synthesis of L. edodes under the conditions used here. Taken together, we show here that illumination of L. edodes W4 with green light for only 1 min/d and at a relatively low intensity of 0.35 W/m2 markedly stimulated the growth yield. This observation is novel.
Discussion We here describe the stimulation of mycelial biomass formation by L. edodes W4 by short light exposure. The phenomenon is novel and striking, since exposure times as short as 1 min/d were sufficient to stimulate biomass production by 50–100 %. The most dramatic stimulation of biomass production was observed with green fluorescent light, amounting to over 100 %. Fluorescent lights are not ‘‘clean’’ light sources and have emission peaks outside the main spectral color, due to the emission lines of the mercury in the these light sources. LED, which provide for better defined emission spectra, also stimulated biomass production, but the effect was less dramatic. Best stimulation was observed with exposure to green LED illumination at 0.35 W/m2 for 1 min/d, resulting in a 1.6-fold increase in biomass production (P \ 0.01). Growth stimulation to a similar extent could also be observed with monochromatic (laser) light of 532 nm. This of course suggests that light sensory systems in L. edodes are active at the level of growth control. Conclusions about the action spectra cannot be drawn from our observations, but 532 nm is clearly within the range of the absorption spectrum of the responsible light sensing system(s). Among the well-known effects of light on fungi are stimulation of carotenoid biosynthesis, sporulation behavior, phototropism, and circadian clock control (Purschwitz et al. 2006; Tisch and Schmoll 2010). However, we could not observe macroscopic or microscopic changes in morphology; morphometric analysis of hyphal growth also did not reveal differences between mycelia grown in the dark or with intermittent illumination. The carotenoid content, was also not altered by illumination.
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In a study of the effect of illumination at different wavelengths on extra- and intra-cellular pigments and biomass production in Monascus purpureus, Isaria farinosa, Emericella nidulans, Fusarium verticillioides and Penicillium purpurogenum, (Velmurugan et al. 2010) did not observe stimulatory effects by light; to the contrary, most illumination schemes reduced biomass and pigment substantially. However, these authors used continuous illumination, which we found decreased the growth of L. edodes (not shown). This contrasts with results obtained by Ramirez et al. (2010) using continuous LED illumination of Phanerochaete chrysosporium at different wavelengths. They found that green LED illumination increased lignin peroxidase production by 20–27 %, while biomass production varied from 23 to -11 %, with no apparent pattern. Continuous or extended illumination was employed in most previous studies and there are essentially no studies available that describe the effects of short light exposure, e.g. 1 min/d. A short light exposure of 5 min did, however, induce conidiation in Trichoderma atroviride, and two blue-light sensors, blue-light regulators 1 and 2 (blr-1 and blr-2), similar to the Neurospora crassa white-collar 1 and 2, were identified (Casas-Flores et al. 2004). As discussed by these authors, many effects of light on fungi may in fact have been missed by using long-time light exposure, which often inhibits growth and metabolic processes rather globally. What causes the remarkable growth stimulation observed in the present study remains unknown. Light reception by fungi is complex involving different chromophores like flavin, retinal, or biliverdin, and a range of associated proteins (Heintzen 2012; Rodriguez-Romero et al. 2010). Conceivably, the short light pulses activate a developmental switch, which is maintained for prolonged times by the cells. Molecular studies on the mechanism of the light response of L. edodes will have to await the release its genome sequence. However, the light-stimulation of biomass production described here should be of immediate interest for biotechnological applications. Acknowledgments This work was supported by a Russian Federation Government grant to leading scientists (contract number 14.Z50.31.0011).
