Carbon utilization profile of a thermophilic fungus, <i>Thermomyces lanuginosus</i> using phenotypic microarray

doi:10.4236/abb.2013.49A004

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Advances in Bioscience and Biotechnology, 2013, 4, 24-32 ABB

http://dx.doi.org/10.4236/abb.2013.49A004 Published Online September 2013 (http://www.scirp.org/journal/abb/)

Carbon utilization profile of a thermophilic fungus,

Thermomyces lanuginosus using phenotypic microarray

Nokuthula Peace Mchunu1,2*, Kugen Permaul1, Maqsudul Alam2,3, Suren Singh1

1Department of Biotechnology and Food Technology, Durban University of Technology, Durban, South Africa

2Centre for Chemical Biology, University Sains Malaysia, Bayan Lepas, Penang, Malaysia

3Advanced Studies in Genomics, Proteomics and Bioinformatics, University of Hawaii, Honolulu, USA

Email: *nokuthula@dut.ac.za

Received 29 June 2013; revised 30 July 2013; accepted 26 August 2013

License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

The thermophilic filamentous fungus, Thermomyces

lanuginosus produces the largest amount of xylanase

reported. In addition to this, it expresses large

amount of other enzymes that have been used indus-

trially or have academic interest. Thus, this fungus

has a potential to be applied for biomass conversion

to produce biofuel or other applications. In this study,

the Biolog system was used to characterize the utilisa-

tion and growth of T. lanuginosus on 95 carbon

sources. The carbohydrates based compounds, both

single sugars and oligosaccharide, showed the best

utilisation profile, with the pentose sugar xylose in-

ducing the highest growth, followed by trehelose, raf-

finose, D-mannose turanose fructose and glucose.

Among oligosaccharides, sucrose had the highest my-

celium formation followed by stachyose, maltose,

maltotriose, glycogen and dextrin. Interestingly the

fungus also grew well on cellobiose suggesting that

this fungus can produce cellulose hydrolysing pro-

teins. D-alanine was the best amino acid to promote

fungal growth while the effect of other amino acids

tested was similar to the control. These results dem-

onstrate the ability of this fungus to grow relatively

well on most plant based compounds thus making this

fungus a possible candidate for plant biomass conver-

sion which can be applied to a number of biotechno-

logical applications including biofuel production.

Keywords: Filamentous Fungi; Thermophilic; Carbon

Source; Hexose; Pentose

1. INTRODUCTION

The importance of fungi and other microorganisms is

widely acknowledged, primarily due to their application

in biotechnology industries as well as the effects they

have on human health. Fungi are able to produce a vari-

ety of biotechnology products which include industrial

enzymes, enzymes used in bioassays or for diagnostics,

antibiotics, and enzymes involved in bioremediation

[1,2]. During industrial application and scientific re-

search, specific metabolic pathways or molecules that are

related to a particular process are studied in depth. This

however can lead to the overlooking of other molecules

or useful products. The invention of genomics has pro-

duced a wealth of data, however to understand those data

one must understand the relationship of genes within an

organism and the interactions of gene products in me-

tabolism.

The area of studying either gene or protein interactions

on a larger scale is a relatively new field as it has spilled

over from genomics. Although high-throughput screens

for bacteria and unicellular fungi (yeast) using knock-out

experiments are used frequently, this technique is labour

intensive and time consuming. Even after obtaining mu-

tants, methods of characterization can be limited or ex-

pensive as in the case of DNA microarrays. Alternative

approaches for the characterization of functional genes

are being developed and advanced [3]. One approach is

to focus on the effect of a particular gene at a cellular

level and to assess how it affects the organism as a whole.

Therefore, phenotypic characteristics that the organism

displays, become markers (for the effect) of a particular

gene with relatively high certainty. Although phenotyp-

ing has been around for some time, it still provides a

very useful way to describe biological differences be-

tween cells. As such, a specific phenotype is the final

goal of any strain enhancement process for new products

or processes. Therefore a good phenotypic assay method

would be beneficiary in functional genomics [4].

Like many organisms, the natural habitat of fungi in-

*Corresponding author.

