Appl Microbiol Biotechnol (2006) 72: 1063–1073DOI 10.1007/s00253-006-0376-8

APPLIED MICROBIAL AND CELL PHYSIOLOGY

Li Miao . Theresa F. N. Kwong . Pei-Yuan Qian

Effect of culture conditions on mycelial growth, antibacterialactivity, and metabolite profiles of the marine-derived fungusArthrinium c.f. saccharicola

Received: 30 November 2005 / Revised: 6 February 2006 / Accepted: 8 February 2006 / Published online: 15 March 2006# Springer-Verlag 2006

Abstract The effects of culture conditions and competitivecultivation with bacteria on mycelial growth, metaboliteprofile, and antibacterial activity of the marine-derivedfungus Arthrinium c.f. saccharicola were investigated. Thefungus grew faster at 30°C, at pH 6.5 and in freshwatermedium, while exhibited higher antibacterial activity at25°C, at pH 4.5, 5.5, and 7.5, and in 34 ppt seawatermedium. The fungus grew faster in a high-nitrogen mediumthat contained 0.5% peptone and/or 0.5% yeast extract,while exhibiting higher bioactivity in a high-carbon mediumthat contained 2% glucose. The fungal growth was inhibitedwhen it was co-cultured with six bacterial species,particularly the bacterium Pseudoalteromonas piscida. Theaddition of a cell free culture broth of this bacteriumsignificantly increased the bioactivity of the fungus.Metabolite profiles of the fungus revealed by gas chroma-tography (GC)-mass spectrometry showed clear differenceamong different treatments, and the change of relative areaof three peaks in GC profile followed a similar trend with thebioactivity variation of fungal extracts. Our results showedclear differences in the optimal conditions for achievingmaximal mycelial growth and bioactivity of the fungus,which is important for the further study on the masscultivation and bioactive compounds isolation from thisfungus.

Introduction

Microbes are vast and largely untapped resources of novel,structurally diverse metabolites. Many of these metabolitespossess highly valuable bioactivities to humans. In general,metabolite biosynthesis in microbes is tightly controlled by

regulatory mechanisms to avoid overproduction; yet, theseregulatory mechanisms often limit the discovery of novelmetabolites and also the yield of bioactive metabolitesthrough fermentation process to undesirably low levels. Agood understanding of the role of culture conditions in thebiosynthesis of metabolites may lead to better exploitationof microbial metabolites.

To find novel compounds with promising bioactivities,many high-cost methods such as high-throughput screen-ing of different biological sources have been employed(Grabley and Thiericke 1999; Maier et al. 1999). Alter-natively, an effective screening process can be achievedthrough systematic manipulation of culture conditions for asmall number of promising organisms. In fact, cultureconditions have a major impact on the growth of microbesand the production of microbial products. As far as cultureconditions are concerned, there is usually a dilemmabetween achieving maximal growth rates and maximalantibiotic yields because conditions that allow fast cellgrowth could be unfavorable to metabolite production(Audhya and Russell 1974; Frisvad and Samson 1991;Chisti and Moo-Young 1993). The yield of bioactivecompounds can sometimes be substantially increased bythe optimization of physical (temperature, salinity, pHvalue, and light) and chemical factors (media components,precursors, and inhibitors) for the growth of microbes(Calvo et al. 2002; Llorens et al. 2004).

On the other hand, the competitive cultivation betweendifferent microbial taxa has been suggested as a new tool toboost the discovery of novel compounds (Mearns-Spragget al. 1997). However, for the studies on marine microbes,so far there has only been one study demonstrated that amarine fungus produced a novel antibiotic compound onlywhen co-cultured with a marine bacterium (Cueto et al.2001). Therefore, antagonistic responses stimulated bychemical signals produced by potential competitorscertainly deserve closer examination.