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References Bahn YS, Xue C, Idnurm A, Rutherford JC, Heitman J, Cardenas ME (2007) Sensing the environment: lessons from fungi. Nat Rev Microbiol 5:57–69 Bisen PS, Baghel RK, Sanodiya BS, Thakur GS, Prasad GB (2010) Lentinus edodes: a macrofungus with pharmacological activities. Curr Med Chem 17:2419–2430 Blumenstein A, Vienken K, Tasler R, Purschwitz J, Veith D, Frankenberg-Dinkel N, Fischer R (2005) The Aspergillus nidulans phytochrome FphA represses sexual development in red light. Curr Biol 15:1833–1838 Bruhn JN, Mihail JD, Pickens JB (2009) Forest farming of shiitake mushrooms: an integrated evaluation of management practices. Bioresour Technol 100:6472–6480 Casas-Flores S, Rios-Momberg M, Bibbins M, Ponce-Noyola P, Herrera-Estrella A (2004) BLR-1 and BLR-2, key regulatory elements of photoconidiation and mycelial growth in Trichoderma atroviride. Microbiology 150:3561–3569 Crosthwaite SK, Dunlap JC, Loros JJ (1997) Neurospora wc-1 and wc-2: transcription, photoresponses, and the origins of circadian rhythmicity. Science 276:763–769 Falandysz J, Borovicka J (2013) Macro and trace mineral constituents and radionuclides in mushrooms: health benefits and risks. Appl Microbiol Biotechnol 97:477–501 Heintzen C (2012) Plant and fungal photopigments. Membr Transp Signal 1:411–432 Hibbett DS, Hansen K, Donoghue MJ (1998) Phylogeny and biogeography of Lentinula inferred from an expanded rDNA dataset. Mycol Res 102:1041–1049 Linden H, Rodriguez-Franco M, Macino G (1997) Mutants of Neurospora crassa defective in regulation of blue light perception. Mol Gen Genet 254:111–118 Medlin L, Elwood HJ, Stickel S, Sogin ML (1988) The characterization of enzymatically amplified eukaryotic 16Slike rRNA-coding regions. Gene 71:491–499 Pocock T, Krol M, Huner NPA (2004) The determination and quantification of photosynthetic pigments by reverse phase high-performance liquid chromatography, thin-layer chromatography, and spectrophotometry. Photosynth Res Protoc 274:137–148 Poyedinok NL, Buchalo AS, Efremenkova OV, Negriyko AM (2005) The light factor in biotechnology cultivation of medicinal mushrooms. Intl J Med Mushroom 7:448–449 Purschwitz J, Muller S, Kastner C, Fischer R (2006) Seeing the rainbow: light sensing in fungi. Curr Opin Microbiol 9:566–571 Ramirez DA, Munoz SV, Atehortua L, Michel FC Jr (2010) Effects of different wavelengths of light on lignin peroxidase production by the white-rot fungi Phanerochaete chrysosporium grown in submerged cultures. Biores Technol 101:9213–9220 Rao JR, Smyth TJ, Millar BC, Moore JE (2009) Antimicrobial properties of shiitake mushrooms (Lentinula edodes). Int J Antimicrob Agents 33:591–592 Rodriguez-Romero J, Hedtke M, Kastner C, Muller S, Fischer R (2010) Fungi, hidden in soil or up in the air: light makes a difference. Annu Rev Microbiol 64:585–610
Biotechnol Lett (2014) 36:2283–2289 Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28: 2731–2739 Tisch D, Schmoll M (2010) Light regulation of metabolic pathways in fungi. Appl Microbiol Biotechnol 85:1259–1277 Velmurugan P, Lee YH, Venil CK, Lakshmanaperumalsamy P, Chae JC, Oh BT (2010) Effect of light on growth,
2289 intracellular and extracellular pigment production by five pigment-producing filamentous fungi in synthetic medium. J Biosci Bioeng 109:346–350 Xu T, Beelman RB, Lambert JD (2012) The cancer preventive effects of edible mushrooms. Antican Ag Med Chem 12:1255–1263 Yoshida Y, Hasunuma K (2004) Reactive oxygen species affect photomorphogenesis in Neurospora crassa. J Biol Chem 279:6986–6993
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ORIGINAL RESEARCH PAPER
Increased mycelial biomass production by Lentinula edodes intermittently illuminated by green light emitting diodes Lubov B. Glukhova • Ludmila O. Sokolyanskaya • Evgeny V. Plotnikov • Anna L. Gerasimchuk • Olga V. Karnachuk • Marc Solioz • Raisa A. Karnachuk
Received: 29 May 2014 / Accepted: 24 June 2014 / Published online: 22 July 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Fungi possess a range of light receptors to regulate metabolism and differentiation. To study the effect of light on Lentinula edodes (the shiitake mushroom), mycelial cultures were exposed to blue, green, and red fluorescent lights and light-emitting diodes, as well as green laser light. Biomass production, morphology, and pigment production were evaluated. Exposure to green light at intervals of 1 min/d at 0.4 W/m2 stimulated biomass production by 50–100 %, depending on the light source. Light intensities in excess of 1.8 W/m2 or illumination longer than 30 min/d did not affect biomass
production. Carotenoid production and morphology remained unaltered during increased biomass production. These observations provide a cornerstone to the study of photoreception by this important fungus.
