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INTERNATIONAL SYMPOSIUM

PRODUCTION OF POLYPORUS SQUAMOSUS BIOMASS IN SUBMERGED CULTURE WITH


ALTERNATIVE CARBON SOURCE
/
OBINEREA DE BIOMAS DIN POLYPORUS SQUAMOSUS N CULTUR SUBMERS CU
SURSE DE CARBON ALTERNATIVE
Ass.Prof. Ph.D. Mariana Ferde, Lecturer Ph.D. Eng. Mirela Dinc
Universities Polytechnic Bucharest, Faculty of Biotechnical Systems Engineering / Romania
Tel: 0729821256; E-mail: marianaferdes@yahoo.com

Abstract: Production of biomass from edible


mushrooms like Polyporus squamosus, dryads saddle,
in submerged cultures is influenced by the type of
utilised carbon source. The aim of this paper was to
test several culture media containing different sugars
in order to obtain biomass from this known fungus.
The fungal strain was cultivated on the orbital
incubator on malt extract broth, potato dextrose broth,
Czapek Dox broth and molasses. The largest amount
of mycelium and the highest biomass yield were
registered for the culture medium containing sugar
beet molasses and salts that favored the growth of
hyphae and thus the accumulation of biomass.

Rezumat: Obinerea de biomas din ciuperci comestibile


cum este Polyporus squamosus, buretele de nuc, n
culturi submerse este influenat de tipul sursei de
carbon utilizate. Scopul lucrrii a fost testarea mai multor
medii de cultur coninnd diferite zaharuri pentru
obinerea de biomas din acest fung cunoscut. Tulpina
fungic a fost cultivat pe un incubator orbital pe mediile
malt extract broth, potato dextrose broth, Czapek Dox
broth i melas. Cea mai mare cantitate de miceliu i cel
mai bun randament de producie s-au nregistrat pe
mediul cu melas din sfecl de zahr i sruri care au
favorizat creterea hifelor i acumularea de biomas
proteic fungic.

Keywords: microbial biomass, Polyporus squamosus,


submerged culture, carbon source

Cuvinte cheie: biomas microbian, Polyporus squamosus,


cultur submers, sursa de carbon

INTRODUCTION
Many biotechnological processes have been
developed to isolate microorganisms able to use organic
material as a source of carbon and energy and to convert
inorganic nitrogen into high-food-value proteins. These
can be used in human foodstuffs or animal feed to
replace traditional plant or animal sources [1,2,9]. The
main criteria for selection of microorganisms are the
nature of the raw material, the nutritional value, protein
content, amino acid balance of biomass, the type of
culture, nutritional requirements, type of separation,
toxicological characteristics, the high specific growth rate,
the ability to develop high cell density [2,5,6,7].
The principal properties of fungi are their ability to use
a large number of complex growth substances such as
sugars, cellulose and starch and easy recovery by simple
filtration, reducing production costs.
The most important aspects of the quality of fungal
biomass relate to: nutritional value of the product, safety
of the product, production of protein concentrate with low
content of nucleic acid and toxic substances.
Wild mushrooms are becoming more and more
important in our diet for their nutritional, organoleptic, and
pharmacological characteristics. Based on their high
mineral content, low fat, and low calories, the nutritional
values of mushrooms have already been reevaluated.
Additionally, mushrooms have been known to have an
effect on preventing several diseases such as cancer,
hypercholesterolemia, and hypertension.
Many studies have focused on biotechnological
processes for artificial cultivation of mushrooms.
Polyporus squamosus is one of the most important
cultivated mushroom for food purpose and also it is a well
studied white-rot fungus [1,3].
Polyporus squamosus belongs to the family
Polyporaceae and grows on dead wood of various tree
especially walnut, but several species are found on other
substrates [8,10,11]. Polyporus squamosus is an edible
mushroom that has a desirable aroma and taste and
adds texture to a meal [4].