Arthrinium saccharicola, first reported by Stevens(1917), is usually found on deteriorated sugarcane. Themycotoxin β-nitropropionic acid produced by A. sacchar-icola can cause the damage of the central nervous system in

L. Miao . T. F. N. Kwong . P.-Y. Qian (*)Coastal Marine Laboratory, Department of Biology,Hong Kong University of Science and Technology,Clear Water Bay,Kowloon, Hong Kong SAR, Chinae-mail: boqianpy@ust.hkTel.: +852-2358-7331Fax: +852-2358-1559

human beings. Our previous study demonstrated promisingantibacterial activity of a marine-derived fungus A. c.f.saccharicola in primary screening (Miao and Qian, 2005).The objective of this study was to optimize the cultureconditions of A. c.f. saccharicola for enhanced bioactivecompound production. Specifically, we empirically inves-tigated the effects of temperature, salinity, pH, culturemedium composition, and competitive cultivation withbacteria on the mycelial growth, bioactivity, and metaboliteprofile of A. c.f. saccharicola.

Materials and methods

Isolation and cultivation of fungus

The fungal strain A. c.f. saccharicola was isolated fromseawater in a mangrove habitat in Yung Shue O, HongKong (22°19′N, 114°16′E). A. c.f. saccharicola wascultured in 30 ml of glucose peptone yeast extract (GPY)culture medium (2% glucose, 0.5% peptone, and 0.5%yeast extract) in 100% seawater, at 25°C with rotation(100 rpm) for 10 days before chemical extraction. Toexamine the effects of culture conditions (salinity, temper-ature, pH, and culture medium composition) on fungalgrowth and antimicrobial bioactivity, parameters of cultureconditions were changed one at a time according to theexperimental design described below while all othercultivation parameters remained unchanged.

Chemical extraction of fungal cultures

Fungal cultures were filtered through a four-layer auto-claved cheesecloth. The mycelia were washed with auto-claved distilled water and then freeze-dried. The spentculture medium was extracted with equal volumes of ethylacetate twice. Afterwards, the organic fractions werecombined and ethyl acetate was removed at reducedpressure. The residues were weighed and then redissolvedin ethyl acetate for later user.

Measurement of fungal growth

The fungal growth was determined according to the dryweights of mycelia.

Measurement of fungal antibacterial activityusing disc diffusion assay

Antibacterial assay of the fungal extracts was performedusing a standard disc diffusion assay (Acar 1980) withreplication (n=3). Bacteria Pseudoalteromonas spongiae(Lau et al. 2005) and Vibrio vulnificus were served as atarget because these two bacteria were found sensitive tothe minor changes in bioactivity of fungal extracts in theprimary screen. A 250-μg fungal extract was loaded onto a

sterile filter paper disc (6 mm in diameter). The paper discwas air-dried and placed onto the nutrient agar plate thathad already been inoculated with a lawn of target bacteria.After incubation for 24 h at 30°C, the antibacterial activitywas evaluated by measuring the width of the growthinhibition zones from the edge of each filter paper using adissecting microscope.

Analysis of fungal metabolite profiles usinghigh-performance liquid chromatography (HPLC)

Ten microliters of fungal extracts (50 μg μl−1) weresubjected to HPLC for metabolite profile analyses. HPLCwas performed using aWaters system connected to a dual λabsorbance detector (Waters 2487) using a reverse phasecolumn on the analytical scale (Merck, Lichrospher-100,RP-18, endcapped, 250×4.6 mm, particle size 5 μm). Themobile phase was applied as the linear gradient. Thesolvent was degassed for at least 30 min before use inchromatography. The chromatographic parameters are thefollowing: Detection—PDA 206 nm; mobile phase—Awater, B acetonitrile; 0∼20 min 90% A∼90% B, 20∼25 min90% B∼90% A, 25∼30 min 90% A; flow rate—1 ml min−1.

Analysis of fungal metabolite profiles usingchromatography-mass spectrometry (GC-MS)

The fungal extracts were dissolved in 100 μl of DCM andthen transferred to a Sep-Pak C18 Cartridges (Waters). Thecartridge was first eluted with 3 ml Milli-Q water and then3 ml ACN. The ACN fraction was dried in vacuo at 35°C,and the dried extract was then dissolved in 1 ml ACN. GC-MS was performed using aWaters system (Varian/CP-3800and Varian/Saturn 2200) equipped with a relatively non-polar capillary column (CP-Sil 8 CB-MS, 30-m length,0.25-μm film thickness, 0.25-mm I.D.). The injection portwas held at 275°C and the interface at 300°C. Thetemperature was programmed from 75 to 310°C at 10°Cmin−1. Helium was used as the carrier gas.