L. B. Glukhova L. O. Sokolyanskaya E. V. Plotnikov A. L. Gerasimchuk O. V. Karnachuk M. Solioz (&) R. A. Karnachuk Dept. of Plant Physiology and Biotechnology, Tomsk State University, Lenin Prospect 36, 634050 Toms, Russian Federation e-mail: [email protected]
Filamentous fungi can sense light and use it as a signal to physiological and morphological responses and it has been known for many decades that processes, such as sporulation, morphogenesis, phototropism, circadian clock regulation or carotenoid biosynthesis, are influenced by light (for review see Rodriguez-Romero et al. 2010). However, the light-sensor proteins involved in these regulatory processes have only recently been characterized. The first red-light receptor, phytochrome, was characterized in Aspergillus nidulans by Blumenstein et al. (2005). In addition to phytochrome, blue-light and green-light sensing systems have been found in fungi (Bahn et al. 2007; Crosthwaite et al. 1997; Linden et al. 1997). With the advent of genome sequencing, it has become clear that most, if not all, fungi possess multiple light sensors and light-regulated systems which regulate not only enzymes, but also the expression of genes [see Tisch
L. B. Glukhova e-mail: [email protected] L. O. Sokolyanskaya e-mail: [email protected] E. V. Plotnikov e-mail: [email protected] A. L. Gerasimchuk e-mail: [email protected] O. V. Karnachuk e-mail: [email protected] R. A. Karnachuk e-mail: [email protected]
Keywords Fungal biomass Green light Hyphal morphology Intermittent illumination Lentinula edodes Phototropism Shiitake mushroom
Introduction
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and Schmoll (2010) and Rodriguez-Romero et al. (2010) for recent review]. Nevertheless, the study of these complex systems is still in its infancy in most fungi. Light of different wavelengths can potentially be applied to improve the growth yield of biotechnologically important basidiomycetes. Lentinula edodes, the shiitake mushroom, is one of the most cultivated mushrooms in the world (Bruhn et al. 2009). It is known not only for its culinary properties but also for its antitumor and antimicrobial activities (Poyedinok et al. 2005; Bisen et al. 2010; Falandysz and Borovicka 2013; Rao et al. 2009; Xu et al. 2012). However, as in many fungi, the role of light perception is largely unknown. In the present study, we exposed growing mycelia of L. edodes to a range of illumination regimes, from continuous light to short daily exposures of 1 min, using a range of light sources. Short illumination of 1 min/d with green light afforded a marked stimulation of biomass production. This effect is not only biotechnologically of interest but lays a cornerstone for the study of the mechanisms of photoreception by L. edodes.
