development of a growth model system for agaricus bisporus
TRANSCRIPT
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Wageningen UR - Plant Research International - Plant Breeding Department
Development of a growth model system for
Agaricus bisporus
April-October 2010
Maya Hernando Calvo
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Title: Development of a growth model system for Agaricus bisporus. Author: Maya Hernando Calvo Supervisors: Dr. Johan Baars, Patrick Hendrickx Examiners: Dr. Johan Baars, Dr. Richard Visser Carried out at :Plant Breeding department. Plant Research International Wageningen University and Research Center
This thesis has been submitted
in partial fulfillment of the
requirements for the degree of
Master in Plant Science.
Wageningen, October 2010
Cover picture by Alba Castañeda Vera
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Table of Contents
LIST OF TABLES ......................................................................................................... 6
LIST OF FIGURES ....................................................................................................... 7
PREFACE .................................................................................................................... 8
SUMMARY .................................................................................................................10
1 GENERAL INTRODUCTION .................................................................................12
1.1 Objectives of the study .....................................................................................13
2 LITERATURE REVIEW ........................................................................................14
2.1 Mushroom cultivation system ...........................................................................14 2.1.1 Compost .................................................................................................... 14 2.1.2 Spawning/Inoculation ................................................................................... 15 2.1.3 Casing ....................................................................................................... 15 2.1.4 Harvesting .................................................................................................. 16
2.2 Influence of microbial biomass in A. bisporus ...................................................17
2.3 Production of mushrooms in axenic conditions ..................................................17
2.4 Alternatives to compost ....................................................................................18
2.5 Assessing fungal growth ...................................................................................20 2.5.1 Laccase ...................................................................................................... 21 2.5.2 Chitin ........................................................................................................ 21 2.5.3 Ergosterol .................................................................................................. 21
2.6 Nutrition of A. bisporus .....................................................................................22 2.6.1 Carbon ....................................................................................................... 22 2.6.2 Nitrogen ..................................................................................................... 24 2.6.3 Carbon:Nitrogen ratio .................................................................................. 26 2.6.4 Minerals ..................................................................................................... 26 2.6.5 Vitamins and growth factors .......................................................................... 26
2.7 Water ...............................................................................................................26
3 EXPERIMENTS ...................................................................................................28
3.1 Testing different carbon sources for Agaricus bisporus nutrition .......................28 3.1.1 Introduction ................................................................................................ 28 3.1.2 Materials and methods ................................................................................. 29 3.1.3 Results ...................................................................................................... 32 3.1.4 Conclusions ................................................................................................ 36
3.2 Use of Plackett Burman experimental design to evaluate different compounds of the defined media for cultivation of Agaricus bisporus on solid substrates. ...............38
3.2.1 Introduction ................................................................................................ 38 3.2.2 Materials and Methods .................................................................................. 39 3.2.3 Results ...................................................................................................... 40 3.2.4 Conclusions ................................................................................................ 42
3.3 Evaluating the effect of two different pH buffer agents on Agaricus bisporus. ...43
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3.3.1 Introduction ................................................................................................ 43 3.3.2 Materials and Methods .................................................................................. 44 3.3.3 Results ...................................................................................................... 45 3.3.4 Conclusions ................................................................................................ 47
3.4 Testing hydrogel as a solid substrate for Agaricus bisporus ..............................48 3.4.1 Introduction ................................................................................................ 48 3.4.2 Materials and methods ................................................................................. 48 3.4.3 Results ...................................................................................................... 49 3.4.4 Conclusions ................................................................................................ 49
3.5 Testing a new hydroponic method for Agaricus bisporus cultivation suitable for nutritional investigations ..........................................................................................50
3.5.1 Introduction ................................................................................................ 50 3.5.2 Materials and methods ................................................................................. 51 3.5.3 Results ...................................................................................................... 53 3.5.4 Conclusion .................................................................................................. 55
4 GENERAL CONCLUSION AND RECOMMENDATIONS FOR FURTHER RESEARCH .....56
5 REFERENCES .....................................................................................................58
APPENDIX I. COMPOSITION OF THE DEFINED MEDIA USED FOR THE EXPERIMENTS
TESTING DIFFERENT CARBON SOURCES. ...................................................................62
APPENDIX CALIBRATION FOR THE CONVERSION RATE AREA ERGOSTEROL TO DRIED FRUIT BODIES BIOMASS. ..........................................................................................64
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List of Tables
Table 1Estimated raw materials needed depending on the formula. Source: (Van Griensven,
L.J.L.D. 1988) ....................................................................................................... 14
Table 2 Summary of the sequence of activities in the commercial mushroom production Source:
(J.J.P. Baars 1996) ................................................................................................. 16
Table 3 Substrate used for mushroom cultivation as alternative to compost by different authors.
........................................................................................................................... 18
Table 4 Carrier and Media used for mushroom cultivation by different authors ...................... 20
Table 5 Values are expressed as percentages compared to growth on glucose. Source: Modified
from Flegg, Spencer and Wood, 1985. ....................................................................... 23
Table 6Concentration of carbon source per treatment ........................................................ 30
Table 7 Concentration per treatment (mM) depending on the type of monomer utilized. Amount
adjusted to obtain the same concentration of C for both types of monomers ................... 31
Table 8 Solute potential (Ψw
) per treatment depending on the type of monomer, hexose or
pentose. ............................................................................................................... 31
Table 9 Concentration of sugars per solution .................................................................... 32
Table 10 Average dry weight of mycelium growing in different carbon sources. Averages over
five replicates. ....................................................................................................... 33
Table 11 Average mycelium dry weight for two different growing periods (3 and 6 weeks).
Average over 6 concentrations. ................................................................................ 35
Table 12 Dry weight of mycelium (mg) produced in 21 days for two types of carbon sources,
monomers and polymers, in three different concentrations. Averages over five types of
monomers in X replicates, and four types of polymers. ................................................ 36
Table 13 Distribution of the high and low concentrations according with the Pluckett-Burman
experimental design. .............................................................................................. 39
Table 14 Plackett-Burman design of the experiment showing the combinations of nutrients
under study........................................................................................................... 41
Table 15 Main effects on A. bisporus mycelium growth according with the Plucket-Burman
experiment design.................................................................................................. 42
Table 16 Characteristics of the buffers used in the experiment. ........................................... 44
Table 17 Composition of the nutrient solution ................................................................... 45
Table 18 Water content of different horticulture substrates ................................................ 48
Table 19. Results of the ergosterol assay. Average over 2 samples ...................................... 54
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List of Figures
Figure 1 Yields on monomeric nitrogen sources. Yield on asparagine (181± 60 mg) was set as
100%. Source: (Johan J. P. Baars et al. 1994) ........................................................... 25
Figure 2 Petry dishes with 21days old colonies showing the promoting growing effect of the
addition of compost extract. .................................................................................... 33
Figure 3. Above, average of mycelium dry weight for different sugars in six different
concentrations after three weeks. Below, average of mycelium dry weight for different
sugars in six different concentrations after six weeks. .................................................. 34
Figure 4 Weight of dry mycelium produced in 21 days with three different concentration of
sugars. Averages over three replicates*. Bars showing LSD (p<0.05). ........................... 35
Figure 5; Example of a nutrients solution used in mushroom nutrition research. .................... 38
Figure 6 Pictures of the colony development under different treatments according with the
Pluckett-Burman experiment design. ......................................................................... 41
Figure 7 Effect of buffer concentration on colony diameter. Increasing buffer concentrations from
left to right ........................................................................................................... 45
Figure 8. Effect of the buffer concentration on colony diameter and on pH of the media. ......... 46
Figure 9 Schema of soaking for the different treatments. ................................................... 52
Figure 10. Example of the growing container and the soaking technique ................... 52
Figure 11 Malformed mushroom ..................................................................................... 53
Figure 12 Changes in the percentage of water content in time for three different submersion
frequencies. .......................................................................................................... 54
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Preface
From the moment I decided to write my Master Thesis on Mushroom production I knew it was a
way to finish an unresolved matter. As granddaughter and daughter of mushroom growers, as
agronomist and as a scientist I was obligated to go in deep into mushroom science, at least
once. Besides that, I had the curiosity to study the science behind the practice, to which I am so
familiar with, but also to get in contact with the science that will change the practice. I realized
that six months is not enough time to reveal all the secrets of this field but thanks to this
opportunity I discovered an interesting topic of study full of challenges and opportunities.
During these 6 months in PRI I enjoyed the skilled training and help of Patrick Hendrickx and
the reviews and comments of Johan Baars. I benefited also from the critical observations of
Anton Sonnenberg. I am very grateful to you all for guiding me during this research.
I must express my gratitude to my friend Alba, for her original and always helpful and
encouraging comments. Also my boyfriend Coen deserves my recognition for his support during
this thesis and during my stay in The Netherlands. And in general; to all those who made from
my two years at WUR such a highly enriching experience: Thank you!.
However nothing of this would have been possible without the confidence and support of my
family who believes in me more than I do myself.
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Summary
Compost is the conventional substrate used for the production of the white common mushroom
(Agaricus bisporus (J.E. Lange) Imbach). However compost is a problematic substrate due
mainly to its environmental impact (odors and limitations in disposal). Consequently it is
advisable to decrease the amount of compost used, by increasing the efficiency of the current
substrate or by replace it for another substrate. To achieve that it is necessary to know the
nutritional needs of A.bisporus. The complexity of compost and the difficulty to obtain
mushrooms under laboratory conditions difficult nutritional research. For that, new production
systems have to be designed that allow us to study the mushroom nutrition at fructification
phase. This thesis is part of the project System Innovation in the Production of Button
Mushrooms which aims to development a growing system in which mushroom production takes
place in an inert carrier with nutrients supplied by a chemically defined media.
In this thesis we have studied several factors which need to be optimized prior to develop an
hydroponic system for A. bisporus. Those factors were: formulation of the nutrients media, pH
control and design of the production system.
The defined nutrient media has to be optimized to produce enough mycelium biomass to sustain
a significant production of mushrooms. Different carbon sources in different concentrations were
tested. We have shown that complex sugars, such as guar gum produce significant more
biomass that simple sugars. It was also observed that monosaccharides perform remarkably
better when mixed. Those findings can be applied to formulate supplements to improve compost
productivity.
A buffer agent has to be included in the nutrient media to regulate pH. We evaluated the effect
on mycelium of three different buffer agents (BisTris-HCL, BisTris-Citric acid and MES-KOH).
MES-KOH resulted the buffer with less inhibitory effect on growth.
An new hydroponic design was tested. The inert carrier was submerged in the nutrients media
periodically. However the aerial mycelium was negatively affected by the contact with the liquid,
delaying the colonization of the carrier and limiting fructification. More work is required to
optimize the cultivation system. Further research must be focused on how to manipulate the
nutrition during the cropping cycle without wetting the mycelium.
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1 General Introduction
The white button mushroom, Agaricus bisporus (J.E. Lange) Imbach., is worldwide cultivated
and consumed. The Netherlands is one of the main producers and exporters of mushrooms in
Europe. The Dutch mushroom sector is characterized by high-tech growing systems and
innovative solutions. Its productivity is among the highest in Europe and it is a reference point
for the global mushroom growing industry.
Currently mushroom production is carried out in compost which is a pasteurized mixture of
manure, straw and gypsum which shows a high selectivity for mushroom growth. Compost
technology has advanced enormously in the last decades increasing yields and efficiency.
However this substrate is not exempt from inconveniences:
- Price: Compost is made out of sub-products from other agriculture activities with relatively
affordable raw material prices. However the labour and time required for its production and
transport costs make the compost an expensive substrate. Currently compost represents about
40% of the total production cost in mushroom production in the Netherlands (Johan Baars,
personal communication).
- Disposal: Once compost is not longer viable it has to be disposed in special dumping sites
according with the regulations of animal manure. It can also be sold as soil amendment but it
requires first a transformation process which may take between 9 months to 3 years, which
increases production cost.
- Animal diseases: Compost is mainly made out of animal manure. Manure availability, and
consequently compost availability, is highly sensible to restrictions due to animal disease
outbreaks.
- Efficiency: Compost is renewed when the production is not longer viable (2-3 harvests) despite
the fact that the nutrients in it are not totally exhausted. It is calculated that 35% of the
nutrients in compost are not being used during the mushroom production.
- Renovation: Renew of compost is usually made every four weeks. This compulsory break
interrupts the production cycle, decreases efficiency and increases labour costs.