Effects of salinity and temperature on fungal growthand bioactivity

The effects of salinity on mycelial growth and bioactivitywere studied by growing fungus in GPY media made ofthree different salinities, 0, 17, and 34 ppt at 25°C. Theeffects of temperature were studied by growing fungalcultures in GPY medium at 15, 20, 25, 30, and 35°C in thesalinity of 34 ppt.

Effects of pH value on fungal growth and bioactivity

The effects of pH value were studied by growing the fungalcultures in GPYmedia at seven different pH values rangingfrom 3.5 to 9.0. GPY medium was adjusted to acid and

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alkaline pH by the addition of 50 mM citrate phosphatebuffer (pH 3.5 to 6.5) or 50 mM Tris–HCl buffer (pH 7.5 to9.0), respectively. To minimize the effect of differentextents of theMaillard reaction (browning reaction betweencarbohydrates and protein/amino acid) at high temperatureunder different pH values, the GPY medium and the bufferwith different pH values were autoclaved separately andthen mixed. The final pH values of the medium (mean ofthree replicates) were 3.68±0.02, 4.35±0.01, 5.40±0.01,6.27±0.01, 7.41±0.03, 8.24±0.01, and 8.68±0.02.

Effects of nutrient concentration on fungal growthand bioactivity

The effects of nutrient concentrations on mycelialgrowth and bioactivity were examined by adjustingthe concentrations of glucose, peptone and yeastextract in media as follows: A (2% glucose + 0.5%peptone + 0.5% yeast extract), B (2% glucose + 0.5%peptone), C (2% glucose + 0.5% yeast extract), D(0.2% glucose + 0.5% peptone + 0.5% yeast extract),E (2% glucose + 0.05% peptone + 0.05% yeastextract), and F (0.2% glucose + 0.05% peptone +0.05% yeast extract).

Effects of co-cultivation with bacterial competitorson fungal growth and bioactivity

The fungal strain was grown in 25 ml of GPY medium andcultured at 25°C for 5 days. Co-cultivated bacterial strainswere kept in 5 ml of GPY medium for 2 days and wereharvested using centrifugation (5,000 rpm, 4,500×g,15 min). Bacterial cell pellets were resuspended in 5 mlof fresh GPY medium and the supernatant was filteredthrough a 0.22-μm pore membrane (Millipore, Bedford,MA, USA) to remove the remaining cells. The resuspendedbacterial cells and the cell-free supernatant were individu-ally added into 5-day-old fungal cultures (three replicateseach). Afterwards, the fungal–bacterial cultures werecultured for an additional 5 days. A 5-day-old fungalculture added with 5 ml of freshly autoclaved GPYmediumwas used as a control.

In a preliminary experiment, the following 14 bacterialstrains that had originally been isolated from natural marinebiofilms in Hong Kong waters were tested: B1 (Vibrioharveyi), B2 (Rhodovulum sp.), B3 (P.spongiae), B4(Vibrio sp.), B5 (Shewanella algae), B6 (V. fluvialis), B7(Micrococcus luteus), B8 (Staphylococcus haemolyticus),B9 (V. vulnificus), B10 (S. aureus), B11 (V. halioticoli),B12 (P. piscida), B13 (Moraxella phenylpyruvica), andB14 (Loktanella hongkongensis) (Lau et al. 2002; Lee andQian 2003; Lau et al. 2004). Based on the findings of thepreliminary experiments, three bacterial strains (S. algae, V.vulnificus, and L. hongkongensis) were used to study theeffects of bacterial cell density and co-culture duration onfungal growth and bioactivity. Different numbers of thebacterial cells (108, 109, and 1010 representing 1×, 10×, and

100× treatment, respectively) were added into 5-day- or 7-day-old fungal cultures (three replicates each), which werecultured for an additional 5 or 3 days, respectively.

Statistical analyses

Statistical analyses were carried out using the SPSSstatistical package. The differences among treatments ineach experiment were compared using one-way analyses ofvariance (ANOVA) followed by Tukey’s test. The differ-ences between the treatment and control in the co-cultivation experiment were compared using one-wayANOVA followed by Dunnett’s test. In all cases, thethreshold for significance was 5%.