Materials and methods
Biotechnol Lett (2014) 36:2283–2289
12 day old mycelial cultures and incubated at 22–24 °C and 70–80 % relative humidity. Cultures were grown either in the dark or under the illumination regimes described under Results and discussion. To obtain different illumination levels, cultures were placed at different distances to the light sources. The following illumination schemes were used: blue, green, and red fluorescent lights at illumination intensities at the culture surfaces of 0.44, 0.88, 1.77, 3.1 and 6.66 W/m2. Blue, green, and red LED with maxima at 465, 515 and 645 nm, respectively, were used at 0.4 W/m2. Green laser light of 532 nm was used at 0.4 W/m2. Exposure times were as detailed under Results. Light intensities at the culture surfaces and spectral characteristics of the light sources were determined using an AvaSpec-2048-2 optical fiber spectrophotometer (Avantes, Netherlands), using the instrument software. Biomass determination For biomass measurement, the cultures were grown for 12 days. Mycelium was collected from the surface of solid medium, air dried and the dried mycelium was weighted. The radius of each colony was measured at four fixed points. For statistical analysis, 20–40 measurements were taken for each type of treatment.
Strain characterization A commercial strain of L. edodes, W4, was purchased from Fungi Perfecti, Olympia, WA. The 18S rRNA sequence was determined by PCR amplification with the primers 50 -AACCTGGTTGATCCTGCCAGT and 50 -TGATCCTTCTGCAGGTTCACCTAC (Medlin et al. 1988), followed by DNA sequencing of the product of 1,727 base pairs. The sequence was deposited in GenBank under accession number of KM015456. Phylogenetic analysis was performed with the program MEGA5, using the maximum likelihood method, using the default parameters (Tamura et al. 2011).
Quantification of mycelial carotenoid accumulation Intracellular pigments were determined essentially as described (Yoshida and Hasunuma 2004). Briefly, 40 mg fresh mycelium was extracted with 3 ml methanol for 30 min at 60 °C. After centrifugation for 10 min at 20,0009g, the pellet was re-extracted with 3 ml acetone for 30 min at 50 °C. The combined methanol and acetone fractions were re-centrifuged as before and spectral absorption was determined from 300 to 500 nm. Carotenoid content was calculated using an extinction coefficient E1 % of 2,500 cm-1 (Pocock et al. 2004).
Strains and growth conditions Hyphal morphology Mycelia were grown in Petri dishes 9 cm diam. containing 20 ml solid Czapec medium: 4 % (w/v) yeast extract, 3 % (w/v) glucose, 0.2 % NaNO3, 0.1 % KH2PO4, 0.05 % KCl, 0.05 % MgSO47H2O, and 10 mg FeSO4 l-1. Petri dishes were inoculated with
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Hyphal diameters were assessed from growth plates by transferring 0.5 mm of the top agar layer to a microscope slide and photographing it with a Carl Zeiss AxioStar microscope, fitted with a digital
Biotechnol Lett (2014) 36:2283–2289
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Fig. 1 Light emission spectra of light sources used in this study. a blue fluorescent lamp (kmax = 460 nm), b green fluorescent lamp (kmax = 545 nm), c red fluorescent lamp
(kmax = 660 nm), d blue LED (kmax = 465 nm), e green LED (kmax = 515 nm), f red LED (kmax = 645 nm), g green laser (kmax = 532 nm)
camera. Plates were inspected for complete mycelia removal. From each of five independent experiments, 30–50 images representing different hyphen densities were taken and on each image, 30–80 random measurements of hyphen diameters were performed. The measurements were calibrated with a calibration slide with 10 lm divisions, photographed at the same magnification.