INTRODUCERE
Un mare numr de procese biotehnologice au avut ca
scop izolarea microorganismelor capabile s utilizeze
diferite substrate organice ca surs de carbon i energie
i s transforme azotul anorganic n biomas proteic cu
valoare nutritiv ridicat. Acest tip de proteine pot fi
utilizate n alimentaia omului i n furajarea animalelor i
pot nlocui sursele conventionale provenite din plante i
animale [1,2,9]. Principalele criterii de selecie a
microorganismelor sunt natura materiilor prime, valoarea
nutritiv, coninutul de protein i aminoacizi, tipul culturii,
cerinele nutriionale, modul de separare din mediul de
cultur, caracteristicile toxicologice, viteza maxim
specific de cretere,
proprietatea de a dezvolta
densiti celulare ridicate [2,5,6,7].
Principala caracteristic a fungilor este capacitatea lor
de a utiliza un mare numr de substane complexe de
cretere cum sunt zaharurile, celuloza i amidonul, de a fi
uor separate prin filtrare i un pre sczut.
Cea mai important calitate a biomasei fungice se
refer la valoarea sa nutritiv, sigurana i obinerea de
concentrate proteice cu coninut sczut de acizi nucleici
i substane toxice.
Ciupercile devin din ce n ce mai importante pentru
dieta omului datorit caracteristicilor nutriionale,
organoleptice i farmacologice. Avnd n vedere
concentraia optim de minerale, valorii sczute a
lipidelor i coninutului caloric, valoarea nutriional a
ciupercilor este reevaluat. n plus, se pare c unele
ciuperci pot preveni unele boli cum sunt forme de cancer,
hipercolesterolemia i hipertensiunea.
Au fost efectuate numeroase studii privind procesele
biotehnologice de cultivare artificial a ciupercilor.
Polyporus squamosus este una din cele mai importante
ciuperci cultivate n scop alimentar implicate i n
putrezirea materialului lemnos [1,3].
Polyporus squamosus face parte din familia
Polyporaceae i crete n natur pe diferite specii de
arbori n special nuc, dar unele specii pot utiliza i alte
1

INTERNATIONAL SYMPOSIUM
substrate [8,10,11]. Polyporus squamosus are arom i
gust plcute i confer textur alimentelor [4].
MATERIAL AND METHOD
Fungal strain
The biomass production was performed using a fungal
strain of Polyporus squamosus from the culture collection
of Laboratory of Microbiology, Faculty of Biotechnical
Systems Engineering. The strain was cultured in tubes
o
on potato agar medium and preserved at 4 C.
Culture media
Several liquid culture media were used for the fungal
grow: malt extract broth (Merck), potato dextrose broth
(Difco), Czapek Dox broth (Oxoid) and a medium
containing sugar beet molasses 100.g/L, ammonium
nitrate 3 g/L, ammonium phosphate 1 g/L. The fungal
strain was kept as stock culture in tubes on agar slant
medium.
Fungal growth and biomass development
To estimate the growth rate on solid medium, the
Polyporus strain was inoculated in Petri dishes onto
potato dextrose agar medium and the diameter of colony
was measured each day. The growth rate was calculate
as diameter of colony (mm) / time (hours).
Biomass production was carried out in 500 mL
o
Erlenmeyer flasks, on a rotary shaker, at 25 C, 150 rpm,
for 72 hours. The samples were collected every day and
the amount of wet biomass, the dry matter (using a
o
thermobalance at 103 C), the total soluble solids
(refractometer method), the pH were analyzed. The
microscopic aspect of cultures after 72 hours of growth
was described for all culture media.

MATERIALE I METODE
Tulpina fungic
Producerea de biomas a fost efectuat cu o tulpin
fungic de Polyporus squamosus din colecia de culturi a
Laboratorului de Microbiologie din Facultatea de Ingineria
Sistemelor Biotehnice. Tulpina a fost cultivat n
eprubete pe mediul potato dextrose agar i conservat la
o
4 C.
Medii de cultur
Pentru creterea microorganismului au fost folosite
cteva medii de cultur lichide: malt extract broth
(Merck), potato dextrose broth (Difco), Czapek Dox broth
(Oxoid) i un mediu coninnd melas din sfecl de zahr
100 g/L, azotat de amoniu 3 g/L i fosfat de amoniu
1 g/L. Cultura stoc pentru tulpina fungic a fost pstrat
n slanturi pe medii agarizate.
Creterea fungic i dezvoltarea biomasei
Rata de cretere a tulpinii de Polyporus s-a determinat
pe mediul potato dextrose agar n cutii Petri prin
msurarea zilnic a diametrului coloniei. Rata de cretere
a fost calculata ca diametrul coloniei (mm) / timp (ore).
Producerea biomasei a fost efectuat n flacoane
o
Erlenmeyer de 500 mL pe un incubator rotativ la 25 C,
150 rpm timp de 72 de ore. Au fost recoltate zilnic probe
n care s-a determinat cantitatea de biomas umed,
o
biomasa uscat (la termobalan la 103 C), substanele
solubile totale (prin metoda refractometric) i pH. Pentru
toate mediile de cultur a fost descris aspectul
microscopic al culturilor.