- Odors: Compost generates unpleasant odors during the composting process and when it is
discarded as spent substrate. As a result, the presence of compost manufacturers and
mushroom farms close to urban areas is heavily restricted.
These problems linked to compost production, utilization and dispose have encouraged the
search for alternatives. Other substrate formulas for A.bisporus have been previously studied
(Block et al. 1958)(Sánchez & D.J. Royse 2001)(Bechara et al. 2006b). Lower yields,
contamination problems and elevated adaptation costs hamper the conversion to this ‘free-
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compost’ systems. It can be said that at the moment there is not an economically viable
alternative to the traditional manure-straw base compost for A. bisporus.
Therefore, improving efficiency in compost utilization is a key factor to increase productivity and
decrease production cost. Compost is a complex matrix difficult to study and analyze. It is
necessary to perform research on the nutritional requirements of A. bisporus to understand
which factors are limiting and how compost can be improved. The nutritional needs at the
mycelium stage have been broadly investigated but the needs at fructification remain unknown
due to the difficulty of obtaining mushrooms in laboratory conditions.
This thesis is part of the project “System Innovation in the Production of Button Mushrooms”
funded by the Dutch Ministry of Agriculture. One of the objective of the project is to design a
nutritional model for A. bisporus. The initial step is to design a growing system for A.bisporus
suitable for nutritional studies in which the fungus is able to grow and produce fruit bodies. The
current approach aims for a hydroponic system with an inert carrier as physical support where
nutrition is provided by a defined media. Nutrition investigations are then possible by variations
in the media.
1.1 Objectives of the study
- Evaluate the mycelium growth in several nutrient solutions suitable for mushroom production
under hydroponic conditions. Determine, with the results, the defined media for subsequent
treatments.
- Evaluate the mycelium growth in different inert carriers suitable for mushroom production
under hydroponic conditions. Results will determine the inert carrier to be used in the second
phase.
- Evaluate different pH control measures to be applied in mushroom production under
hydroponic conditions.
- Evaluate the mycelium growth and the mushroom yield in a hydroponic system where the inert
substrate is soaked into the nutrient solution.
- Identification of weak points in the design and elaboration of a list of recommendations for
future studies.
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2 Literature review
2.1 Mushroom cultivation system
In order to develop a “compost free” growing system it is highly interesting to look at the
current system.
2.1.1 Compost
The substrate used for Agaricus bisporus commercial cultivation worldwide is mushroom
compost. This compost is a very complex substrate with unique chemical, physical and
microbiological properties highly selective for mushroom production. It is obtained as result of a
carefully controlled organic matter degradation process.
Mushroom compost formulation varies slightly depending on the availability of raw materials. In
the Netherlands, the main ingredients for compost preparation are: straw-rich horse manure ,
chicken manure, wheat straw, gypsum and water. There are two main formulas depending on
the availability of horse manure, horse manure compost and straw compost. Quantities of each
ingredient are modified depending on the characteristics of the raw material in order to adjust
compost formula and avoid deficiencies or excesses, especially in nitrogen content (Table 2.1).
Table 1Estimated raw materials needed depending on the formula. Source: (Van Griensven, L.J.L.D. 1988)
Horse manure
compost Straw compost
Horse manure* (kg) 1000 0
Straw (kg) 0 1000
Chicken manure (kg) 100 800
Gypsum (kg) 30 85
Water** (l) 300-900 5000
Compost resulting (kg) 900-1300 3000
* includes straw used as bedding material
** water required highly dependent on climate conditions
Moisture content is also an important factor in composting. The optimum moisture at the end of
the composting process is 71-74%, which corresponds to 65-69% at inoculation (Van Griensven,
L.J.L.D. 1988).
Moisture content also determines the quantity of air in the compost heap. Higher water content
implies less porosity of the compost heap which compromises the air renewal. Composting is an
aerobic process which relies on a wide range of microorganisms. During the decomposition
process CO2 is generated. If there is no aeration it may lead to anaerobic conditions which
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induces fermentation and restricts microbial activity. During the composting process the piles
are mixed and watered periodically to achieve a compost of uniform and good quality.
An important step in mushroom compost preparation is pasteurization. Mushroom compost is
pasteurized to destroy undesirable organisms and to create a substrate with the smallest
number of competitors as possible for the mushroom. Mushroom compost is inserted in
pasteurization tunnels where the temperature in compost is maintained at 45ºC for 4-5 days
(Van Griensven, L.J.L.D. 1988). The heat treatment reduces the amount of mesophilic
microorganism in the substrate. Thermophilic microfauna, although able to survive the heating
process, will be deactivated when the temperature drops to cultivation values (22-24ºC). The
compost is pasteurized not sterilized since the presence of certain microorganism in compost is
essential. Mycelium grows about twice as fast in compost than it does in any other substrate,
however if the compost is sterilized, growth rate is halved (Van Griensven, L.J.L.D. 1988). The
growth stimulation effect by the microflora is not fully understood.
2.1.2 Spawning/Inoculation
Compost is inoculated with the fungus by adding mushroom spawn. Spawn is a organic matrix
colonized with the mushroom fungus. The most widely used inoculum carrier is grain (rye, millet
or wheat). The grain is sterilized and inoculated with a pure culture of the fungus. Some other
substances such as calcium carbonate and calcium sulfate are added to regulate pH and to
prevent stickiness. The grain is incubated in sterile conditions until it is fully colonized.
Spawn is incorporated into the substrate right after the composting phase is finished. If
spawning is delayed it increases the risk of appearance of competitive fungi. The amount of
grain spawn added to the substrate varies widely. Spawn applications of 2,5%-4% of the weight
substrate are commercially used (Bechara 2007)., although it depends on the grain used. The
grain serves as temporal source of nutrients until the mycelium is adapted to the new
environment.
After spawning, the temperature in the compost has to be maintained between 24-27ºC and a
relative humidity of 90-95% for about 14 days to obtain a successful compost colonization.
(J.J.P. Baars 1996).
2.1.3 Casing
One unique fact of mushroom cultivation is the requirement of a layer of soil covering the
compost called casing layer. In the Netherlands the most commonly used casing soil is a
mixture of peat (80%) and spent lime (20%) with a moisture content of 60-65%. It is placed on
the compost once this is colonized, approximately 2 weeks after spawning. The casing soil will
promote the formation of mycelium aggregates which will develop as fruiting bodies. Uncased
compost produces hardly any mushrooms (Van Griensven, L.J.L.D. 1988).
Besides the properties of casing soil to promote sporophore initiation it is also necessary to
protect compost from desiccation and against pest and diseases. One third of the water in
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mushrooms comes from the casing soil (Kalberer 1987). From the casing to the harvest of the
first fruit bodies takes usually about 18 to 21 days (Flegg, P. B.; Spencer, D. M.; Wood, D. A.
1985) depending on the cultivation conditions.
How casing soil promotes A. bisporus fructification is one of the big mysteries in mushroom
production. It has been proven that sterile casing soil is not effective, so fructification is directly
related with living biomass in the casing soil. However, sterile casing soil with addition of
activated carbon promotes fructification as non sterile casing soil does (Long & Jacobs 1974).
Activated carbon is a charcoal product which is widely used for gas adsorption. Consequently It
has been suggested that the biomass in the casing soil removes volatile metabolites which
inhibit fructification.
From the time of casing, growing rooms require a reduction of the temperature and an
increasing in the ventilation. This is essential to obtain fruit bodies, since it has been prove4 that
certain volatile metabolites and high CO2 concentrations inhibit fructification (D. A. Wood 1976a).
2.1.4 Harvesting
Mycelium will colonize the casing soil and it will form hyphae aggregates, called pins. The first
mushrooms can be harvested one week after pinning.
Typically from the same compost three harvests or flushes are picked. The time between two
consequent flushes is approximately 8 days. The first flush usually provides the highest yield,
40% of the total, and the best quality product. The decrease in yield between successive flushes
may be due to the depletion of certain nutrient or to the accumulation of toxic metabolites in the
substrate.
Table 2 Summary of the sequence of activities in the commercial mushroom production Source: (J.J.P. Baars 1996)
Time table Activities
Preparation of compost
day 1 to 21 Phase I composting; easily degradable substrate are consumed
day 22 to 32 Phase II composting; pasteurization of the compost (6 hrs at 56-60 C) and conditioning
Spawning and colonization of compost
day 32 to 46 Introduction into the compost and colonization of the compost by A. bisporus at 24-27 C
Casing and development of fruit bodies
day 46 to 56 Application of the casing layer and subsequent colonization by A. bisporus
day 56 to 63/66 Initiation of fruit body development, growth of fruit bodies and picking of the first flush.
day 63/66 to 71/77 Development and picking of the second flush.
day 71/77 to 80/86 Development and picking of the third flush.
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2.2 Influence of microbial biomass in A. bisporus
A. bisporus has a close relation with the microbial biomass in compost. It has been reported that
A. bisporus is able to feed on dead microbial cells found in compost (Flegg, P. B.; Spencer, D. M.;
Wood, D. A. 1985). Besides that, it has also been observed an association with the living
microbial biomass remaining in the substrate (Straatsma et al. 1989). Scytalidium
thermophilum is a thermophilic fungi commonly found in mushroom compost. It multiplies
rapidly during the pasteurization and conditioning phase of composting and survives when the
temperature drops. Bechara (2006a) proved that although S.thermophilum is a thermophilic
fungus, it is able to grow at mushroom cultivation temperatures (19-24ºC). As a result of its
metabolisms S. Thermophilum produces carbon dioxide, degrades complex molecules and
secretes growth promoting compounds to the substrate. It also stimulates mycelium growth in
sterile compost (Straatsma et al. 1989). The growth promoting effect of other fungi, mesophilic
and thermophilic, habitually present in compost has been also researched. However S.
thermophilum resulted the most effective of them (Straatsma & Samson 1993).
The response of S.thermophilum varies depending on the substrate. The growth stimulation
effect of S.thermophilum is highly significant on sterile compost but in grain based substrates
the response is less marked and depends on the type of grain. Oat grain based substrate
responded more favorably to the addition of S.thermophilum than rye or millet grain
formulations (Bechara 2007).
How this fungus stimulates A. bisporus growth is unclear. Some theories point at the fact that
the biomass of the fungi can function as a concentrated source of nitrogen, phosphorus and
minerals. Other reason might be that S.thermophilum secretes enzymes which promote the
degradation of complex molecules increasing the amount of nutrients available for A. bisporus.
Other hypothesis point at the influence of S.thermophilum in gas exchange. A combination of all
the hypothesis seems the most likely explanation (Wiegant et al. 1992).
2.3 Production of mushrooms in axenic conditions
Most strains of A. bisporus do not produce fruiting bodies in agar media (Long & Jacobs 1974).
The development of hypha aggregates is stimulated by i) changes in the environment like the
reduction in temperature and in CO2 concentration and ii) by the presence of a casing layer. The
casing layer has specific microbiological, physical and chemical properties to induce fructification.
The bacteria Pseudomonas putida is present in casing layer and it is the responsible of inducing
fructification (Long & Jacobs 1974). Besides this microbiological component for a successful
fructification an appropriate density, texture and humidity of the casing layer is essential. All
these specific requirements, among many others, move cultivation of A. bisporus away from
traditional laboratory conditions and practices.
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2.4 Alternatives to compost
As stated before, mushroom compost entails a wide range of problems. Looking for an
alternative substrate has been the research topic for many scholars. As a result there are
several studies reporting A. bisporus production in other substrates than compost. There are
two main approaches: experiments looking for a formulation of solid nutrients (Table 3), and
experiments in which an inert support material is impregnated with a defined nutrients media
(Table 4).
Table 3 Substrate used for mushroom cultivation as alternative to compost by different authors.
One of the first studies on this topic was made by Block (1958). His alternative to conventional
manure compost was a composted mixture of sawdust, hay and chicken manure. The yields
although acceptable in weight basis (kg mushroom/kg substrate) if compared with compost,
were lower in area basis (kg mushroom / m2 surface). Till in 1962 decided to eliminate the
composting process in mushroom substrate production. In his experiment, straw, peat, calcium
carbonate, cottonseed meal and soya bean were mixed, watered and sterilized. Mycelium
growth was significantly slower but the yields were higher. However the high operational cost
made it not viable on commercial scale. In 1971 James P. San Antonio produced mushrooms in
aseptically colonized rye grain covered afterwards with a non-aseptic layer of casing soil. He
concluded about his method that was a “reliable mean to obtain fruit of the cultivated
mushroom in the laboratory” however he still showed preference for “Till substrate”.