OPEN ACCESS

N. P. Mchunu et al. / Advances in Bioscience and Biotechnology 4 (2013) 24-32 25

fluences what phenotype it will display. The natural en-

vironments of fungi involve many factors including, nu-

trients, physical factors and other organisms. The nutri-

ents are the major contributor of phenotypic characteris-

tics, thus assessing nutrient requirements is vital. In the-

ory, a complete phenotyping assay will involve a combi-

nation of hundreds of carbon source, nitrogen sources,

phosphate, sulphur and other nutrients. This will push the

boundaries with assay numbers of hundreds of thousands

when including other physical factors such as tempera-

ture, pH and O2. Such scales are not feasible for most

laboratories due to labour and cost restrictions. The in-

troduction of the Phenotypic MicroArray System (PM)

from Biolog Incorporated (Harvard, California) offers a

viable screening option for most researchers and indus-

tries. The Biolog system is designed for high throughput

screening of different basic nutrient sources, additives

required for growth and antagonistic compounds for nu-

merous microorganisms including filamentous fungi. The

phenotypic assays are designed from a physiological

perspective to survey in vivo function of diverse path-

ways including both metabolic and regulatory pathways.

Included in the tests are basic cellular nutritional path-

ways for C, N, P, and S metabolism, pH growth range

and regulation of pH control, sensitivity to NaCl and

various other ions, and sensitivity to chemical agents that

disrupt various biological pathways. The FF database

also analyzes fungal growth via turbidimetric analysis

(Biolog, Inc, CA). Analysis of both color development

and turbidity provides for extremely accurate identifica-

tions to the species level [5,6].

One of the most desired characteristics of numerous

industries is the ability of an organism to utilize any plant

biomass. Optimizing plant biomass conversion is a pre-

dominant factor identified for improving the production

of an economical biofuel production. One of the obsta-

cles however, is finding a suitable organism that is capa-

ble of converting different carbohydrate compounds and

that has biological and physiological characteristics to be

able to fit in this process. An organism that has a poten-

tial to be applied in this area is T. lanuginosus. Thus

thermophilic filamentous fungus produces a wide range

of thermostable enzyme including a large group of car-

bohydrate hydrolyses. These enzymes include: amylase,

glucoamylase, xylanase, lipase, phytase, protease and

chitinase [2]. These thermostable enzymes can be applied

in different industries including the food industry for the

production of sugar syrup, animal feed industry, pulp and

paper industry and bioremediation/bio-conversion of

waste industry [7]. Based on this organism’s ability to

produce carbohydrate hydrolases and other useful en-

zyme like lipases, it has been proposed that T. lanugino-

sus may contain previously unidentified proteins that

have ability to act on the different carbohydrate material

and this can be anaylsed using the Biolog system. FF

MicroPlate is specifically designed for the testing of car-

bon utilisation in filamentous fungi and yeast, including

species from the genera Aspergillus, Penicillium, Fusa-

rium, Alternaria, Mucor , Gliocladium, Cladosporium,

Paecilomyces, Stachybotrys, Trichoderma, Zygosaccha-

romyces, Acremonium, Beauveria, Botryosphaeria, Bo-

trytis, Candida, and Geotrichum (Biolog, Inc.). This arti-

cle discusses the use of Phenotypic MicroArray using the

FF MicroPlate to assess the ability of T. lanuginosus to

utilize different carbon sources.

2. MATERIALS AND METHODS

The experiments were performed by growing T. lanugi-

nosus on 2% malt extract agar at 50˚C for 5 - 7 days until

spore formation was visible. Global carbon assimilation

proﬁles were evaluated by using Biolog FF MicroPlate

(Biolog, Inc., Hayward, CA). The FF MicroPlate test

panel contains 95 wells, each with a different carbon-

containing compound, and one well with water as control.

The inoculum for the 96 well FF plates for the biology

system was prepared by first soaking a sterile swab then

gently rolling over the plate. The spores were suspended

in 16 ml of FF inoculum media supplied by Biolog in

glass tubes the mixed gently by hand. The spore suspend-

sion used was approximately 75% transmittance at 590

nm using the Biolog Tubidometer. 100 µl of the spore

suspension was added to each well and microplates were

incubated at 50˚C. Sample were done in triplicates and

readings were taken using the Biolog Microstation, at 2 h

intervals until 68 h. Water and tween 80 were used as

controls.

Biolog software was used to measure growth or bio-

mass at the absorbance of 750 nm, while assimilation

(general uptake and usage) was evaluated at 490 nm by

measuring the formation of a reddish-orange colour.

Joining Cluster Analysis was used to group carbon

sources utilized by T. lanuginosus using the Minitab 16

software (Minitab Inc.) and was applied to identify the

different groups of carbon sources from the experimental

data set. The joining cluster analysis was designed by

means of the Euclidean distance with complete linkage.