Results

Effects of salinity and temperature on fungal growthand bioactivity

A. c.f. saccharicola grew faster in freshwater (0 ppt) than inseawater of 17 or 34 ppt (Fig. 1a, Table 1). In addition,changes in salinity altered the color of the mycelia fromlight pink in full strength of seawater, and grey pink in 50%of seawater to black in freshwater. Although the fungalgrowth increased with a decrease in salinity, the bioactivityof this fungus changed in the opposite direction. Highsalinity condition (34 ppt) promoted the antibacterialactivity of this fungus (Fig. 1b).

The growth of A. c.f. saccharicola increased whiletemperature was increased from 15 to 30°C (Fig. 2a), butfungal growth dropped to the lowest at 35°C. Theantibacterial activity of the fungus increased while tem-perature increased from 15 to 25°C. Antibacterial activitydecreased at 30°C and was totally inhibited at 35°C(Fig. 2b, Table 1).

Effects of pH value on fungal growth and bioactivity

A. c.f. saccharicola grew under a wide range of pHconditions (from 3.5 to 9.0). The pH of culture mediumchanged slightly during the initial culture period. By day 5,the pH values in different treatments changed to 3.49±0.03,4.25±0.02, 5.26±0.02, 6.04±0.04, 6.73±0.0.05, 7.6±0.10,and 8.19±0.04. At the end of the experiment, the finalpH values were at 3.61±0.01, 4.35±0.02, 5.00±0.04,5.69±0.05, 5.05±0.06, 6.35±0.12, and 7.21±0.05. Thegrowth of A. c.f. saccharicola was the slowest at pH3.5. Fungal growth increased with pH up to themaximum growth at pH 6.5 and then decreasedgradually at pH larger than 6.5 (Fig. 3a, Table 1).

A. c.f. saccharicola showed different antibacterialactivity when cultured under different pH conditions(Fig. 3b, Table 1). The fungus had higher bioactivitywhen grown at pH 4.5, 5.5, and 7.5. No bioactivity was

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detected when the fungus was cultured under alkalineconditions.

Effects of nutrient concentration on fungal growthand bioactivity

The nutrient concentration greatly affected the growth of A.c.f. saccharicola. The fungus grew two times faster inhigh-nutrient media (medium A) than in low-nutrientmedia (medium D and F). The growth of A. c.f.saccharicola was enhanced by high concentrations ofpeptone and yeast extract (medium Avs D) but not glucose(medium A vs E). Yeast extract appeared to be moreimportant than peptone in supporting the growth of thefungus (medium B vs C) (Fig. 4a, Table 1).

The antibacterial activity of A. c.f. saccharicola washigher after the fungus was cultured in media with highglucose concentration (medium A, B, C, and D) but thebioactivity was greatly reduced when the fungus wascultured in the media with high concentrations of peptone

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Fig. 1 The mycelial growth (a), antibacterial activity (b), andmetabolite production (c) of Arthrinium c.f. saccharicola cultured inGPY media with different salinities (0, 17, and 34 ppt). Antibacterialactivity is indicated by the size of growth inhibition zones created onthe lawn of two target bacteria, Pseudoalteromonasspongiae andVibrio vulnificus. The test concentration of fungal extract was250 μg disc−1. Metabolite production is indicated by the relativearea of three peaks in GC-MS profiles. Data plotted are means±SDof three replicates per treatment

Tab

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Factors

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galgrow

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activ

ity

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Pseud

oalteromon

asspon

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Salinity

(ppt)

017

3434

170

340

17Tem

perature

(°C)

3025

2015

3525

3020

1535

2530

2015

35pH

6.5

8.5

5.5

9.0

7.5

4.5

3.5

4.5

7.5

5.5

3.5

6.5

8.5

9.0

7.5

4.5

5.5

3.5

6.5

8.5

9.0

Medium

AC

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DF

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means

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%glucose+0.5%

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%glucose+0.5%

yeastextract,D

:0.2%

glucose+0.5%

pepton

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yeastextract,E

:2%

glucose+0.05

%peptone+0.05

%yeastextract,

F:0.2%

glucose+0.05

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%yeastextract

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and high yeast extract but low glucose concentration(medium E). Although the fungus grew much faster in highthan in low-nutrient media (medium A vs F), there was nodifference in bioactivity (Fig. 4b, Table 1).