Other types of mushroom such as Shiitake (Lentinula edodes), are commercially grown on
pasteurized grain or sawdust-based substrates. There have been some attempts to adapt those
substrates to mushroom production. In 2001 Sanchez and Royse cultivated A. bisporus brown
form (Portabella) in shiitake substrate (a non composted substrate base on oak sawdust). They
obtained yields comparable with those obtained at commercial scale for this strain. Some years
Author/s Substrate
(Block et al. 1958) Gum wood sawdust, hay straw and chicken manure (composted)
(Till 1962) Chopped straw, ground straw, white peat, calcium carbonate,
cottonseed meal and soya bean meal (non composted).
(San Antonio 1971) Rye grain and calcium carbonate
(Sánchez & D.J. Royse
2001)
Oak sawdust, millet, rye, peat, alfalfa meal, soybean flour, wheat
bran, and calcium carbonate (non composted)
(Bechara et al. 2006a) Millet grain and oilseeds (niger, safflower, and soybean)
(Mamiro et al. 2007) 50% spent mushroom compost 50% non composted substrate
from (Sánchez & D.J. Royse 2001)
(Jose E. Sanchez et al.
2008)
Pangola grass, sorghum grains, sawdust, corncobs, hydrated lime,
pulverized limestone and CaSO4
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later Bechara experimented with different non composted grain based substrates. He mixed
grain (millet or rye) with perlite obtaining once again acceptable yields comparable with
compost systems. Millet grain resulted to be a better substrate than rye grain and the optimal
combination was 75/25 (grain/perlite).
One of the main problems mushroom farms have to this face up is the disposal of many tonnes
of spent mushroom compost (SMC). Mamiro in 2007 tried to find a way to reuse SMC. She
mixed Shiitake substrate with SMC in ratio 1:1 and supplemented with a mineral preparation
specific for mushroom growing. The system produced reasonably good yields of the brown strain
of A. bisporus from 50-100% compared to conventional systems.
Another group of studies looking for alternatives to compost is directed to design a system in
which the physical support is inert and the nutrients are added to that support usually as a
liquid (Table 4).
The first attempt was made on 1972 by Smith and Hayes. They soaked an inert carrier of
vermiculite and pumice stone with a medium of malt and straw extract. The results were not
fully satisfactory. They tried an inert carrier of sphagnum peat mixed with solid nutrients such
as calcium, cellulose or soya meal soaked with cereal extract. The yields were significantly
higher than with the liquid media. Wood on 1976 measured the primordial formed in an axenic
culture made out of cellulose, glucose, xylan and other salts with compost extract using
vermiculite as carrier.
There are two previous studies reporting A.bisporus production under hydroponic conditions in
which all the nutrients are supplied by a chemically defined liquid media which is periodically
renewed. Firstly, Aksy and Gunay in 1999 designed a system in which mushroom were grown in
perlite and volcanic tuff. Nutrients were added by a defined medium consisting of sucrose (2%),
ammonium phosphate and potassium nitrate among other ingredients. The medium was
pumped into the containers in a close cycle circuit. They claimed to have reached “commercially
viable” yields. To research on the carbohydrate metabolism of A. bisporus Hammond and
Nichols designed a growing system with vermiculite as carrier impregnated with a solution made
of straw, soya flour, glucose and water. The efficiency of this system is unknown because
evaluating mushroom yields was outside of the scope of this experiment.
In 2006, Bechara studied three different hydroponic systems, open cycle, close cycle and non
circulating (Bechara et al. 2006a). The inert carrier used was perlite and once the mycelia fully
colonized the perlite, a sterile casing layer was overlain over the perlite. Two carbon sources
were tested ,sucrose and dextrin. The dextrin solution performed significantly better than the
sucrose solution. Even though the substrate, containers and other inputs were sterilized, these
experiments were heavily affected by contaminants from the starting phase. He made use of
antimicrobial compounds (Topsin and tetracycline) which decreased the growth of contaminants
only in the dextrin based treatments.
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Table 4 Carrier and Media used for mushroom cultivation by different authors
Author/s Carrier Media
(Smith & Hayes 1972) Vermiculite and pumice stone Malt extract, straw extract
(Smith & Hayes 1972) Partially inert sphagnum peat
Solids (2g calcium, 10gr cellulose,
3,2 casein / soya meal and cereal
straw)
(D. A. Wood 1976b) Vermiculite
Cellulose, Glucose, Xylan, Peptone,
basal salts and aqueous mushroom
compost extract
(Hammond & Nichols
1976)
Vermiculite
or Chalk/peat mixture Straw, soya flour, glucose, water
(Aksu & Günay 1999) Volcanic tuff and perlite
Sucrose, ammonium phosphate
and potassium nitrate + essential
minerals
(Bechara et al. 2006a) Perlite Sucrose and dextrin
2.5 Assessing fungal growth
In order to evaluate the efficacy of a nutrient solution or a growing system it is required that
reliable measurements of the mycelium biomass are generated. In liquid cultures this is possible
by drying and weighing the mycelium produced. However when the mycelium is growing in solid
media, biomass measurements get difficult. Once the substrate is colonized it is not possible to
separate substrate and mycelium.
The efficiency of different growing systems is traditionally assessed by the mushroom yield per
kilogram of substrate. However, several studies consider it more reliable to look at the biological
efficiency BE = fresh weight of mushrooms/ dry weight substrate (BE=fw/dw) or dry weight
mushrooms/dry weight of substrate (BE = dw/dw.). For the white button mushroom the
average BE (fw/dw) is 50-70% while BEs of 70-90% are considered very good (Sánchez & D.J.
Royse 2001).
In the experiments with alternatives substrates commented before the ranges of BE (fw/dw)
vary considerable. With A. bisporus growing in Shiitake substrate Sanchez and Royse (2001)
obtained a BE of 77%. The experiment by Sanchez (2008) with Pangola grass he got 48.1%,
Bechara got the highest BE with the non circulating hydroponic system, 168% It is clear that
this parameter favors the hydroponic systems since the dry weight of the substrate its
significantly lower than with organic carriers.
In experiments only looking at the vegetative phase, growth of mushroom mycelium in solid
substrates has been traditionally assessed by observation methods. Measurements such as the
diameter of a colony growing in a petri dish or the distance covered by the hyphae front in a
21
given substrate growing in a glass tube (Flegg, P. B.; Spencer, D. M.; Wood, D. A. 1985). These
techniques provide useful comparative data, but they are not reliable since they do not consider
the density of mycelial growth.
To get more accurate data some indirect methods to measure the biomass produced were
developed, such as determination of the CO2 generated or the nutrients consumed. These
indirect methods are not specific for fungal biomass so the results may be affected for the
presence of contaminants. Currently biochemical methods are being applied for the assessing of
the mycelium growth. The most important methods are Laccase, Chitin and Ergosterol
measurement. The first one is based on estimation of the enzymes secreted by the fungi and
the other two are based on measurements of specific chemical compounds exclusive from fungi
cells, such as Ergosterol or Chitin (Matcham et al. 1985a). It is important to notice that none of
the current methods for measuring fungal biomass is able to differentiate between A. bisporus
and contaminant fungi, specially if it has similar features and metabolism than the mushroom
does.
2.5.1 Laccase
This biochemical method is based on the relation between Laccase, one of the enzymes secreted
by A. bisporus for its nutrition, and dry weight of mushroom mycelium. This analytical method
was the first indirect technique based on measuring compounds exclusively generated by fungi
(Dijkstra et al. 1972). The big advantage of this method is that the extraction and assay are
easy to perform.
Currently the reliability of this method is under discussion, since the levels of laccase, as
commented before, decrease significantly at the start of the fructification phase. Besides, when
looking at samples in vegetative phase the results may not be accurate either, since proteases
may be secreted during mycelium senescence and degrade the laccase (Matcham et al. 1985b).
2.5.2 Chitin
The aminoglucan chitin is the main component of the fungal cell walls and in the exoskeleton of
arthropods. Fungal chitin is linked to other polysaccharides which do not occur in the arthropods
(Peter 2005).
Chitin essay is one of the most popular indirect methods to estimate fungal biomass. The main
criticism towards this method is the fact that the chitin content of mycelium varies with the age
(Sharma et al. 1977) . Furthermore, this method cannot be use to compare mycelium growing
in different media without calibration (Roche et al. 1993), since the substrates may influence
the outcome significantly. Fungal chitin determination is a laborious technique and the precision
is highly dependent on the conversion factors which relate the chitin content with the mycelial
biomass.
2.5.3 Ergosterol
22
Ergosterol is a membrane lipid found almost exclusively in fungi. Consequently relating
ergosterol content with fungi biomass is a highly specific method for fungi. This technique was
developed to measure fungal contaminants in grains (Seitz et al. 1977). Later, it has been
adapted for the determination of fungal biomass in many substrates, from plant material to
indoor environment. In the first stages it was only used to assess living fungal biomass since
ergosterol is prone to degradation once the hyphae are dead. However it has been proven
(Mille-Lindblom et al. 2004) that samples if kept refrigerated can be analyzed months after
sampling.
In the Ergosterol assay it is required that a correlation between ergosterol content and fungal
biomass is determined for each of the species and for each of the substrates tested. So in every
analysis a reference has to be included to calculate the conversion factor. Besides that the
ergosterol content may vary during different fungal growth stages (Niemenmaa et al. 2008),
which is requires another conversion factor to be included. Furthermore, sterols endogenous to
rye grain have similar gas chromatography retention times as those from fungi (Matcham et al.
1985a). Consequently, the current ergosterol technique is not applicable to growing medium
inoculated with rye grain spawn, or in case no grain should be included in the sample.
Overall the ergosterol assay has been proven to be a sensitive and reliable indicator for fungal
growth. Therefore it will be the method we use for biomass measurement in solid substrates
during the course of this thesis.
2.6 Nutrition of A. bisporus
A. bisporus is a heterotrophic organism; therefore all the nutrients must be supplied
by the substrate or the growing media. The growth requirements of A.bisporus are
satisfied by the following four classes of chemical compounds (D.A. Wood, T.R.Fermor 1985).
2.6.1 Carbon
All fungi depend on organic carbon. Carbon is the qualitative an quantitative most important
nutritional element. A. bisporus is a saprophytic fungus so its sources of carbon are the
decomposed remains of living organism. It can be in form of small molecules such as proteins or
nuclei acids or in the form of structural materials, such as cellulose or lignin. Fungi have
efficient enzymatic systems for degrading complex molecules. However the more complex the
molecule becomes, more difficult is to degrade it and more energy is required.
Nevertheless fungi are capable of adjusting their metabolism. They prefer easily metabolizable
carbon sources over less readily metabolized carbon sources (Wannet 1999). This preference for
easily metabolizable carbon sources is known as glucose repression. This mechanism represses
the enzymes required for the degradation of less favorable carbon compounds, resulting in the
preferential utilization of glucose (Griffin 1996) . It is an energy saving response, since no
energy is spent on the synthesis of other catabolic enzymatic systems. This effect has been
studied on some filamentous fungi such as Aspergillus nidulans (Flipphi et al. 2003), (Bailey &
Arst 1975) or Trichoderma (Strauss et al. 1995)
23
Carbon requirements during the vegetative phase differ from the requirements at fructification.
This has been proven by measuring the concentration of several enzymes secreted by the
mycelium growing in compost (D. A. Wood & Goodenough 1977). Laccase concentration,
enzyme related with the degradation of lignin, increases during mycelial growth and then
declines rapidly at the start of the fructification phase.In contrast cellulase activity increases
significantly at fruiting. It has been suggest that this is a two step process. In the vegetative
phase the need for carbohydrates is lower and more constant. During that process the lignin,
which protects the cellulose fibers, is degraded. At fructification there is a need to obtain high
levels of mono and disaccharides for the fast mushroom construction. Then the cellulase activity
increases and degrades the exposed cellulose fibers (D. A. Wood & Goodenough 1977).