Out of the 95 compounds used in this analysis for the

purpose of this study, only compound belonging to car-

bohydrates and amino acid groups will be discussed in

details (Figure 1).

3. RESULTS

3.1. Cluster Analysis of Carbon Source

Assimilation and Growth Profiles

Carbon source utilization profiles for T. lanuginosus

were analyzed using cluster analysis. The data generated

was divided into 4 distinct clusters for assimilation

N. P. Mchunu et al. / Advances in Bioscience and Biotechnology 4 (2013) 24-32

A1 Water A2 Tween

A3 N-

Acetyl-D-

Ga l act o sa

mine

A4 N-

Acetyl-D-

Glucosam

ine

A5 N-

Acetyl-D-

Mannosa

mine

Adonitol

Amygdali

A8 D-

Arabinos

A9 L-

Arabinos

A10 D-

Arabitol

A11

Arbutin

A12 D-

Cellobios

B1 α-

Cyclodext

rin

B2 β-

Cyclodext

rin

Dextrin

B4 i-

Erythritol

B5 D-

Fructose

B6 L-

Fucose

B7 D-

Galactose

B8 D-

Ga l acturo

nic Acid

Gentiobio

B10 D-

Gluconic

Acid

B11 D-

Glucosam

ine

B12 α-D-

Glucose

Glucose-

1-Phosph

ate

Glucuron

amide

C3 D-

Glucuroni

c Acid

Glycerol

Glycogen

C6 m-

Inositol

C7 2-

Keto-D -

Gluconic

Acid

C8 α-D-

Lactose

Lactulose

C10

Maltitol

C11

Maltose

C12

Maltotrios

D1 D-

Mannitol

D2 D-

Mannose

D3 D-

Melezitos

D4 D-

Melibiose

D5 α-

Methyl-D-

Galactosi

D6 β-

Me t hyl-D -

Ga l actos i

D7 α-

Me t hyl-D -

Glucoside

D8 β-

Methyl-D-

Glucoside

Palatinos

D10 D-

Psicose

D11 D-

Raffinose

D12 L-

Rhamnos

E1 D-

Ribose

Salicin

Sedohept

ulosan

E4 D-

Sorbitol

E5 L-

Sorbose

Stachyos

Sucrose

E8 D-

Tagatose

E9 D-

Trehalose

E10

Turanose

E11

Xylitol

E12 D-

Xylose

F1 γ-

Amino-

butyric

Acid

Br omosu

ccinic

Acid

Fumaric

Acid

F4 β-

Hydroxy-

butyric

Acid

F5 γ-

Hydroxy-

butyric

Acid

F6 p-

Hydroxyp

henyl-ace

tic Acid

F7 α-Keto

glutaric

Acid

F8 D-

Lactic

Acid

Methyl

Ester

F9 L-

Lactic

Acid

F10 D-

Malic

Acid

F11 L-

Malic

Acid

F12

Quinic

Acid

G1 D-

Sacchari

c Acid

Sebacic

Acid

Succinam

ic Acid

Succinic

Acid

Succinic

Acid

Mono-

Methyl

Ester

G6 N-

Acetly-L-

Glutamic

Acid

Alaninami

G8 L-

Alanine

G9 L-

Alanyl-

Glycine

G10 L-

Asparagi

G11 L-

Aspartic

Acid

G12 L-

Glutamic

Acid

H1 Glycyl-

L-

Glutamic

Acid

H2 L-

Ornithine

H3 L-

Ph e n yla la

nine

H4 L-

Proline

H5 L-

Pyrogluta

mic Acid

H6 L-

Serine

H7 L-

Threonine

H8 2-

Amino

Ethanol

Putrescin

H10

Adenosin

H11

Uridine

H12

Adenosin

e-5'-

Monopho

sphate

Figure 1. 95 Carbon sources found in FF MicroPlate from Biolog, Inc.

(Figure 2) and for biomass (Figure 3). The analysis for

general assimilation showed that cluster I and II contain

carbon sources that lead to very slow biomass formation.

The most dominant compounds in these clusters are

amino acids, except for alanine, and organic acids, esters,

alcohols, phosphorylated sugars, rare sugars, rare poly-

mers, a nucleotide and aromatics groups. Water (control)

was grouped in cluster II not I as it had higher assimila-

tion rate. The trend was similar when growth was ana-

lyzed with exception that cluster I was bigger than clus-

ter II (Figure 3). Amino acids and some carbohydrates

are also identified to give slow formation of biomass in

these clusters. The other difference was that water had

moved down to cluster I while tween 80 shifted up to

cluster II.