RESULTS
Polyporus squamosus strain developed onto potato
o
dextrose agar at 25 C forms white, fluffy colonies, with a
growth rate of 0,57 mm/h. The growth and the aspect of
Polyporus colony are shown in figure 1.

REZULTATE
Tulpina de Polyporus squamosus dezvoltat pe
o
potato dextrose agar la 25 C formeaz colonii albe,
pufoase, cu o vitez de cretere de 0,57 mm/or.
Creterea i aspectul coloniei de Polyporus sunt
prezentate n figura 1.

100

80
60

40

Diameter,
mm

20
0
a

Fig.
The growth
0 12 day
4 dayof6Polyporus
day colony (a) and the morpho-colonial aspect on potato dextrose agar medium
(English language)

In liquid culture media, the accumulation of fungal


biomass has started from day 1, correlated with the
depletion of solid soluble substances. For each culture
medium the dry biomass, the total soluble solids and pH
values are presented in figure 2.

n medii lichide acumularea de biomas fungic a


nceput din prima zi, proces corelat cu epuizarea
substanelor solide solubile. Pentru fiecare mediu de
cultur valorile biomasei uscate, substanei solide
solubile totale i pH-ului sunt prezentate n figura 2.

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2.5 DW, g/200mL

Malt extract broth


Potato dextrose broth
Czapek broth
Molasses

2
1.5
1
0.5

0
0h

24 h

48 h

Time

72 h

6 TSS %
5
Malt extract broth
Potato dextrose broth
Czapek broth
Molasses

4
3
2
1
Time

0
0h

24 h

48 h

72 h
b

Malt extract broth


Potato dextrose broth
Czapek broth
Molasses

pH 4
2

0
0h

24 h

48 h

72 h

Time
c

Fig. 2 - Variations of the average values of dry biomass,(a), total soluble solids (b) and pH (c) during the growth of Polyporus squamosus
strain on malt extract broth, potato dextrose broth, Czapek Dox broth and molasses
((English language))

The culture media used in this experiment contain


different amounts of carbon sources: in malt extract
medium the main carbon source is represented by
maltose and glucose; potato dextrose broth contains
potato infusion and glucose; in Czapek Dox medium the
unique carbon source is sucrose. The molasses medium
is more complex because of the variable composition of
sugar beet molasses,with a content of about 50% sugar,
minerals (calcium, phosphorus, potassium, sodium,
magnesium), amino acids, organic acids, betaine, other
compounds.
The wet biomass was easily separated from culture
medium by filtration (an obvious technological
advantage) and the results for dry biomass determined
using a thermobalance are shown in figure 1a.
As can be seen in this figure, the micelial growth
begins from the first day. The Polyporus fungus is
characterised by a rapid growth, a short lag phase and a
short generation time. The previous studies have
demonstrated that for a longer culture time (in this case
72 hours), the fungal biomass accumulation slows, and
the texture of mycelium is altered.