and edible. Genome sequences are, unfortunately, not available for any of these strains/species but it is clear that the cultivar of L. edodes used in this study is a typical representative of the shiitake family (Fig. 1). To identify phototropic responses of L. edodes mycelia, a range of different light sources was employed to test light effects on biomass formation, morphology and carotenoid content. Fig. 1 shows the spectral characteristics of the light sources used. Blue, green, and red fluorescent lights provided relatively broad-range illumination with maxima at 460, 545, and 660 nm, respectively. While fluorescent lights are very cost-effective, they have the disadvantage of containing intense bands at other wavelength (cf. Fig. 1a–c). These bands are due to emission lines of the mercury vapor, used to produce light in these lamps. Blue, green, and red LED, on the other hand, provide narrower-band spectra with maxima at 465, 515, and 645, respectively, and are free of intense
Results A commercially-cultivated shiitake strain, L. edodes W4, was used to investigate the effect of light on this fungus. Phylogenetic analysis of the 18S rRNA sequence revealed that L. edodes W4 was closely related to strains FJ379277, FJ379282, and FJ379282 with sequence similarities of 99.4, 99.9, and 99.9 %, respectively. All these strains are commercially used
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Fig. 3 Dry biomass of L. edodes mycelia grown under exposure to different LED light sources at 0.35 W/m2 and 1 min/d exposure. Other details of the experiment are described under Materials and methods. The error bars indicate the standard error of three independent experiments and the asterisk a statistically significant difference of P \ 0.01
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Fig. 2 Dry biomass of L. edodes mycelia grown under exposure to different fluorescent light sources at varying exposure times and intensities. a blue fluorescent light, b green fluorescent light, c red florescent light. Light intensities were as follows: 0.44 W/m2 (black bars), 0.88 W/m2 (grey bars), 1.77 W/m2 (hatched bars), 3.1 W/m2 (stippled bars), 6.66 W/m2 (open bars). Other details of the experiment are described under Materials and methods. The error bars indicate the standard error of three independent experiments
wavelength bands (Fig. 1d–f). Laser light, finally, can provide essentially monochromatic illumination, (Fig. 1g).
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Biomass production of L. edodes W4 under illumination with blue, green, and red fluorescent lights for 1–60 min per day and at intensities ranging from 0.44 to 6.66 W/m2 was determined. Exposure of L. edodes culture to blue fluorescent light at intensities of 0.44–1.77 W/m2 for 1–30 min/d stimulated biomass production by approximately 100 % (Fig. 2a; P \ 0.01), compared to dark controls. This effect declined at higher light intensities or longer exposure times. Essentially the same observation was made under illumination with green fluorescent light (Fig. 2b). However, blue fluorescent light has a strong emission band in the green at 546 nm due to a mercury emission line (cf. Fig. 1a). Illumination with red fluorescent light also produced significant growth stimulation, but in a much narrower range of exposure times and light intensities (Fig. 2c). Overall, a roughly two-fold stimulation of biomass production by L. edodes could be observed by relatively short, lowlevel intermittent illumination with fluorescent light. (Fig. 2). To obtain more precise information on the wavelength characteristics of the light-stimulation of biomass production by L. edodes, LED with narrower and cleaner emission spectra were employed. Under these conditions, it became apparent that the best stimulatory effect was produced by green LED light of 0.35 W/m2 for 1 min/d, which led to 1.6 times the biomass compared to dark control cultures (Fig. 3).
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The over 50 % increase in growth yield observed under these conditions was statistically highly significant (P \ 0.01). In contrast, the effects of illumination with red or blue LED light were not or only marginally significantly. The positive effect on growth by green light was corroborated by exposing cultures to green laser light of wavelength 532 nm under comparable conditions. Biomass accumulation was similarly stimulated by nearly 50 %, namely from (44.3 ± 4.2) mg to (62.1 ± 3.1) mg under illumination by green laser light for 1 min/d (P \ 0.01). (Fig. 3). To obtain information about the possible reasons for the increase in biomass when L. edodes was grown with green light illumination, the hyphal morphology was assessed. There was a large variation in hyphae diameters, as apparent from microscopic examination. However, quantitative morphometric analysis revealed that hyphae diameters did not differ significantly between cultures grown in the dark and those exposed to green light (Fig. 4). Hyphae diameters differed widely, from \1 to [7 lm; approx. 50 % of the hyphae displayed diameters from 3 to 4.9 lm. There were also no other apparent morphological differences between light and dark grown hyphae. (Fig. 4). Light induction of carotenoid biosynthesis has been described in many fungi and has received extensive attention in Neurospora crassa (Rodriguez-Romero et al. 2010). It was thus of interest to look at the influence of illumination on carotenoid content. In carotenoid extracts of hyphae, there was a single peak
at (440 ± 2) nm, corresponding to 41 ± 2 ng of carotenoid per mg of fresh mycelium; this value did not significantly vary between extracts from hyphae grown either in the dark or exposed to green light. Thus, there was no detectable light effect on carotenoid synthesis of L. edodes under the conditions used here. Taken together, we show here that illumination of L. edodes W4 with green light for only 1 min/d and at a relatively low intensity of 0.35 W/m2 markedly stimulated the growth yield. This observation is novel.