Cluster III (assimilation) showed good assimilation for

T. lanuginosus. This cluster contained mainly carbohy-

drates which are monosaccharide (sorbose, galactose,

arabinose, ribose fucose and rhaminose), disaccharides

(Lactose and Lactoluse), oligosaccharides and polysac-

charides (cyclodextrine, tagose, gentibiose amd meli-

biose), some amino acids (asparagine and alanyl-glycine)

and alcohol (sorbitol, glycerol, Maltitol and xylitol).

Cluster IV contained carbon sources that enabled the

fastest growth and included several monosaccharides,

oligosaccharides (xylose, glucose, raffinose, glucose,

fructose, cellobiose, maltose arabitol, NA-glucosamine,

etc.). Growth analysis revealed that compounds found in

Cluster III and IV were similar to those found in assimi-

lation analysis cluster IV, however the cluster proportions

were different. In growth analysis, most of the carbon

sources clustered in group III, while only three carbon

sources found in cluster IV (xylose, NAG and sucrose)

which were classified as yielding higher biomass. Sur-

prisingly, cellobiose also showed good biomass produc-

tion and was clustered in group III.

3.2. Hexoses and Pentoses

Analysis of these 95 carbon sources was done further by

analyzing carbon sources that fell into the following spe-

ific groups, hexose and pentose, oligosaccharides and c

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N. P. Mchunu et al. / Advances in Bioscience and Biotechnology 4 (2013) 24-32 27

Figure 2. Joining cluster analysis applied to 95 carbon sources based on their assimilation and utili-

zation of carbon sources by T. lanuginosus measured at 490 nm using the Biolog system (the standard

deviation for absorbance values was an average of 0.041).

amino acid based compound and the rest were not as-

sessed further. Analysis of hexose and pentose utiliza-

tion revealed maximal assimilation of xylose followed by

trehalose, NAG and mannose (Figures 3 and 4). Xylose

exhibited 15% more assimilation than the second best

compound trehalose with absorbance values of 3.1 and

2.6, repectively (Figure 4). Fructose, raffinose, glucose

nd turanose also showed good general assimilation. The a

N. P. Mchunu et al. / Advances in Bioscience and Biotechnology 4 (2013) 24-32

Figure 3. Joining cluster analysis applied to 95 carbon sources based on the growth of T. lanu-

ginosus on these carbon sources. Groth was measured at 750 nm using the Biolog system.

biomass production showed that again xylose produced

the highest biomass followed by NAG and trehalose

(absorbance values, 1.4, 1.36 and 1.23, respectively, Fig-

ure 5). These were followed by glucose, mannose and

raffinose with absorbance above 2, among the better

hexose and pentose sugars. Water assimilation was meas-

ured at 1.36 and for tween 80 at 1.11. Nevertheless when

the effect on growth was analyzed, tween 80 showed

better biomass promotion than water with absorbance of

0.68 and 0.33, respectively.

3.3. Oligosaccharides

In oligosaccharide analysis, sucrose exhibited the best

assimilation followed by maltotriose, stachyose, maltose,

extrin and glycogen (Figure 6). Cellobiose also showed d

N. P. Mchunu et al. / Advances in Bioscience and Biotechnology 4 (2013) 24-32 29

Figure 4. Assimilation of monomeric sugars (hexose and pentose) by T. lanuginosus SSBP. The as-

similation was measured at an absorbance of 490 nm for 68 hours at 2 hour intervals.

Figure 5. Growth of T. lanuginosus SSBP in monomeric sugars (hexose and pentose). The growth

was measured at an absorbance of 750 nm for 68 hours at 2 hour intervals.

Figure 6. Assimilation of oligosaccharides by T. lanuginosus SSBP. The assimilation was measured

at an absorbance of 490 nm for 68 hours at 2 hour intervals.

N. P. Mchunu et al. / Advances in Bioscience and Biotechnology 4 (2013) 24-32

relatively good general assimilation. Water assimilation

was lower than most of common carbohydrates while the

assimilation of rare occurring carbohydrate compounds

was even lower than water and tween 80 (sedoheptulose

and gentibiose). In biomass production sucrose again

produced the most biomass followed by maltose, glyco-

gen, maltose, stachyose, palantiose, cellobiose and dex-

trin (Figure 7). Again common carbohydrate compounds

supported more biomass production in T. lanuginosus

than rare compounds.