Mediile de cultur utilizate n experiment conin diferite


cantiti de substane sursa de carbon: n mediul cu
extract de mal principala surs de carbon este
reprezentat de maltoz i glucoz; mediul potato
dextrose broth conine infuzie de cartof i glucoz; n
mediul Czapek Dox zaharoza este unica surs de
carbon. Mediul cu melas este mai complex din cauza
compoziiei variabile a melasei din sfecl de zahr, cu un
coninut de aproximativ 50% sucroz, minerale (calciu,
fosfor, potasiu, sodiu, magneziu), aminoacizi, acizi
organici, betain, ali compui.
Biomasa umed a fost uor separat prin filtrare din
mediul de cultur (un avantaj tehnologic evident);
rezultatele pentru biomasa uscat determinat la o
termobalan sunt prezentate n figura 1a.
Dup cum se poate observa din figur, creterea
miceliului ncepe din prima zi. Fungul Polyporus se
caracterizeaz printr-o cretere rapid, o faz de lag
scurt i un timp de generaie mic. Studiile anterioare au
demonstrat c pentru un timp de dezvoltare mai lung (n
acest caz 72 de ore), acumularea de biomas fungic
ncetinete i textura miceliului este alterat.
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INTERNATIONAL SYMPOSIUM
The values of substrate content were recorded as TSS
(total soluble solids, refractometric) and are presented in
figure 1b. The consumption of nutrients begins in the first
24 hours and it is significant increased in the last day.
The pH values in culture liquid differ considerably
depending on the composition of media, but the
variations are in neutral area (figure 1c).
In table 1 the values of biomass (g/L), substrate
consumption (g/L) and biomass yield are shown. The dry
biomass (g/L) varied considerably for each culture
medium with different carbon source: in potato dextrose
broth containing glucose and infusion of potato as main
carbon source the determined value was 4.3 g/L (the
lowest amount). For sucrose in molasses as carbon
source, the dry biomass was 9.8 g/L .
The highest yield ( biomass / consumed substrate),
29% is recorded for the molasses medium, containing
water, molasses and salts. In the malt extract medium the
growth was poor. Thus, to produce large amount of
fungal biomass the use of cheap culture media like sugar
beet molasses represents an effective way.

Valorile concentraiei substratului au fost nregistrate


ca TSS (substane solide solubile, refractometric) i sunt
prezentate n figura 2b. Consumul de nutrieni ncepe din
primele 24 de ore i este semnificativ crescut n ultima zi.
Valorile pH n lichidul de cultur difer considerabil n
funcie de compoziia mediului, dar variaiile se situeaza
n zona neutr (figura 1c).
In tabelul 1 sunt prezentate valorile biomasei ((g/L),
consumului de substrat (g/L) si ale randamentului n
biomas. Biomasa uscat variaz considerabil pentru
fiecare mediu de cultur cu diferite surse de carbon: n
potato dextrose broth coninnd glucoz i infuzie de
cartof ca surs principal de carbon valoarea
determinat a fost 4,3 g/L (cea mai mic valoare). Pentru
zaharoza din melas ca surs de carbon biomasa uscat
a fost 9,8 g/L.
Cel mai bun randament (biomasa / substrat
consumat), 29% s-a nregistrat pentru mediul cu melas
coninnd ap, melas i sruri. n mediul cu extract de
mal creterea a fost slab. Astfel, pentru a produce
cantiti mari de biomas fungic utilizarea unui mediu de
cultur ieftin ca melasa din sfecl de zahr reprezint o
modalitate eficient.

Table 1 (English language)


Values of wet and dry biomass (after 72 hours), substrate consumption and biomass yield (English language)
Culture medium

Wet biomass (g/L)

Dry biomass (g/L)

Czapek Dox broth


Potato dextrose broth
Malt extract broth
Molasses

36.95
23.55
22.5
61.25

7.66
3.27
4.32
9.86

Substrate
consumption (g/L)
27
13
19
34

Biomass yield (%)


28.3
25.1
22.7
29.0

Imaginile miceliului de Polyporus squamosus


prezentate n figura 2 demonstreaz o cretere bun
pentru toate substratele, dar cele mai bune condiii par
s fie n cazul mediului cu melas. Hifele formeaz
structuri filamentoase care faciliteaz filtrarea biomasei
dup cum se poate vedea n primele 4 imagini (a, b, c,
d). Pe lamele microscopice miceliul fungic se prezint
ca numeroase hife vegetative cu diametrul de 1-5 m,
care se extind continuu prin vrfurile extreme. Asocierile
hifale sunt vizibile n special n mediul de cultur cu
melas (figura 2h).