Effects of co-cultivation with bacterial competitorson fungal growth and bioactivity

In the co-cultivation experiment, among the 14 testedbacterial strains, six species (P.spongiae, S. algae, V.vulnificus, V. halioticoli, P. piscida and L. hongkongensis)showed detectable effects on the growth and bioactivity ofA. c.f. saccharicola (Fig. 5). Compared to the control, thefungus growth was significantly increased when spentculture media of S. algaewere added into the fungal culturemedia, while the fungus growth was significantlydecreased when cultivated in the presence of the cells ofP. piscida (Fig. 5a). The bioactivity of the fungus was

significantly higher than that in the control when spentculture media of certain bacteria, such as P. spongiae,P. piscida, and L. hongkongensis), were added into thefungal culture media (Fig. 5b).

The cell density of S. algae and V. vulnificus in the co-cultivation did not affect the antibacterial activity of thefungus. However, the fungus showed stronger bioactivityafter co-cultivation with 109 cells of L. hongkongensis thanwith 108 or 1010 cells of this bacterium (Fig. 6). The timing(day 5 or day 7) for the addition of bacterial cells into thefungal cultures did not cause a significant effect on thebioactivity of A. c.f. saccharicola.

HPLC profile of A. c.f. saccharicola metabolites

Growth of A. c.f. saccharicola in different conditions(temperature, salinity, pH, nutrient concentration, and co-cultivation) resulted in similar metabolite profiles. Therewere several major peaks (e.g., larger peak areas withretention times of 2–4 min and 16 min and some smallerpeak areas with retention times of 10–14 min) in the HPLCprofiles of all treatments. Differences in metabolite profiles

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00

Fig. 2 The growth (a), antibacterial activity (b), and metaboliteproduction (c) of Arthrinium c.f. saccharicola cultured in GPYmedium at different temperatures (15, 20, 25, 30, and 35°C).Antibacterial activity is indicated by the size of growth inhibitionzones created on the lawn of two target bacteria, Pseudoalteromonasspongiae and Vibrio vulnificus. The test concentration of fungalextract was 250 μg disc−1. Metabolite production is indicated by therelative area of three peaks in GC-MS profiles. Data plotted aremeans±SD of three replicates per treatment

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Fig. 3 The growth (a), antibacterial activity (b), and metaboliteproduction (c) of Arthrinium c.f. saccharicola cultured in GPYmedium at different pH (pH 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, and 9.0).Antibacterial activity is represented by the size of growth inhibitionzones created on the lawn of two target bacteria, Pseudoalteromonasspongiae and Vibrio vulnificus. The test concentration of fungalextract was 250 μg disc−1. Metabolite production is indicated by therelative area of three peaks in GC-MS profiles. Data plotted aremeans±SD of three replicates per treatment

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among the different treatments were expressed as therelative percentage of the peak area of those major peaks,especially the peaks with retention times of 2–4 min and16 min. Subfractions from HPLC were then collected andtested for their antibacterial activity. The active fractionswere the peaks with the retention time of 2–4 min (strongeractivity) and 16 min (weaker activity).

GC-MS profile of A. c.f. saccharicola metabolites

There are several major peaks in the GC-MS profile(Fig. 7) and the relative peak area of these peaks varied indifferent treatments. There was a relationship between thepeak area (peak 1, 2, and 3, labeled in Fig. 7) and fungalbioactivity. The peak area was decreased with decreasingbioactivity. Because the relative areas of peak 1, 2, and 3have been plotted in panel c of the first four figures, therewas only one GC-MS profile of fungal metabolites shownin Fig 7. In salinity experiment, the relative area of peak 1and 2 in 34 ppt treatment was much larger than in 0 and17 ppt treatments, while area of peak 3 in 34 ppt treatmentwas much smaller than other the two treatments (Fig. 1c).In temperature experiment, the relative area of peak 1, 2,and 3 in 25 and 30°C treatments were significantly largerthan that in other treatments, and all the three factions couldhardly be detected in 35°C treatment (Fig. 2c, Table 2). InpH experiment, the relative area of peak 1, 2, and 3 in pH7.5 treatments were significantly larger than that in othertreatments (Fig. 3c, Table 2). In nutrient experiment, therelative area of peak 1, 2, and 3 in medium E was thesmallest among all the treatments (Fig. 4c). Four co-culturesamples (B3 medium, B9 cell, B12 medium, and B12 cell)were selected to test their GC profile because the antibac-terial activities of these samples were significantly differentfrom the control. The GC profiles of these co-culturesamples were relatively simple than that in other experi-ments, and peak 3 was not detected in all these co-culturesamples. The antibacterial activity of B3 and B12 mediatreatments were higher than that of control, and the relativearea of peak 1 in these two treatments was also much largerthan that in control (Fig. 8).