Some previous studies have been carried out to examine carbon nutrition of Agaricus bisporus
in the vegetative phase (Styer 1928)(Treschow 1944), (Humfeld 1948), (Dijkstra et al. 1972),
(D. A. Wood 1976a) (table 5). Although the methods differed remarkably among the studies
(different growing conditions, nutrients media, strain, C:N and pH) these experiments provided
some guidelines on A.bisporus carbon nutrition. The general pattern is that monosaccharides
(glucose, fructose, xylose, malate) are better carbon sources than disaccharides. While glucose
appears as a good carbon sources in the five studies it is not in all cases the best. Xylose and
fructose appeared as better options for Treschow and Dijkstra respectively.
Table 5 Values are expressed as percentages compared to growth on glucose. Source: Modified from Flegg, Spencer and Wood, 1985.
Styer, 1930
Treschow, 1944
Wood, 1976
Manning and
Wood, 1983
Dijkstra, 1972
Glucose Dense 100 100 100 100
Fructose 95 83 78 134
Galactose Medium 73 43 26 40
Xylose Dense 132 76 24 72
Arabinose Medium 33 22
Mannitol None 20 93 18 38
Mannose 39
Trehalose 24 83
Glycerol 7
Maltose Dense 73 43 56 71
Sucrose 68 33 79 29
Cellobiose 53 38
Lactose 18 53
Oxalate 100
Malate 116
Tartrate 32
Soluble starch 62
Succinate Sparse
24
In contrast, mushroom compost which is an excellent substrate, is lacking on those simple
sugars and carbohydrates are bound in complex insoluble polymers. As pointed out before,
A.bisporus is capable of excreting the enzymes necessary for degrading those polymers.
Although that requires energy it gives the fungi a competitive advantage over other
microorganism which are not able to feed on complex polymers. Consequently, the lack of
monosaccharides in compost makes it a selective substrate for fungi growth.
Concentration
The early studies of Treschow (1944) devised an optimum concentration for growth on glucose
between 0.02M and 0.1M. The ratio between carbon and the other nutrients was also studied by
Treschow who observed that with increasing amounts of Calcium the mycelium is able to
tolerate increasing amounts of other nutrients. One important ratio is C/N ratio which will be
discussed later (section 2.3.1) . In following studies the concentration of glucose in the synthetic
media was raised. Dijkstra used 0.17M for his comparative studies. It is important to point out
that in some of these studies compounds such as casein (Dijkstra et al. 1972) were used as
nitrogen source. Casein is a protein which can be degraded and used as carbon source by the
fungi (Kalisz et al. 1986)
Treschow (1944) reported a strong inhibitory effect in mycelium growth with sugar
concentration above 150. How this inhibitory effect is regulated is unclear. It can be a
consequence of high osmotic pressure, which increases above tolerable levels the water
potential. Or it can be that the metabolism is overloaded by the elevated presence of easily
degradable carbohydrates. This effect has been reported in yeast and it is known as Crabtree
effect (Crabtree 1928). In Saccharomyces cerevisiae a slowing in the respiration occurs at high
glucose concentration. Glucose is quickly metabolized by glycolysis and the NADH generated is
oxidized by fermentation rather than by respiration, even under aerobic conditions. As results of
this fermentation yeast produces alcohol aerobically (Griffin 1996). Whether the Crabtree effect
also appears in other types of fungi is still under discussion.
Carbon nutrition and mycelium morphology
Carbon is the main nutrient for fungi. The availability or scarcity of it determines its growth rate
and development. Besides it also determines its morphology. A rich and complex medium
reduces the length of the hyphae growth and increases branch frequency resulting in a dense
colony. A fast degradable medium will result in longer hyphae creating a bigger but sparser
colony (Gow & Gadd 1994). Due to that, colony diameter measurement is not an accurate
method for assessing fungal growth when testing different carbon sources.
2.6.2 Nitrogen
The study of A. bisporus nitrogen requirements has received less attention than carbon
requirements.
25
Treschow (1944) in his early studies on nutrition of the cultivated mushroom tested several
nitrogen sources. The first finding was the fact that NO3 cannot be utilized by A. bisporus. He
reported also a significant drop in the pH in the medium when using ammonium salts i.e.
(NH4) 2SO and NH4NO3. Consequently growth yields on ammonia salts depend on the buffering
capacity of the medium
In later studies with complex substances it has been demonstrated that proteins such as casein
are efficiently utilized by the white button mushroom as sole carbon and nitrogen source (Kalisz
et al. 1986). Baars (1994) tested different monomeric nitrogen sources, the results are
summarize in figure 1. Good growth was obtained with the amino acids asparagine and
glutamine. However their transaminated forms, aspartate and glutamate performed poorly.
0 20 40 60 80 100 120
Asparagine
Glutamine
Glycine
Alanine
Allantoin
Uric acid
Urea
Arginine
Glutamate
Aspartate
Glucosamine
Proline
Pheylalanine
Thymine
Cysteine
Nitrate
Control
Nitr
og
en S
ou
rce
Yield (%)
Figure 1 Yields on monomeric nitrogen sources. Yield on asparagine (181± 60 mg) was set as 100%. Source: (Johan J. P. Baars et al. 1994)
In compost, the nitrogen sources available for A. bisporus are protein or peptide nitrogen bound
to the lignin fraction and the microbial protein synthesized during composting process .
Besides, A. bisporus and other basidiomicetes are able to use microbial biomass as sole source
of both nitrogen and carbon (Fermor & D. A. Wood 1981).
Nevertheless it is clear that nitrogen has a direct relation with mushroom yield. The
supplementation of compost during cropping with nitrogen rich substances, such as soybean or
oilseeds, is a common practice by growers. The increase in yield due to these supplements is
significant, although only if is done before the second crop (D. J. Royse & J. E. Sanchez 2007) .
26
2.6.3 Carbon:Nitrogen ratio
The Carbon:Nitrogen (C:N) ratio of the growing media and the compost is a decisive factor for A.
bisporus nutrition. The optimal C:N ratio ranges from 5 to 20 (Dijkstra 1976) . High nitrogen
content (low C:N ratio) induces the production of free ammonia which is harmful for mycelium
growth. High C:N ratio has fewer consequences but implies that the shortage of nitrogen is
limiting fungi growth.
2.6.4 Minerals
There is a wide range of minerals required for mushroom nutrition. Their low concentration and
the interactions occurring between them make difficult to determine optimal doses. One of the
essential minerals is Phosphorus, since it is involved in many metabolic processes. But high
concentrations result harmful. Besides, Potassium, Sulphur, Magnesium, Calcium and Iron play
an essential role and should be included in the defined media (D.A. Wood, T.R.Fermor 1985).
2.6.5 Vitamins and growth factors
A.bisporus growing in defined media requires the addition of small concentrations (10-10 – 10-06
M) of biotin (B7) and thiamine (B1) for growth. In compost, those requirements are fulfilled by
the B-complex vitamins produced by the microbial population (D.A. Wood, T.R. Fermor 1985).
2.7 Water
Water is essential in mushroom production. Mushrooms contain more than 90-95% water and
restrictions in water supply have a negative effect on mushroom production (Kalberer 1987).
Commonly water requirements are addressed by the percentage of water content in the
substrate. But this is not a reliable measurement of water availability for mycelium growth.
Water can be highly concentrated with solutes or it can be bound by the substrate impeding
water uptake by the organism. Water availability needs to be determined by other parameters
such as water activity or water potential Ψw. Currently few studies address the water
requirements for mushroom production in values of water potential, so real necessities remain
unknown.
To define a nutrients solution is important to know the fungi optimal water potential. The
objective is to formulate a nutrient rich solution but without inhibiting growth due to water
stress. Mycelium growth under water stress due to reduced osmotic potential decreases the
nutrient uptake with the consequent effect on mycelium growth .
27
In order to absorb water, the osmotic potential of mycelial cells has to be lower than the water
potential of the substrate. Kalberer (1987) studied water potential in mushroom and casing soil
in different phases of cultivation without taking into account matrix potential. The osmotic
potential of the press juice of the mushrooms was about -0.8MPa. In the casing soil the water
potential was -0.26 MPa, and in compost -1.4 Mpa.
On 2004 Molloy (Molloy 2004) looked at mycelium growth in solutions with experimentally
altered water potential. She observed that the fungus tolerates lower water potentials when
growing in compost than when growing in nutrients media (malt extract).
Magan et al., (1995) tested the effect of osmotic potential and the matrix potential on linear
growth rate of three strains of Agaricus bisporus (C63 (=U3), C43 and C81). The osmotic
potential Ψπ
was altered by adding KCl to the medium (3% maltextract agar). They found
optimal growth at Ψπ values between -0.5 and -1.0 MPa. At Ψ
π values lower than -1.5 MPa the
linear growth rate decreased strongly. At Ψπ-values lower than -2.0 the linear growth rate was
lower than 1 mm per day.
During this thesis the potential of different nutrients solutions will be calculated in order to
evaluate the effect of the osmotic pressure in mycelium growth.
28
3 Experiments
The research has been focused on the optimization of three factors key to develop an
hydroponic system for A.bisporus; formulation of a defined media, pH control and evaluation of
a new hydroponic design.
- Nutrient solution: Big amounts of mycelium are required for the production of a single
sporophore. The defined media has to be able to generate in short time enough mycelium to
sustain fructification. Achieving a successful fructification is vital to research the nutritional
demands of the fruit bodies. Therefore big part of this research is focused on improving the
biomass production by optimizing the nutrient solution. Sections 3.1 and 3.2
- pH: Control of the acidification is important when working with artificial media. The decrease in
the pH due to the acids excreted by the fungus can inhibit its own growth. A buffer agent is
required for a continue mycelium production. Section 3.3
- Production system design: We are looking for an hydroponic system suitable for nutritional
studies at fructification, that implies:
- It is able to produce mushrooms (fructification phase) - All the nutrients used by the fungi can be measured - The nutrition can be modified during the growing phase - Can be replicated with comparable results (all the factors considered)
In this preliminary phase of the design we were looking at the possibility to supply nutrients by
immersing the growing container into the growing media. Section 3.4 and 3.5
3.1 Testing different carbon sources for Agaricus bisporus nutrition
3.1.1 Introduction
Carbon is the main nutrient for fungi; therefore it is the critical nutrient to optimize in order to
formulate an effective growing media. Despite all the research done in A. bisporus carbon
nutrition, few general rules can be deducted (Section 2).
During the years, the optimal concentration of sugars in the growing media has been a point of
discussion. Treschow in 1944 found inhibition due to high glucose concentration above 150 mM.
However some researchers formulated solutions with higher glucose concentrations; Hammond
(1975) formulated a solution for his experiments with 222mM of glucose and Dijkstra (Dijkstra
1976) with 166mM.
In addition, some scholars have reported sensitivity of A. bisporus to high initial sugar
concentrations (Sharma et al. 1977)(Dijkstra et al. 1972). Few scientific data is available on
this concentration effect on A. bisporus. It has been observed that the sugars tolerance
29
increases with the presence of some nutrients like calcium or phosphate (Sharma et al.
1977)(Dijkstra 1976). The osmotic pressure of the solution is also a factor to consider in the
formulation of a growing media. Solutions excessively concentrated will decrease the water
potentials, limiting the uptake of water and nutrients.
Complex sugars need to be degraded by the enzymatic system to be available for the fungus.
The sugars result of the degrading process are liberated gradually to the medium. Therefore
with complex carbon sources it is not expected an accumulation of readily degradable sugars,
which could prevent the concentration effect.
Therefore, we considered important to test the optimal sugar concentration for the standard
defined media and the growing conditions to be used during this project. We decided to analyze
the response of A. bisporus to different carbon sources in different concentrations and in several
combinations to set a reference point for the formulation of the media. For that, we designed
the following three experiments:
1.- Testing different carbon sources and combinations among them.
2.- Testing different concentration of monomers.
3.- Testing different concentration of complex sugars.
3.1.2 Materials and methods
Experiment 1 Testing different carbon sources and combinations among them
Three monomers and one disaccharide and combinations among them four, were tested as sole
carbon sources. Besides, 5ml of compost extract were included in one treatment. They were
carried out 12 different treatments with five replicates for solid media and with five replicates
for liquid media.
Preparation of compost extract
Compost Phase II was obtained from a commercial producer. 1 kg of compost was mixed with
2liters of water and let it stand for 1hour before boiling for 5 minutes. The mixture was
centrifuged for 1,5hours at 350 rpm. It was passed through a sieve and separated in different
recipients and centrifuged again for 45 mins at 2000rpm (Centrifuge: Heraeus Multifuge 3 Plus,
rotor: TTH-750 High-Capacity). The liquid was filtered though a filter paper and the sediment
discarded. The extract was autoclaved at 121ºC for 20 mins.