The images of Polyporus squamosus mycelium


shown in figure 2 demonstrate a good growth on all the
substrates, but the best conditions seems to be the
molasses culture medium. The hyphae form filamentous
structures that facilitate the filtration of biomass as can
see in the first 4 images (a, b, c, d). On microscopic
slides the fungal mycelium consists of numerous
vegetative hyphae, 1-5 m in diameter, that extend
continously at their extreme tips. Especially in molasses
culture medium the hyphal associations are visible
figure 2h).

e
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Fig. 2 Images of fungal biomass produced in malt extract broth (a, e), potato dextrose
broth (b, f), Czapek Dox broth (c, g) and molasses (d, h) media (a, b, c, d mycelium in liquid media, e, f, g, h hyphae on microscope
camera)
(English language)

In all liquid media, Polyporus squamosus formed white


cotton-like micelium and increased the viscosity of
culture medium (figure 2 a, b, c, d). This fungus has a
high grow rate and produces large amounts of biomass
in culture media containing sugars as carbon source.
The metabolic versatility of the genus Polyporus
makes this fungal group can be grown in most varied
conditions and in different nutritive media.

n toate mediile lichide, Polyporus squamosus


formeaz miceliu cu aspect de fibre de bumbac i
determin creterea vscozitii (figura 2 a, b, c, d).
Fungul are o rat de cretere nalt i produce cantiti
mari de biomas n mediile de cultur coninnd zaharuri
ca surs de carbon.
Versatilitatea metabolic a genului Polyporus face ca
fungii din acest grup s poat fi crescui n cele mai
variate condiii i n diferite medii nutritive.

CONCLUSIONS
Microorganisms such as bacteria, algae and fungi
have been considered as protein sources. Culture of
fungi for its nutritional value using carbon substrates
such as molasses is well known and best accepted by
consumers.
Polyporus squamosus is a edible mushroom with a
good taste and flavor that can be grown in submerged
culture on nutritive media.
A Polyporus squamosus strain was cultivated to
produce microbial biomass using different carbon
sources: glucose, maltose, sucrose, potato infusion,
molasses contained in four culture media.
The dry weight, pH, total soluble solids values were
analyzed for: malt extract broth, potato dextrose broth,
Czapek Dox broth and molasses culture medium. The
o
fungal strain was grown on an orbital shaker, at 25 C,
150 rpm, for 72 hours.
The mycelium was easily separated from the culture
and then dry to obtain a biomass used in food industry
and in feedstuff.
The highest value of biomass yield (29%) was
recorded for molasses culture, containing sucrose as
carbon source, ammonium nitrate and ammonium
phosphate.
On microscopic slides, the fungal culture consists of
vegetative hyphae with 1-5 m in diameter that forms
thicker filaments especially in molasses medium.

CONCLUZII
Microorganismele ca bacteriile, algele i fungii sunt
considerate ca surse de proteine. Cultura fungilor pentru
valoarea lor nutritiv utiliznd surse de carbon ca melasa
este cunoscut i cel mai bine acceptat de consumatori.
Polyporus squamosus este o ciuperc comestibil
cu un gust i arom bune, care poate fi crescut n
cultur submers pe medii nutritive.
O tulpin de Polyporus squamosus a fost cultivat n
scopul producerii de biomas utiliznd diferite surse de
carbon: glucoz, maltoz, zaharoz, infuzie de cartof,
melas coninute n patru medii de cultur.
Valorile biomasei uscate, pH-ului, substanelor
solubile totale au fost analizate pentru urmtoarele
medii de cultur: malt extract broth, potato dextrose
broth, Czapek Dox broth i melas. Tulpina fungic a
o
fost crescut pe un incubator orbital la 25 C, 150 rpm
timp de 72 ore.
Miceliul a fost separat din cultur i apoi uscat pentru
obinerea de biomas utilizat n industria alimentar i
n furajarea animalelor.
Cea mai mare valoare a randamentului n biomas
(29%) s-a nregistrat pentru mediul de cultur cu melas,
coninnd zaharoz ca surs de carbon, azotat de
amoniu i fosfat de amoniu.
Pe preparatele microscopice, cultura fungic const
din hife vegetative cu diametrul de 1-5 m, care
formeaz filamente mai groase n special n mediul cu
melas.

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[2]. Archer D.B., Connerton I.F., MacKenzie D.A., (2008)
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