Discussion

Several studies reported that certain bacterial species couldbe induced to produce antibiotics in the presence of othermicrobes (Patterson and Bolis 1997; Mearns-Spragg et al.1998), which suggests that microbial antagonism might beused as a new tool to screen for novel compounds.However, there were still few studies that documented theco-culture effects on microbial growth and their bioactivity.In this study, we co-cultured A. c.f. saccharicola with 14fouling bacterial species and found that the fungus grewfaster when the spent culture media of six bacterial specieswere added into the fungal cultures compared to thecontrol, while the fungus grew slower when the cells ofthese bacterial species were added (Fig. 6). In addition,

some spent bacterial culture media exhibited inductiveeffects on the antibacterial metabolite production of thefungus. Similar results were also reported by Burgess et al.(1999). As bacteria produce a variety of exo-polysaccha-rides, enzymes, lipids, vitamins, and other unidentifiedgrowth factors (Zientz et al. 2004), it is possible that someof these bacterial metabolites in the spent culture mediumpromoted fungal growth and bioactivity while bacterialcells competed with the fungus for nutrients. Especiallywhen the competitive bacterial strain had a high growthrate in the fungal culture, the fungal growth and bioactivitywere extensively inhibited under the fierce competition ofresources by the bacterium, such as B12 P. piscida (Fig. 5).At the termination of the experiment, the cell density ofP. piscida was about ten times higher than that of otherbacteria (data not shown).

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Fig. 4 The growth (a), antibacterial activity (b), and metaboliteproduction (c) of Arthrinium c.f. saccharicola cultured in mediacontaining different nutrient concentrations. Growth is indicated bymycelial dry weight. Antibacterial activity is indicated by the size ofgrowth inhibition zones created on the lawn of two target bacteria,Pseudoalteromonas spongiae and Vibrio vulnificus. The testconcentration of fungal extract was 250 μg disc−1. Metaboliteproduction is indicated by the relative area of three peaks in GC-MSprofiles. Data plotted are means±SD of three replicates pertreatment. A: 2% glucose + 0.5% peptone + 0.5% yeast extract,B: 2% glucose + 0.5% peptone, C: 2% glucose + 0.5% yeast extract,D: 0.2% glucose + 0.5% peptone + 0.5% yeast extract, E: 2%glucose + 0.05% peptone + 0.05% yeast extract, and F: 0.2%glucose + 0.05% peptone + 0.05% yeast extract

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Halotolerant marine fungal species have evolved uniquemetabolism to cope with the salinity change. The growth ofsome marine-derived fungal species increases with in-creasing salinity in culture media while antimicrobial

activity sometimes reaches the maximum in mediacontaining 25–50% seawater (Masuma et al. 2001; Bugniand Ireland 2004). In this study, we found that A. c.f.saccharicola grew faster at low salinity than under at high

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Fig. 5 The growth (a) and antibacterial activity (b) of Arthrinium c.f. saccharicola cultured in GPY medium in the presence of eitherthe cells (cell) or spent culture medium (med.) of bacterialcompetitors B3 (Pseudoalteromonas spongiae), B5 (Shewanellaalgae), B9 (Vibrio vulnificus), B11 (V. halioticoli), B12 (P. piscida)and B14 (Loktanella hongkongensis). Antibacterial activity is

indicated by the size of growth inhibition zones created on thelawn of two target bacteria, P. spongiae and V. vulnificus. The testconcentration of fungal extract was 250 μg disc−1. Data plotted aremeans±SD of three replicates per treatment. “*” indicates valuessignificantly different from the control (Dunnett’s test, p<0.05)