Preparation of the growing media
The concentration of the different carbon sources was adapted to get the same amount of
carbon atoms in the final concentration for all the treatments (Table 6(Sharma et al. 1977)).
The remaining components in the media were formulated to satisfy the requirements for
mushroom growth (APPENDIX I). The solid media had an agar content of 2% w/v. Media was
30
autoclaved (120ºC for 20 mins) and 2,5 ml/l of sterile phosphate were added afterwards. 25 ml
were poured per petry dish and per growing container. The inoculation was carried out by an
agar plug of 5mm diameter from the outside perimeter of a 10 days old colony. Petry dishes and
growing buckets were placed in the growing room (24ºC, 80%HR) for 3 weeks.
Table 6 Concentration of carbon source per treatment
Treatment number
Glucose (g/l)
Xylose (g/l)
Fructose (g/l)
Maltose (g/l)
Compost extract (ml)
I 18.02
II 18.02
III 18.02
IV 18.02
V 9.01 9.01
VI 9.01 9.01
VII 9.01 9.01
VIII 9.01 9.01
IX 9.01 9.01
X 9.01 9.01
XI 4.5 4.5 4.5 4.5
XII 4.5 4.5 4.5 4.5 5.0
Colony diameter was measured in the petry dishes 3, 6, 10, 14 and 21 days after inoculation.
Biomass dry weight was in liquid cultures was measured by filtrating, drying (stove 120ºC,
24hrs) and weighing mycelium produced in the liquid media after 21 days.
Experiment 2 Testing different concentration of monomers.
Five carbon sources, glucose, fructose, xylose, ribose and arabinose were tested in six different
concentrations, ranging from 54g/l to 1g/l. Molarities were adjusted to obtain the same carbon
concentration per treatment (Table 7). Two harvest dates were performed; at 3 and 6 weeks
after inoculation. The solute potential of every solution used was calculated (Table 8) with the
following equation.
Solute potential
The solute potential of every solution ψS is given by the equation:
where
i = the ionization constant. For non-electrolytes i = 1.
M = molar concentration expressed in mol/L.
iMRTs −=ψ
31
R = the universal gas constant (R = 8.314*10-3 MPa L/mol K)
T = the absolute temperature (K)
Table 7 Concentration per treatment (mM) depending on the type of monomer utilized. Amount adjusted to obtain the same concentration of C for both types of monomers
Treatments
I II III IV V VI
Carbon Source Molar
Weight mM mM mM mM mM mM
hexoses Glucose, Fructose 180.16 300 200 100 50 20 10
pentoses Xylose, Ribose,
Arabinose 150.13 360 240 120 60 24 12
Table 8 Solute potential (Ψw
) per treatment depending on the type of monomer, hexose or
pentose.
Treatments
I II III IV V VI
Cabon Source Molar
Weight MPa MPa MPa MPa MPa MPa
hexoses Glucose, Fructose 180.16 -0.74 -0.50 -0.25 -0.12 -0.05 -0.02
pentoses Xylose, Ribose,
Arabinose 150.13 -0.89 -0.59 -0.30 -0.15 -0.06 -0.03
Experiment 3 Testing different concentration of complex sugars
In this experiment five complex carbon sources were tested in three different concentrations.
The complex sugars used were Sucrose, Raffinose, Pectins from apples, Guar gum and Arabic
gum. The concentrations were adjusted to get 7.2, 14.4 and 21.6 gr of carbon, in order to
compare the results with the data from the harvest after 3 weeks from previous experiments.
The final concentration of the complex sugar was 16.5, 33 or 50 gr/l depending on the
treatment.
Defined media was prepared containing all the elements necessary for mushroom growth apart
from carbon source (APPENDIX I). For every treatment 100ml of solution were prepared by
adding a sole carbon source to the defined media and autoclaved (121 ºC 20 mins). After
cooling, sterile phosphate was added (2.5ml/l) and 25ml of the nutrients solution was poured
per container. The experiment was done in three replicates.
32
Table 9 Concentration of sugars per solution
SOLUTION 1 SOLUTION 2 SOLUTION 3 7,2 grC/l* 14,4 grC/l 21,6 grC/l
Carbon sources g/mol g. C/mol g. sugar/ L g. sugar/ L g. sugar /L
Sucrose 342.30 144 17.11 34.23 51.34
Raffinose 504.42 216 16.18 32.36 48.54
Pectin n/a n/a 16.50** 33.00** 50.00**
Guar gum n/a n/a 16.50 33.00 50.00
Arabic Gum n/a n/a 16.50 33.00 50.00
* 7.2 g. C/L is equivalent a 100mM of glucose or 120mM Xylose ** As estimation for comparable amounts of C
The cultures were inoculated with a liquid suspension of mycelium. 520mg of fresh mycelium
growing on agar overlaid with a cellophane disk were scraped off the cellophane and added to
100ml of sterile water. After mixing for 20 s in a blender, 1ml of the suspension was added per
container. The strain used was the commercial hybrid A15. The growth containers were placed
in a warm room at 24ºC and 80% HR.
After 21 days cultures were harvested on filter paper. Some cultures growing in dense media
(pectin from apples and guar gum) required a centrifugation prior to be filtered. They were dried
overnight in a stove (120ºC) and weighted.
The treatment with guar gum at the highest concentration could not be harvested due to
excessive viscosity of the media.
3.1.3 Results
Experiment 1
The combination of Glucose, Xylose, Fructose, Maltose with sterile Compost extract (treatment
XII) showed the highest colony growth and the highest dry weight. The addition of the compost
extract had a significant improvement effect in comparison to the same combination without the
extract (Figure 2). In general combinations of different sugars performed better than single
sugars, with the exception of maltose. Maltose is a disaccharide so its response was expected to
be similar than combinations of two monomers. (Table 10).
33
Table 10 Average dry weight of mycelium growing in different carbon sources. Averages over five replicates.
Carbon sources Average of dry weight
(mg)
Glucose, Xylose, Fructose, Maltose and sterile Compost extract 83.93 a
Glucose and Fructose 34.93 b
Fructose and Maltose 27.28 b,c
Glucose, Xylose, Fructose and Maltose 17.87 c
Maltose 14.48 c,d
Glucose and Maltose 12.38 d
Xylose and Maltose 12.23 d
Glucose and Xylose 9.92 d
Xylose and Fructose 9.78 d
Glucose 5.53 d
Xylose 4.50 d,f
Fructose 3.40 f
Means followed by different letters indicate significant differences P< 0.05.
Figure 2 Petry dishes with 21 days old colonies showing the promoting growing effect of the addition of compost extract.
Experiment 2
Glucose and fructose were the only two sugars which showed significant differences in growth
among the six concentrations after 21 days (Figure 2, above). The highest growth was obtained
with the highest concentration 300mM of glucose (49.7mg). For three of the five monomers
tested the growth was scarce in all the concentrations without remarkable differences between
the highest and the lowest.
The remarkable growth promoting effect of compost extract demands further research (Figure
2).
For the treatments with a growing period of 6 weeks there were significant differences in all the
sugars except for ribose (Figure 2, below). Glucose provided the highest growth (75.27mg) with
Glucose, Xylose, Fructose, Maltose and sterile compost extract
Glucose, Xylose, Fructose and Maltose
34
200mM. There was a slightly decline (not significant) in production of biomass with the highest
concentration for fructose and glucose.
However mycelium growth did not appear to be heavily affected by the low solute potential. The
highest biomass production occurred at the potentials of -0.74MPa for 3 weeks and -0.50MPa for
6 weeks. Mycelium of A. bisporus was able to grow in media with solute potentials below -
0.80MPa. Nevertheless the solute potential was only calculated for the sugars, not for the
complete media, however the remaining nutrients are present in low concentrations to affect
significantly to the solute potential.
Figure 3. Above, average of mycelium dry weight for different sugars in six different concentrations after three weeks. Below, average of mycelium dry weight for different sugars in six different concentrations after six weeks.
0.0
20.0
40.0
60.0
80.0
100.0
120.0
Fructose Glucose Ribose Xylose Arabinose
3 weeks
bio
mas
s d
ry w
eig
ht
(mg
) 300/360mM
200/240mM
100/120mM
50/60mM
20/24mM
10/12mM
0.0
20.0
40.0
60.0
80.0
100.0
120.0
Fructose Glucose Ribose Xylose Arabinose
6 weeks
biom
ass
dry
wei
gh
t (m
g)
300/360mM200/240mM100/120mM
50/60mM20/24mM10/12mM
35
Glucose and Fructose produced significantly (P< 0.05) more mycelium in six weeks than in three
For xylose and ribose there was not difference in the production between the longer and the
shorter period. Unexpectedly, for arabinose the mycelium yield for 6 weeks was significantly
lower than for 3 (Table 11).
Table 11 Average mycelium dry weight for two different growing periods (3 and 6 weeks). Average over 6 concentrations.
Average of dry weight
weeks
Carbon source 3 6
Arabinose 4.42 a 3.04 b
Fructose 13.81a 33.29 b
Glucose 24.7 a 59.31b
Ribose 6.50 7.22
Xylose 8.94 13.11 Values followed by different letter in the same row indicate significant differences (P< 0.05)
Experiment 3
Figure 4 Weight of dry mycelium produced in 21 days with three different concentrations of sugars. Averages over three replicates*. Bars showing LSD (p<0.05). *Due to technical problems, there were not enough replicates of the guar gum treatment for
carrying our an statistical analysis of guar gum not possible for having only 2 replicates of each treatment Mycelium growing in pectin and guar gum showed the highest biomass production. The high
viscosity of these media allowed surface growth besides submerged growth.
Higher sugar concentrations resulted in higher biomass production except for raffinose where
there were not significant differences among the three treatments.
0.0
50.0
100.0
150.0
200.0
250.0
300.0
350.0
Arabic Gum Guar gum Pectin Raffinose Sucrose
Bio
mas
s d
ry w
eig
h (
mg
)
treatment 16.5 gr/l
treatment 33 gr /L
treatment 50 gr C/L
36
The high viscosity of the media hampered the sampling process. In future experiments cultures
on complex sugars should be grown on solid media.
The result of this experiment can be compared with some of the results obtained in the
experiment number 2 where five monomers where tested. The general media used, the growing
conditions and the harvesting and measurements were the same. The dry weight produced by
the polymers is from 4 to 7 times higher than the biomass produced by the monomers in the
same time (Table 12).
Table 12 Dry weight of mycelium (mg) produced in 21 days for two types of carbon sources, monomers and polymers, in three different concentrations. Averages over five types of monomers and four types of polymers all in 3 replicates.
3.1.4 Conclusions
A. bisporus grows better in a mixture of different carbon sources than in a sole carbon source.
The reasons of this preference for the mixtures over the single sugars remains unclear. The
effects is notable in the case of the combination of two similar hexoses, as glucose and fructose.
Their mixture increases mycelium production in about ten times when compared with the single
sugar.
Hexoses are better carbon sources for mycelium growth than pentoses. This contradicts the
results from Treschow (1944) but agrees with the results from Dijkstra (1976) and (Manning &
D. A. Wood 1983).
A. bisporus mycelium is able to growth at concentrations of 360 mM (-0.89MPa) without being
affected by the inhibitory effect of high osmotic potentials. That confirms the results of Magan N.
et al. (1995) who found optimal growth at Ψπ values between -0.5 and -1.0 MPa (Section 2.7).
Polymers are good carbon sources for mycelium growth. From the results we can deduct that
A.bisporus has preference for the more complex sugars. Polymers require a degradation process
before being assimilated. This degradation process requires the synthesis and liberation of many
different enzymes. For that reason it is surprising that the utilization of polymers is more
favorable than the utilization of monomers. On the other hand A. bisporus is not used to the
direct uptake of simple sugars from the substrate. Simple sugars are hardly present in nature. It
might be that the gradually liberation of monomers from the degradation process keeps the
Mycelium dry weight (mg)
Concentration of sugar 50-54g/l 33-35g/l 16.5-17g/l
monomers 20.29 mg 13.65 mg 10.27 mg
polymers 102.62 mg 92.28 mg 41.07 mg
37
metabolic system of the fungus in equilibrium. Therefore, it is possible that the presence of
readily available sugars overloads the metabolism in the same way that has been reported in
other fungi (section 2).