Discussion We here describe the stimulation of mycelial biomass formation by L. edodes W4 by short light exposure. The phenomenon is novel and striking, since exposure times as short as 1 min/d were sufficient to stimulate biomass production by 50–100 %. The most dramatic stimulation of biomass production was observed with green fluorescent light, amounting to over 100 %. Fluorescent lights are not ‘‘clean’’ light sources and have emission peaks outside the main spectral color, due to the emission lines of the mercury in the these light sources. LED, which provide for better defined emission spectra, also stimulated biomass production, but the effect was less dramatic. Best stimulation was observed with exposure to green LED illumination at 0.35 W/m2 for 1 min/d, resulting in a 1.6-fold increase in biomass production (P \ 0.01). Growth stimulation to a similar extent could also be observed with monochromatic (laser) light of 532 nm. This of course suggests that light sensory systems in L. edodes are active at the level of growth control. Conclusions about the action spectra cannot be drawn from our observations, but 532 nm is clearly within the range of the absorption spectrum of the responsible light sensing system(s). Among the well-known effects of light on fungi are stimulation of carotenoid biosynthesis, sporulation behavior, phototropism, and circadian clock control (Purschwitz et al. 2006; Tisch and Schmoll 2010). However, we could not observe macroscopic or microscopic changes in morphology; morphometric analysis of hyphal growth also did not reveal differences between mycelia grown in the dark or with intermittent illumination. The carotenoid content, was also not altered by illumination.
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In a study of the effect of illumination at different wavelengths on extra- and intra-cellular pigments and biomass production in Monascus purpureus, Isaria farinosa, Emericella nidulans, Fusarium verticillioides and Penicillium purpurogenum, (Velmurugan et al. 2010) did not observe stimulatory effects by light; to the contrary, most illumination schemes reduced biomass and pigment substantially. However, these authors used continuous illumination, which we found decreased the growth of L. edodes (not shown). This contrasts with results obtained by Ramirez et al. (2010) using continuous LED illumination of Phanerochaete chrysosporium at different wavelengths. They found that green LED illumination increased lignin peroxidase production by 20–27 %, while biomass production varied from 23 to -11 %, with no apparent pattern. Continuous or extended illumination was employed in most previous studies and there are essentially no studies available that describe the effects of short light exposure, e.g. 1 min/d. A short light exposure of 5 min did, however, induce conidiation in Trichoderma atroviride, and two blue-light sensors, blue-light regulators 1 and 2 (blr-1 and blr-2), similar to the Neurospora crassa white-collar 1 and 2, were identified (Casas-Flores et al. 2004). As discussed by these authors, many effects of light on fungi may in fact have been missed by using long-time light exposure, which often inhibits growth and metabolic processes rather globally. What causes the remarkable growth stimulation observed in the present study remains unknown. Light reception by fungi is complex involving different chromophores like flavin, retinal, or biliverdin, and a range of associated proteins (Heintzen 2012; Rodriguez-Romero et al. 2010). Conceivably, the short light pulses activate a developmental switch, which is maintained for prolonged times by the cells. Molecular studies on the mechanism of the light response of L. edodes will have to await the release its genome sequence. However, the light-stimulation of biomass production described here should be of immediate interest for biotechnological applications. Acknowledgments This work was supported by a Russian Federation Government grant to leading scientists (contract number 14.Z50.31.0011).
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