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Fig. 6 The antibacterial activity of the fungus Arthrinium c.f.saccharicola cultured in the presence of the cells of bacterialcompetitor Loktanella hongkongensis at different densities. The testconcentration of fungal extract was 250 μg disc−1. “1×”, “10×” and“100×” represented bacterial density at 108, 109, and 1010 cells in30 ml of fungal culture. The fungus was cultured axenically for 5 or

7 days before co-cultivation with bacterial competitors for additional5 or 3 days, respectively. “5–5d” indicates 5 days of axeniccultivation before 5 days of co-cultivation; “7–3d” indicates 7 daysof axenic cultivation before 3 days of co-cultivation. Data plotted aremeans±SD of three replicates per treatment

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salinity and showed higher antibacterial activity in a 34-pptseawater medium. This suggests that A. c.f. saccharicolamight originate from terrestrial or freshwater environmentand that antibacterial metabolite production might havebeen enhanced as a response towards the presumablystressful high salinity conditions.

The pH value of the growth medium can also have asignificant effect on the production of secondary metabo-lite (Hays et al. 1997; Yakoby et al. 2000; Singh 2002). Forexample, the production of alkaline protease by the fungusRhizopus oryzae had a 4.2-fold increase under acidicconditions with 5 μl ml−1 Tween 80 (Banerjee et al. 1992).Two optimal pH levels (4.5 and 7.5) were recorded forbioactive metabolite production in this study. This is thefirst time that dual pH optimum was reported in fungi. Thedual pH optima might be due to the production of morethan one active compound by the fungus, while somecompounds might be produced most favorably at pH 4.5and some at around pH 7.5. The dual pH optima might alsobe due to a different carbon source derived from the acidicand alkaline buffers that were used to adjust the culturemedia to pH 4.5 and 7.5, respectively. Conversely, at pH6.5, the medium led to the fastest fungal growth but to alow antibacterial activity. This might be due to the fact thatthe fungus utilized nutrient and energy sources for thegrowth-supporting primary metabolism, leading to over-proliferation of the fungal mycelium and a delayedsecondary metabolism. Drastic decrease in pH value ofthe culture medium after 10 days of cultivation indicatedthat the fungus produced many acidic metabolites after thelog phase. Fungal growth in the first 5 days was slow underhigh pH conditions but increased significantly after 7 days,suggesting that the production of acidic metabolites in thelater cultivation decreased the pH in the culture medium toneutral and subsequently increased the fungus growth.Although the mycelial weights obtained from the pH 8.5and 9.0 treatments were similar to those from othertreatments after 10 days of cultivation, the fungalbioactivity in these two treatments was undetectable. Thismight be due to a delayed metabolite production caused bya delayed mycelial growth or due to a reduced productionof bioactive metabolite under alkaline conditions.

The components of culture media may affect secondarymetabolite production in fungi (Bode et al. 2002). Glucose,phosphate, or ammonium at high concentrations isgenerally regarded as repressors of secondary metabolism(Masuma et al. 1983; Zhang et al. 1996). Many carbon ornitrogen substrates that are quickly metabolized and, thus,sustaining maximum cell growth rates can inhibit second-ary metabolite production (Gallo and Katz 1972). Thecarbon/nitrogen ratio can also affect the type and yield ofmetabolite synthesis (Bruckner and Blechschmidt 1991;Candau et al. 1992). For instance, Garbayo et al. (2003)reported that nitrogen limitation induced carotenoid bio-synthesis in immobilized Gibberella fujikuroi myceliaunder nonlimiting carbon conditions. In this study, mediawith high peptone and/or yeast extract concentrations led toa faster growth of A. c.f. saccharicola but inhibited itsantibacterial activity when glucose was limited. In contrast,the fungus grew slower but had relatively high antibacterialactivity in the medium with low peptone, low yeastextracts, and high glucose concentration. These resultsindicated that yeast extract and peptone, which providepredominantly nitrogenous nutrients, were more importantfor mycelial growth of A. c.f. saccharicola while glucose,as a carbon source, was more important for bioactivecompound production. Highest nitrogen/carbon ratio (me-dium E) substantially inhibited the bioactivity, suggestingthat active metabolites produced by this fungus might bederived from the glucose metabolism.