The utilization of complex sugars is advisable not only for the fast production of biomass but
also because it decreases the risk of contaminations. The degradation required by the polymers
limits the range of organism able to feed in those compounds. However the viscosity that some
polymers create in the nutrients solution may limit the utilization of those compounds in an
hydroponic system with nutrients supplied by a liquid media.
38
3.2 Use of Plackett Burman experimental design to evaluate different compounds of
the defined media for cultivation of Agaricus bisporus on solid substrates.
3.2.1 Introduction
The nutrient requirements of A. bisporus are highly complex and the growing media is a
reflection of this complexity. There are many different compounds in a defined growing media
(Figure 6). To formulate an optimized solution, all the ingredients have to be evaluated and
optimized, which is a laborious work. As a result, the chemically defined nutrient media being
used for mushroom research at the moment are based on previous experiences and on trial and
error approach more than based science principles. For laboratory experiments not involving
nutritional research, easily available media such as malt extract or potato-starch extract are
commonly used.
Figure 5; Example of a nutrients solution used in mushroom nutrition research. (Wardle & Schisler 1969)
The first studies in mushroom nutrition were already testing different growing media. In 1872
Duggar grew mushroom mycelia on filter paper saturated with various nutrients solution
(Duggar 1872). The studies of Treschow in 1944 resulted in the formulation of a chemically
defined growing media for A. Bisporus which is still currently utilized. Besides several scholars
who carried out nutritional studies formulated their own chemically defined nutrients solution.
The nutritional studies were carried out by modifying one factor of the standard solution keeping
the rest constant.
During this thesis several experiments have been carried out to find the most efficient carbon
source in the adequate concentration (see section 3.1). This research has been done by
modifying only one factor in the nutrients solution; the carbon source. The next step is to test
the bests of these carbon sources in combinations among them. Besides it is also of interest to
test their performance with several nitrogen source. This experiment with the traditional
39
factorial design will require high number of experimental units, making it expensive and time
consuming. For that reason a Plackett-Burmann experimental design (Plackett & Burman 1946)
was selected for this experiment.
Plackett-Burmann matrices are experimental designs in which the estimation of K main effects
can be done by K+1 runs. The runs/treatments have to be multiple of four, and every of the
factors under study have to take two values: low level of the factor (-1) and high level of the
factor (1). This design allows a statistical analysis of the results in which the main effects can
be obtained. The factor with the highest positive main effect is the most contributing to the end
value. Plackett-Burman designs have been proved to be an efficient, reliable and rapid method
for screening several nutrient sources for a growing media. It has been used to evaluate
nutrient requirements in many other fungi types (Srinivas et al. 1994)(Yu et al. 1997)(Ghanem
et al. 2000).
This experiment was designed as first step before optimizing the most relevant constituents with
the Surface Response Methodology.
3.2.2 Materials and Methods
In this experiment the 8-runs Placket Burman experimental design matrix was used to evaluate
the relative importance of four carbon sources and two nitrogen sources on A. bisporus
mycelium growth. A “dummy” variable was included to measure the standard error of the
experiment. A dummy does not involve any changes in the treatmetns but it is included in the
statistical analysis as the other variables. The main effect of the dummy should be zero,
however it hardly is.
Table 13 Distribution of the high and low concentrations according with the Pluckett-Burman experimental design.
RUNS glucose fructose guar gum pectin asparagine casein dummy
1 + - - + - + +
2 + + - - + - +
3 + + + - - + -
4 - + + + - - +
5 + - + + + - -
6 - + - + + + -
7 - - + - + + +
8 - - - - - - -
The base medium (APPENDIX I) was modified by replacement of the carbon and nitrogen
sources with the corresponding amounts of the substances under study according with the P-B
design (Table 14). Eight different solutions were prepared separately with a common agar
40
content of 1.2% w/v. After autoclaving (121ºC 20 min), sterile phosphate was added (2.5ml/l).
Twenty five ml of solution were poured per petri dish. Twelve replicates were prepared of each
run. Once solidified the media, the media were covered with a sterile cellophane. The inoculation
was carried out by placing on the cellophane one agar plug (5mm diameter) extracted from the
edge of a 10 days old colony. The strain used was the commercial hybrid A15.
After 21 days the cultures were harvested. The cellophane was separated from the agar and
weighed. Afterwards the mycelium was scraped from the cellophane, placed on a filter paper,
dried in a stove (120 ºC, 48 hours) and weighed.
Statistical analysis
The main effect was estimated as the difference between both averages of measurements made
of the high level (+1) and at the low level (-1) (Yu et al. 1997). The significance of each variable
was determined by applying the students t-test (Plackett and Burman,1946).
( )NMME iixi ∑∑ −+ −=
=xiE main concentration effect
=+iM average of the measurement at high level
=−iM average of the measurements at low level
=N number of trials (8)
The experimental error was estimating by calculating the variance of the dummy variable. The
standard error (SE) was the square root of the variance. The individual effect for every
treatment was divided by the SE for the calculation of the t-value. P values were obtained by
performing a Student’s t-test for each treatment
SEExt xiii /=
=ii xt t-value for the specific treatment i
3.2.3 Results
Among the carbon sources, sucrose and guar gum had the highest promoting effect in mycelium
growth. In contrast, the presence of pectin was detrimental for the fungus under these
conditions.
Referring to the nitrogen sources, casein was favorable for the fungi and asparagine was
harmful. Asparagine has been reported to be a good nitrogen source (see section 2),
consequently the negative effect was unexpected.
41
Even though the nutrient sources were the same in different concentrations, the performance of
the colonies showed remarkable differences, both in size and morphology (Figure 6).
Figure 6 Pictures of the colony development under different treatments according with the Pluckett-Burman experiment design.
Table 14 Plackett-Burman design of the experiment showing the combinations of nutrients under study
Treatment 1 Treatment 2 Treatment 3 Treatment 4
Treatment 5 Treatment 6 Treatment 7 Treatment 8
Treatment glucose
g/l fructose
g/l guar
gum g/l pectin
g/l asparagine
gr/l casein gr/l dummy
1 20 5 5 20 1 5 0
2 20 20 5 5 3 1.5 0
3 20 20 20 5 1 5 0
4 5 20 20 20 1 1.5 0
5 20 5 20 20 3 1.5 0
6 5 20 5 20 3 5 0
7 5 5 20 5 3 5 0
8 5 5 5 5 1 1.5 0
42
Table 15 Main effects on A. bisporus mycelium growth according with the Plucket-Burman experiment design
Factor under study Main effects t-value p values Significance
level
glucose 12.913a 10.464 0.0016 P< 0.05
fructose 5.602 a 4.540 0.0204 P< 0.05 c
guar gum 12.676 a 10.272 0.0017 P< 0.05
pectin -33.450 b -27.107 0.0001 P< 0.05
asparagine -20.526 b -16.634 0.0003 P< 0.05
casein 18.431 a 14.936 0.0005 P< 0.05
dummy -1.234 -1.000 0.5626 ns.
a Indicates a significant positive effect b Indicates a significant negative effect c Non significant at P< 0.01
3.2.4 Conclusions
Glucose and guar gum are good carbon sources for mycelium growth. The presence of pectin is
not favorable for A. bisporus in a mixture with other carbon sources. However, pectin have
performed satisfactory in other experiments with liquid medium (see section 3.1). The
difference of this response between growth in solid and in liquid media demands further
investigations.
With this experiment it is confirmed that casein is a favorable nitrogen source for A. bisporus.
Casein is a protein which can be used by the fungus as sole carbon and nitrogen source (Kalisz
et al. 1986), besides casein has a phosphorus content of 0.85% which might also contribute to
the positive effect in mycelium growth.
Previous experiences reported asparagine as an excellent nitrogen source for A. bisporus
cultures (J.J.P. Baars 1996). The negative effect of asparagine in this case might be due to the
instability of asparagine on heating. In this experiment the media was autoclaved with all the
nutrients included except for the phosphate. In future experiments is advisable to sterilize
asparagine separately by a non-heating method.
It can be concluded that sucrose, guar gum and casein are effective for A. bisporus mycelium
growth. Therefore their optimal concentration can be optimized by the Surface Response
Methodology. Placket Burman design has been proved to be a good tool to scan different
compounds for the formulation of a nutrients solution.
43
3.3 Evaluating the effect of two different pH buffer agents on Agaricus bisporus.
3.3.1 Introduction
Agaricus bisporus has high pH sensitivity. The optimal growing value ranges between 6-7
(Treschow 1944). Above and below these values mycelium growth is strongly affected and even
inhibited. Mycelium growing in compost and in defined media excretes organic acids such as
oxalic acid, as a result of its metabolism (Tsao 1963). These acids produce a drop in the pH that
in conventional production systems is controlled by the compost buffer effect.
In a system where A. bisporus is grown on an inert carrier with the nutrients supplied by a
chemically defined medium, pH variations not only depend on the amount of acids excreted by
the fungus, but also on some other factors:
� Type of nitrogen: the utilization of ammonium salts as nitrogen source will result in a
lowering of pH, while a supply of nitrates will increase pH upon utilization levels by A.bisporus.
� Influence of the carrier: most of the inert substrates lower the pH after prolonged incubation
even without the presence of the fungus.
� Sterilization; the pH of the chemically defined medium might vary with the sterilization
treatment.
In the spawn industry pH is controlled with calcium carbonate. In other systems of submerged
culture a degree of pH control is obtained by addition of different proportions of urea and
ammonium salts. Since the ammonia liberated from the urea neutralizes the acidification
associated with the ammonium ion uptake (Raimbault 1998). Currently phosphate is the
common buffer agent used in defined medium in most of fungal research, however it involves
some inconveniences when performing nutritional investigations. Phosphate has a dual purpose;
it is metabolized as nutrient and used as buffer agent. Consequently it is not possible to know
which part is utilized as nutrient and which part is used as buffer agent. Besides, phosphate
forms insoluble salts with divalent metal ions, limiting the availability of those components.
Therefore a buffer is required which is not influencing the total nutrient balance.
On 1966 Norman Good, concerned of the limitations of phosphate as buffer agent, prepared and
tested 12 new buffer agents for biological research. The requirements for an appropriate buffer
were the following:
- pKa between 6-8
- High solubility in water
- Low membrane permeability (high molecular mass)
- Available in pure form
- Resistant to enzyme degradation
- Solubility of the complexes formed with the cations.
44
The buffers formulated by Good are suitable for nutritional investigation, since their interference
with biological systems is expected to be low. There are studies on the effect of some of these
buffering systems on fungi (Child et al. 1973)(Nagahashi et al. 1996). They reported some
effects on hyphae growth and branching caused by different buffering systems. Child (1973)
tested MES and EMTA on 16 different fungal strains finding significant differences on the
response to the buffer agent among the strains. Nagahashi (1996) reported stimulation of
hyphal growth by Tris but inhibition by MES and Phosphate in mycorrhizal fungus (Gigaspora
margarita). The effect of none of Good’s buffers has been reported on A. bisporus. The objective
of this test was to evaluate the effect on A. bisporus mycelium growth of three different buffer
systems in six different concentrations.
3.3.2 Materials and Methods
The two buffers selected for the experiments were MES (4-Morpholineethanesulfonic acid) and
Bis-Tris (Bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane) (Table 16). The selection was
made taking into account the characteristic previously listed of adequate pKa range, low
membrane permeability, high solubility and low interaction with the remaining nutrients.
Table 16 Characteristics of the buffers used in the experiment.
Buffers Mol.Weight Useful pH
Range pKa (at 20) pKa (at 25) Costs/100 g
MES 195.24 g/mol 5.5–6.7 6.16 6.1 € 73.50
Bis-Tris 209.24 g/mol 5.8–7.2 n/a 6.5 € 108.50
MES/KOH, Bis Tris/citric acid and Bis-Tris/HCL were tested at pH 6.8 as part of a chemically
defined 2% (w/v) agar media (Medium composition in Table X) in six different final
concentrations (7.8, 15.6, 31.3, 62.5, 125, 250mM). The bottles were autoclaved (121ºC for
20min) and phosphate was added individually to every bottle (2,5ml/l). 25ml of the solution
were poured per petry dish (90mm diameter). They were made five replicates per treatment.
The inoculation was carried out by a mycelial plug (5mm diameter) from the peripheral edge of
a 10 days colony of strain A15. To test whether buffering capacity has been affected much by
fungal growth, pH of the media was determined both at the start and the end of the experiment.