3.4. Amino A cids

Amino acid analysis, L-alanine displayed the best as-

similation followed by proline, asparagine, and glutamic

acid (Figure 8). Gylcyl-glutamic acid gave the lowest

assimilation even lower than water and tween 80. It was

also noted that although most of the amino acid base

compounds had high assimilation, they were unable to

support significant biomass production. In biomass pro-

duction L-alanine yielded greater biomass when com-

pared to other amino acids (Figure 9). The rest of the

amino acid compounds produced less biomass than

tween 80 but more than water except for threonine which

was lower.

4. DISCUSSION

In nature the ability of a microorganism to use a variety

of compounds is vital for survival in composting envi-

ronment as different substrates are degraded and utilised

by different organisms. Filamentous fungi play a vital

role in this ecological dynamics as they are responsible

for the majority of the hydrolysis [8,9]. T. lanugiosus is

Figure 7. Growth of T. lanuginosus SSBP in oligosaccharide compounds. The growth was measured at an

absorbance of 750 nm for 68 hours at 2 hour intervals.

Figure 8. Assimilation of amino acid based compounds by T. lanuginosus SSBP. The assimilation was

measured at an absorbance of 490 nm for 68 hours at 2 hour intervals.

N. P. Mchunu et al. / Advances in Bioscience and Biotechnology 4 (2013) 24-32 31

Figure 9. Growth of T. lanuginosus SSBP in amino acid based compounds. The growth was measured at

an absorbance of 750 nm for 68 hours at 2 hour intervals.

among those fungal organisms that thrive in such envi-

ronments with an added ability to survive high tempera-

ture which is only for a select few eukaryotic organisms

[10]. The analysis of carbon source assimilation and

utilization for biomass production in this organism re-

vealed a similar profile to other filamentous fungi studies

of this nature where glucose, xylose, trehalose and NAG

produced high biomass in Trichoderma reesei and As-

pergillus niger [5]. Although the clusters in these studies

were similar to our findings, closer analysis of Cluster IV

revealed that for T. lanuginosus, xylose is the preferred

sugar compared to glucose. This concurs with reports

that T. lanuginosus has the most powerful system for

xylanase production and xylose utilization and thus it

was expected that xylose would produce the most bio-

mass and have the highest assimilation [11-13]

However, the most interesting finding was the high

cellobiose utilization as this organism is well reported as

a cellulose free organism. T. lanuginosus has been pre-

viously described as non-cellulolytic and it was sug-

gested that it probably relies on commensal relationships

in composts with cellulolytic fungi [13-15]. In this study,

growth on cellobiose suggests that this fungus produces

enzymes that have cellulose related activity. This is in

agreement with unpublished data on genome sequencing

of this fungus revealing that 8 predicted genes are with

the possibility of having cellulose activity. Of the 8 genes,

3 were similar to Trichoderma reesei cellulases and the

others to Aspergillus kawachi [16,17].

Trehalose also produced good biomass and assimila-

tion in T. lanuginosus. The suggested reason for this is

that trehalose is used by the organisms as an energy

source; however there is a more important reason in

thermophilic organisms. Trehalose has been widely re-

ported as a part of the physiological adaptation to various

environmental stresses e.g. high temperature, in yeasts

and filamentous fungi [18]. NAG also had high assimila-

tion and biomass production because it is the building

block of fungal cell walls which contain chitin and also

can be converted to energy molecule, therefore high as-

similation and the ability to support growth were ex-

pected [19]. It was surprising that only one amino acid,

alanine, produced significant biomass. This may be be-

cause alanine is one of the few amino acids that can

transform into glucose and can be used in TCA cycle to

provide energy for the cell, thus it may be preferred by

this fungus to supplement the supply of mineral nitrogen

and energy [20].

In conclusion, this study indicates that T. lanuginosus

is a versatile organism that can utilize a diverse range of

carbon sources, including carbohydrates, amino acids,

carboxylic acids, polymers, aromatics, esters, phos-

phorylated and sugar alcohols. The application of Phe-

notypic Array as a tool of carbon utilization studies is a

quick approach to studying and assessing filamentous

fungi for specific activities.

5. ACKNOWLEDGEMENTS

This study was supported by grants from the National Research Foun-

dation, Republic of South Africa and collaboration with the Centre for

Chemical Biology, University Sains Malaysia. The authors are thankful

to Dr. Alison Winger, a Research Associate in Dept. Biotechnology

and Food Technology Durban University of Technology, for valuable

suggestions and critical evaluation of the manuscript.

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