45
Table 17 Composition of the nutrient solution
Component gram / liter
(of ml/liter)
L-asparagine 3.23
L-phenylalanine 0.62
L-histidine 0.13
L-valine 0.44
DL-methionine 0.15
Glucose 30
MgSO4.7H2O 0.4
CaCl2.2H2O 0.02
FeCl3.6H2O stock solution* 1 ml
Trace element solution* 1 ml
Thiamine-HCl-solution* 1 ml
Tween 80 0,5 ml
Phosphate 2,5 ml
3.3.3 Results
Colony diameter was reduced by increasing buffer concentration (Figure 7). Among the three
treatments MES-KOH had the lowest detrimental impact on mycelium, while BisTris-Citric acid
had the highest.
The pH ranged between 6.0 to 6.8. The lowest pH, corresponded with the sample with the
highest growth. (MES/KOH, 15.6 mM) (Figure 8).
Figure 7 Effect of buffer concentration on colony diameter. Increasing buffer concentrations from left to right
gram / liter
FeCl3.6H2O stock solution* FeCl3.6H2O 10,0
Trace element solution* MnCl2.4H2O 7,20
ZnSO4.7H2O 8,80
CuSO4.5H2O 1,26
Thiamine-HCl-solution* Thiamine-HCl 400 mg
���� Bis-Tris/HCl
���� Bis-Tris/Citric Acid
���� MES/KOH
46
0
10
20
30
40
50
60
70
80
90
0 50 100 150 200 250 300
bis-Tris/citric acid concentration mM
colo
ny
dia
met
er (m
m)
5.8
6
6.2
6.4
6.6
6.8
7
7.2
aver
age
pH
colonydiameter (mm)
pH
0
10
20
30
40
50
60
70
80
90
0 50 100 150 200 250 300
Bis-Tris/HCl concentration mM
colo
ny d
iam
eter
(m
m)
5.8
6
6.2
6.4
6.6
6.8
7
7.2
aver
age
pH
0
10
20
30
40
50
60
70
80
90
0 50 100 150 200 250 300
MES/KOH concentration mM
colo
ny
diam
eter
(m
m)
5.8
6
6.2
6.4
6.6
6.8
7
7.2
aver
age
pH
colony diameter (mm)
pH
Figure 8. Effect of the buffer concentration on colony diameter and on pH of the media.
47
3.3.4 Conclusions
The biological buffers, Bis-Tris and MES, have a strong negative effect on A. bisporus mycelium
growth. The inhibitory effect is higher with BisTris than with MES. HCl is better acid to adjust pH
than Citric acid, since it has lower impact on mycelium development.
We consider two main possible causes for the detrimental effect of the buffer agent on mycelium:
1.- The buffer agent increases osmotic pressure in the media above tolerable levels which can
decrease water availability limiting A. bisporus growth. Even Good’s buffers were formulated
with low membrane permeability, they contribute to the osmotic pressure of the media.
2.- The acid/based used to adjust pH has a major harmful effect on mycelium. That can be seen
in the differences between BisTris/HCL and BisTris/Citric acid.
MES/KOH in the concentration of 15.6mM was the treatment with the highest growth. As a
result, we decided to use MES/KOH (aprox.20mM) as buffer agent in the following experiments
concerning nutritional research.
48
3.4 Testing hydrogel as a solid substrate for Agaricus bisporus
3.4.1 Introduction
White button mushroom is commercially grown in compost, which is a highly nutritive substrate
with an average water content of 68% (Van Griensven, L.J.L.D. 1988). When looking for an
inert substrate to cultivate Agaricus bisporus the water holding capacity is an important factor to
consider. Most of the substrates commonly used in horticulture such as vermiculite or perlite are
not able to hold the required amount of water (see table 18). To increase water holding capacity
of these substrates we evaluated the possibility of including an hydrogel in the inert carrier.
Hydrogels are polymers able to absorb many times their weight in water. They are commonly
used in the pharmacy and cosmetic industry. In horticulture they are used as a conditioner to
improve water retention in the substrate (Hady et al. 1981). The advantage of hydrogel over
other solid media is that hydrogels are able to rehydrate when new media is applied. Besides
they can be sterilized in the stove and kept sterile until they are needed. Despite of its many
uses there are not reported experiences on using absorbent hydrogels as solid media to grow
mycelium.
The objective of this experiment is to evaluate mycelium growth in different concentrations of
hydrogel to define the correct hydrogel content to be added to the substrate.
Table 18 Water content of different horticulture substrates
Substrates* water content at leak out (%vol.)
Perlite 44
Pumice 58
Clay granules 19
*Substrates such as vermiculite or glass wool were not
considered for pH interaction problems.
Substrates such as coir, sawdust or peat were not
considered for nutritional interactions with the media.
Source: International Substrate Manual. (Kipp, J.A., Wever, G. and de Kreij, C. 2000).
3.4.2 Materials and methods
The hydrogel used was Stockosorb™ (cross-linked polyacrylamide). It was sterilized overnight in
a dry stove (120ºC). The nutrient media (Malt extract 6% w/v and MES-KOH buffer 20mM) was
autoclaved for 20 min at 121ºC. Six concentrations of hydrogel were tested in five replicates.
The volume of media per petri dish was 25 ml for all the treatments and the content of hydrogel
was depending on the treatment; 1g, 0.83g , 0.67g, 0.50g, 0.33g, or 0.17g per petry dish
(25ml). After adding the hydrogel it was allowed to absorb the media for one hour before
inoculation. Agar plugs of 5mm diameter from the edge of a 2 weeks old colony of commercial
49
hybrid A15 were used as inoculum. The petri dishes were placed in a growing chamber at 24ºC
with 80% RH.
3.4.3 Results
After 16 days only the treatment with the lowest amount of hydrogel 0.17g/25ml showed visible
mycelium growth. The mycelium grew in the liquid media remaining, since the low amount of
hydrogel was not able to absorb all the media. In the rest of the treatments there was no
colonization of the hydrogel. The only growth observed was from the agar plug used as
inoculum and it disappeared after several days.
Measurements of pH were made to detect possible contaminations which would have dropped
the pH. In addition, to rule out possible problems with the inoculum, the agar point was
transferred to normal agar petry dishes and it performed normally.
The experiment was repeated with only one replicate per treatment and the same results were
obtained.
3.4.4 Conclusions
Mycelium is not able to uptake the liquid from the hydrogel. Only the treatment with a low
amount of hydrogel and as a result, medium remaining in liquid form, showed mycelium growth.
This might be due to binding forces within the hydrogel. It has been observed before in several
studies that significant part of the water stored by hydrogel is apparently not available for plants
(Tripepi et al. 1991). This may be due to the high hydrogel surface tension which impedes the
fungi to extract the media out of the substrate. Furthermore, the polymer surface acts as a
semi-permeable membrane (Johnson 1984) so the liquid absorption depends on the
concentration of the media.
The hydrogel has no toxic compounds for the fungi since the inoculum point transferred to new
agar media maintains the capacity to grow.
50
3.5 Testing a new hydroponic method for Agaricus bisporus cultivation suitable for
nutritional investigations
3.5.1 Introduction
When the fructification phase starts, a change in the nutritional demands occurs. A. bisporus
has to mobilize big amounts of nutrients to satisfy the nutritional demands of the fast growing
sporophores. For that reason, the fungus, in fructification phase, feeds in faster degradable
sources than in vegetative phase. A. bisporus, growing in compost, switches from degrading
lignin to degrade cellulose which is an easier metabolizable source (D. A. Wood & Goodenough
1977).
Details on the fungal nutrient requirements at fructification are rather limited. Mushrooms are
commercially grown on compost. Compost is a complex matrix difficult to analyze and modify.
In the laboratory due to the specific features of A. bisporus (see section 2.3) it is difficult to
obtain fruit bodies in axenic conditions. These two factors obstruct the nutritional investigations
at fructification.
By developing a growing system in which mushroom can be grown on a carrier impregnated
with the nutrients solution (hydroponic system) those nutrients requirements at fructification
can be studied.
There are two relevant reported experiences looking for a hydroponic system suitable for
nutritional research: Smith & Hayes in 1972 and Bechara in 2006. Both got low yields when
trying to grow mushrooms on an inert substrate impregnated with liquid nutrient media.
However when a water holding layer was included, to avoid the presence of free water, systems
improved significantly. Besides that, Smith and Hayes found that the growing systems were
more efficient when, instead of liquid nutrients, solids were used.
The problems to produce satisfactory results with liquid media may be due to many different
reasons:
One possibility is that the excretion of metabolites affects the nutrient media, influencing pH and
altering solution compounds (Flegg, P. B.; Spencer, D. M.; Wood, D. A. 1985). Besides, the
utilization of oxygen by the mycelium could generate the presence of anaerobic conditions which
may be detrimental to mycelium growth (Dijkstra et al. 1972). This is considered in liquid
cultures where usually flasks are shaken to distribute inoculum and also to promote media
oxygenation. Furthermore, in open-cycle systems it was considered the possibility that essential
compounds for the promotion of fructification are flushed out with the renewal of the media
(Bechara et al. 2006a).
Despite of all these inconveniences, the use of liquid media facilitates nutritional research. It is
possible to formulate a chemically defined solution and to modify it during the cropping cycle. It
is also easily adaptable to changes occurring in the system, by varying concentrations or pH.
51
Design of the growing system
This experiment is based on a previous experience performed within the framework of this
project (internal document). In that study several inert carriers were tested as potential carriers
for the cultivation of A. bisporus. Some of the carriers tested were; perlite, vermiculite, glass-
wool, diatomaceous earth, rock wool, PLA (poly-lactic acid) and pumice. To increase the water
holding capacity the addition of two different hydrogels, Fytosorb™ and Stockosorb™, was
evaluated.
It was concluded that the most appropriate inert carrier was a mixture between pumice and
hydrogel (Stockosorb™) in proportion in volume of 60/40 (pumice/hydrogel). Pumice stone
keeps the structure open and the hydrogel absorbs the media. Then the pumice stone is
absorbing the media from the hydrogel gradually.
In this previous experience pumice mixed with hydrogel was impregnated with Malt extract as
medium. Although the production was not always reproducible this system produced comparable
amounts of biomass than compost. Even so, this design did not allow the change of the nutrition
during the growing phase, which is an important requirement for the nutritional investigations.
We tested a new design in which the nutrients solution can be modified during the cropping
cycle. We propose a system in which a water holding carrier is soaked into the nutrients media
regularly. In this way the system aims to combine the benefits of a solid substrate with the
advantages of a liquid media. By immersing periodically the perforated container in the solution
it is expected that the carrier absorbs new nutrients. All the nutrients cannot be replaced since
the substrate is already impregnated with previous media. To solve that, solutions should be
formulated with higher concentrations of the nutrient to be tested to be able to evaluate the
differences.
In this experiment we tested the viability of this new hydroponic system. We also looked at the
effects of different soaking frequencies. For this purpose, 9 perforated buckets were used as
growing containers. A sterile mixture of hydrogel and pumice was used again as inert carrier.
3.5.2 Materials and methods
Hydrogel (Stockosorb™,) was stove-sterilized overnight in glass containers. Grain spawn was
prepared by inoculating sterile millet grain with the commercial hybrid A15colonized agar and
leave it in sterile conditions for 14 days. Malt extract (6% w/w) with 20mM MES/KOH buffer was
prepared in glass bottles. Nine sterilizable cylindrical plastic containers (13.3cm diameter,
1530ml volume) were perforated with a warm iron tool and filled with 300 g. of pumice stone
slightly wet. Once sterilized, 7.5 g. of hydrogel and 5 g. of millet grain spawn were added per
container. After mixing they were placed in a bigger container with the malt extract to let it soak
for 30 minutes. They were left to drain for 15 minutes and then they were weighed. To keep
52
them in sterile conditions they were inserted into a sterile non-perforated container and covered
with a lid. They were incubated at 24ºC and 80% RH.
Three different soaking frequencies were tested. One treatment was soaked only once at the
beginning of the experiment. The second treatment was soaked once every 7 days. The third
treatment was soaked twice a week (Figure 9). Every treatment was repeated three times.
day /trtmt 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
I x
II x x x
III x x x x x x
Figure 9 Schema of soaking frequency for the different treatments.
Depending on the treatment, the containers were
soaked during the experimental period. In the
consequent soakings they were placed into the malt
extract for 15 minutes and after draining off for 5
minutes they were weighed. (Figure 10).
In the fifth week samples were collected for
ergosterol analysis. After sampling they were cased
with a sterile casing soil. Once the mycelium
colonized the casing soil (in two weeks time), the
containers were transported to a growing room at
the Mushroom Experimental farm. The lids were
removed and the casing soil was watered with a
solution of non-sterile casing soil to promote
fructification. The room was ventilated and the
temperature was 19ºC.
Figure 10. Example of the growing
container and the soaking technique Preparation of Ergosterol samples
Ergosterol assay was chosen as indirect method for determination of fungal biomass in this
experiment (see section 2).
Samples were kept at -80ºC until analysis. Before analysis samples were freeze-dried and
grounded. In a 50ml plastic tube, 200 mg of the sample was mixed with 3 ml of
53
methanol/10%KOH. Samples were shaken for 10 min. at 230 rpm and subsequently heated at
80ºC for 60 mins 20 µg/ml of 7-dehydrocholesterol was added as a reference of the efficiency of
the extraction together with 1 ml of distilled water and 2 ml of hexane. After centrifugation 10
min., 2300 rpm, (Centrifuge: Heraeus Multifuge 3 Plus, rotor: TTH-750 High-Capacity) the
supernatant hexane phase, was collected. This extraction was performed twice and supernatants
were combined. Hexane was removed by evaporation under vacuum at 30oC for 1 hour. Extracts
were taken up in one ml of methanol by shaking for 10 minutes. Before HPLC analysis samples
were filtered through a 0.2 µm filter.
Standards were prepared with 1 to 50 µg/ml of purchased Ergosterol (Sigma) and analyzed as
above samples.
The samples were analyzed on HPLC (Waters e2695 Separatins Module) with a reverse phase
C18 column (Alltima HP C18-AQ5u). Mobile phase was 90% methanol and 10% (1:1) 2
propanol/hexane.
3.5.3 Results
Although many pinheads were formed only two mushrooms of 8 and 7 grams (fresh weight)
were harvested. Both of the mushrooms developed in the same container, from the treatment
which was submerged once a week. The moisture in the growing room was insufficient, as
consequence the case soil dried out prematurely although it was watered regularly. That implied
the desiccation of the pinheads and malformation of the developing mushrooms (Figure 11)
The colonization of the case soil occurred faster in the treatment which was not soaked during
the experiment time. The water scarcity in the
substrate may have induced the fast colonization of
the wet casing soil. The colonization of the casing soil
took 3 weeks. In commercial conditions, it takes
around 10 days for the total colonization and for the
initiation of fruit body development. This delay in
colonization was more significant in the treatment with
higher soaking frequency. Consequently we can
conclude that mycelium development was negatively
affected by the immersion.
Figure 11 Malformed mushroom
The water content in the substrate increased due to the immersions (Figure 12). However this
increase was more significant during the first soaks. Lower absorption indicates that the carrier
was close to its maximum water holding capacity .
Although the three growing containers in the same treatment were submerged for the same
amount of time not all three absorbed the same amount of media. These differences are likely
to be due to a technical error. The containers were submerged in big buckets filled with malt
extract media until the same level. However those buckets were different in size. Growing
54
containers submerged in bigger amount of liquid absorbed more media than those submerged in
less media.
40
45
50
55
60
65
70
75
0 5 10 15 20
Days
wat
er c
on
ten
t %
Twice a week
Once a week
Only at the beginning
Figure 12 Changes in the percentage of water content in time for three different submersion frequencies.
Ergosterol assay
According with the results from the ergosterol assay the treatment II had the highest mycelium
content (3.1mg dry mycelium/gr substrate), however it is negligible if compared with the
38,34mg dry mycelium/gr compost as average from 21 samples of compost analyzed at the
same time.
Table 19. Results of the ergosterol assay. Average over 2 samples
mg dry mycelium /gr of substrate
treatment I* 1.158
treatment II 3.170
treatment III 1.681
* only one sample
(Treatment I; submerged only once. Treatment II; submerged 3 times. Treatment III;
submerged 6 times)
Details on the results of the calibration and the calculated formula for conversion rate Ergosterol
content –-> mg mycelium, in APPENDIX II.
55
3.5.4 Conclusion
The negative effect of the soaking on mycelium growth was not expected since Agaricus
bisporus, as many more filamentous fungi, is able to grow in submerged conditions and in moist
solid substrates (Griffin 1996). However it has been reported (Wösten et al. 1999),(Joseph G. H.
Wessels 1993)(Unestam & Sun 1995) that to migrate from the aqueous phase to the aerial
phase some physiological changes are required. The aerial hyphae and fruiting bodies are
coated with a thin layer of hydrophobic proteins (Wösten et al. 1999). The assembly of the
hydrophobic proteins creates a impermeable film which insulates hyphae from the environment
(Joseph G. H. Wessels 1993). Those physiological changes are not reversible. The hydrophobin
assemblage is very stable; once the hyphae is covered with hydrophobic proteins they do not
dissemble (J.G.H. Wessels 2000). There is not migration from aerial phase to liquid phase. The
submersion of the aerial hyphae is unnatural and damaging for its growth. The precise reasons
of this negative effect are still unclear.
The moisture in treatments soaked during the experiments (treatments 2 and 3) may be
excessive for mycelium development. Sanchez and Royse (2001) in their experiments looking
for an alternative substrate to compost, observed that the optimum moisture content in the
shiitake substrate when cultivating A. bisporus ranged from 50 to 52%. These values are
considerably below of the desired values for compost (68%). That shows that A. bisporus does
not have an universal moisture content requirement but it varies depending on the substrate
and growing conditions. Therefore we can conclude that it is not advisable to have compost as
only reference point when looking for alternative substrates or carriers.
Mycelium development may also have been affected negatively by the forced air renewal inside
the substrate. Generally mycelium of Agaricus bisporus during the vegetative stage prefers
environments with high concentrations of carbon dioxide (Long & Jacobs 1974). Soaking the
container may replace the internal air with lower CO2 concentrated air.
The Ergosterol assay showed that the mycelium content in the inert carrier was only about 10%
of what was present in compost. The conversion factor (Ergosterol � biomass dry mycelium )
was calculated from a calibration made with compost and fruit bodies, instead that with pumice
and mycelium. There is the possibility that the pumice has an influence in the ergosterol content
not considered in the calibration with compost. For more accurate conversion factor a calibration
with dry mycelium and inert carrier should be performed. Even though we can consider the
results of the ergosterol method an acceptable calculation of biomass in the substrate and the
results were not very positive for the new production system. Furthermore, ergosterol assay is a
time consuming analysis which requires careful handling. Since we are working under sterile
conditions and A. bisporus is the only living organism present in the substrate, we can perform
easier analysis such as protein content.
56
4 General conclusion and recommendations for further research
In this study we defined and investigated the most important aspects in order to develop a
growth model system for A. bisporus suitable for nutritional research. Three main topics were
highlighted; i) optimization of the nutrients media, ii) control of the media acidification and iii)
design of the growing system.
In first place, different monosaccharides, disaccharides and polysaccharides in different
concentrations and in different combinations were tested as carbon sources for mycelium growth.
Complex sugars, such as pectin and guar gum were the most productive carbon sources.
Besides the mixture of different monosaccharides resulted in a remarkable increase in mycelium
production. These findings in nutrition have potential applications in the developing
supplementations for the current substrate.
As a second step in optimization of the nutrients media it was evaluated the relative importance
of four sugars (glucose, fructose, guar gum, pectin) and two nitrogen sources (casein,
asparagine) on A. bisporus mycelium growth. It was concluded that among those six compounds
casein is the nutrient with the highest promoting growing effect. The sugars glucose and guar
gum produced also a favorable response on the mycelial growth. Consequently the optimization
of these 3 compounds is expected to result in a productive nutrient media for A.bisporus.
For controlling the pH a buffer agent has to be included in the medium. The effect of 3 different
buffer agents (BisTris/HCl, BisTris/Citric acid, MES/KOH) on A. bisporus was evaluated. They all
three had a severe inhibitory effect on mycelium growth. MES/KOH was less detrimental than
the two combinations with BisTris. As a result, nutrient solution should include MES/KOH as
buffer agent in the lowest concentration possible to keep the pH in tolerable levels.
Besides a new growing model for A. bisporus was designed. A perforated growing container
filled with a water holding inert carrier was submerged regularly in malt extract. The experiment
showed the negative effect of submerging aerial hyphae. Mycelium was damaged for the
immersions, which decreased colonization rates and delayed fructification. As a result future
designs should allow us to modifying nutrition without the presence of liquid. Besides the water
content in the inert carrier was excessive for the requirements of A. bisporus under those
circumstances.
More work is needed to understand the necessities of A. bisporus in alternative substrates.
Having compost as a point of reference may disturb the investigations since compost has a very
unique characteristics, nutritionally, physically and microbiologically. The alternative substrate
has to sustain A. bisporus fructification but does not have to be a copy of compost. As example,
the average water content in compost is about 69%, this value has been proven excessive in
other types of substrates.
There are many promising compounds which have not been tested as potential nutrients for A.
bisporus. By making use of different experimental designs (i.e. Plucket-Burman) and
57
methodologies (i.e. Surface Response Methodology) it is possible a rapid scanning and
optimization of a wide range of different compounds.
Finally, the search for an alternative substrate will have to include a study of adaptability of
different strains to the new substrate. Mushroom compost has been formulated and improved
for years to meet the demands of the white button mushroom. Besides breeders and growers
have created and selected A. bisporus strains with the best performance in compost. It can be
said that they have been shaped to fit with each other. High yielding strains in compost may not
be the most adequate strains in a compost-free system.
58
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APPENDIX I. Composition of the defined media
Mw (g/mol) Endconcentration in medium Urea 60.06 10.00 mM
MES 195.24 20.00 mM
MgSO4 120.37 1.00 mM
CaCl2 * 2H2O 147.02 0.50 mM
Na2EDTA 372.20 0.13 mM
CuSO4 * 5H2O 249.68 1.00 mM
CoCl2 * 6H2O 237.93 1.00 mM
ZnSO4 * 7H2O 287.54 5.00 uM
MnCl2 197.91 5.00 uM
H3BO3 61.83 4.80 uM
KI 166.01 2.40 uM
(NH4)6Mo7O24 1235.86 52.00 nM
FeSO4 * 7H2O 278.02 25.00 uM thiamine HCL 337.30 0.50 uM D+biotine 244.31 0.10 uM
63
64
APPENDIX Calibration for the conversion rate area ergosterol to dried fruit bodies
biomass.
The conversion rate (ergosterol/dry matter) was calculated by adding specific amounts of dry tissue from fruit bodies to 200mg of dried compost. At the moment of doing the analysis dry mycelium was not available so dry tissue instead of dry mycelium was utilized for the calibration.
The area of ergosterol was corrected by including a factor representing the recovery fraction of the extraction for each sample (Rf). To calculate Rf a fixed amount of dehydrocholesterol was added per sample and by comparing the recovered amount of dehydrocholesterol with the amount added, the recovery fraction of the extraction (Rf)can be calculated. It is assumed the same Rf for ergosterol and for dehydrocholesterol. Table 1.- Detail on the results of the Ergosterol assay.
Sample Weight (mg)
Area ergosterol
rec. fraction*
Area ergosterol corrected
mg mycelium /sample
mg dry mycelium
/ gram substrate
1B pumic 199.75 7400 0.19 38116 0.23130 0.04620 2B pumic 196.90 24261 0.25 98862 0.59992 0.11812 2C pumic 212.30 54666 0.47 115222 0.69919 0.14844 3A pumic 207.90 9095 0.18 51317 0.31140 0.06474 3B pumic 204.70 14133 0.22 62873 0.38153 0.07810
*Table 2.- Calculation of the recovery fraction
Sample Weight (mg) Area 100% rec.
dehyd. rec. fraction dehyd. 1B pumic 199.75 60423 311228 0.19 2B pumic 196.9 76376 311228 0.25 2C pumic 212.3 147659 311228 0.47 3A pumic 207.9 55160 311228 0.18 3B pumic 204.7 69960 311228 0.22
Calibration formula: Where
y = 164793x y = area ergosterol corrected x = mg mycelium
Calibration curve. Area ergosterol vs. mg dry fruit body
y = 164793x + 56295
R2 = 0.9881
0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
0 5 10 15 20 25 30 35 40
mg dry fruit body
Are
a er
gost
erol