Several reports convincingly demonstrated that theprofiles of fungal secondary metabolites varied underdifferent culture conditions (Wang et al. 1998). Althoughthe bioactivity varied among different treatments in allexperiments in this study, the HPLC profiles of the ethylacetate extracts of these treatments were similar. However,we found that the relevant percentage of different peakareas of major peaks differed among the differenttreatments and in most cases, the higher the antibacterialactivity of the treatment, the larger the peak area with aretention time of 2–4 min. By collecting the fractionseluted from HPLC and testing their antibacterial activity,we confirmed that the fraction with a retention time of 2–4had strong activity. These results indicated that the changein bioactivity among different treatments might be

Fig. 7 GC profile of extractsfrom Arthrinium c.f. sacchari-cola. Column: nonpolar capil-lary column (CP-Sil 8 CB-MS,30-m length, 0.25-μm filmthickness, 0.25-mm I.D.). Tem-perature program: 75 to 310°Cat 10°C min−1. Carrier gas:Helium

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attributed to different amounts of the major bioactivecompounds produced by the fungus rather than newcompounds being produced. It might be also because thatthe sensitivity of HPLC was not enough to detect somedifference in chemical profile whereas this differenceactually plays a role in the different bioactivity oftreatments.

Although HPLC profiles of the extracts of differenttreatments were similar, their GC profiles showed signif-icant differences. The change of relative area of three peaks(labeled in Fig. 7) followed the similar trend with thebioactivity variation in different treatments, suggesting thatthese three compounds might be the active component inthe extracts. However, the change of relative area of thethree peaks did not always match well with the bioactivityvariation of fungus extracts, for instance, although theantibacterial activity of pH 4.5 treatment was stronger thanthat of other pH treatments (besides pH 7.5 treatment),peak 1 and 2 could hardly be detected in this treatment. Itindicated that there might be other active compounds in thefungal extracts but could not be detected using GC-MS.Compound 1 (peak 1) production in the acidic pHtreatments was very low compared to other treatments(Fig. 4c). It might be due to the inhibitive effect of citratephosphate buffer on the production of this compound.

In conclusion, culture conditions (temperature, pH,salinity, and medium component) and competitive cultiva-tion with bacteria had detectable effects on the mycelialgrowth and bioactivity of A. c.f. saccharicola. The bestcondition for the fungal growth (i.e., at 30°C, at pH 6.5, infreshwater medium) was different from the best conditionfor the fungal bioactivity (i.e., at 25°C, at pH 4.5, pH 5.5, orpH 7.5, in 34 ppt seawater medium). However, it must bepointed out that these results were based on the single-factor designed experiments, and the optimal conditions for

Bacterial treatment

control B3 med. B9 cell B12 med. B12 cell

Rel

ativ

e pe

ak a

rea

(%)

0

20

40

60

80

100

120

140

Peak 1Peak 2

*

Fig. 8 Metabolite production of Arthrinium c.f. saccharicolacultured in GPY medium in the presence of either the cells (cell)or spent culture medium (med.) of bacterial competitors B3(Pseudoalteromonas spongiae), B9 (Vibrio vulnificus) and B12(P. piscida). Metabolite production is indicated by the relative areaof peak 1 and 2 in GC-MS profiles. Data plotted are means±SD ofthree replicates per treatment. “*” indicates values significantlydifferent from the control (Dunnett’s test, p<0.05)T

able

2Sum

maryof

multip

lecomparisontest(Tuk

ey’sHSD)results

ontheeffect

ofsalin

ity,temperature,pH

andcultu

remedia

onthemetabolite

prod

uctio

n(peak1,

2and3in

GC

profile)of

Arthrinium

c.f.saccha

ricola

Factors

Relativearea

Peak1

Peak2

Peak3

Salinity

(ppt)

340

1734

170

017

34Tem

perature

(°C)

2530

2015

3530

2520

1535

3025

2015

35pH

4.5

7.5

3.5

5.5

6.5

9.0

8.5

7.5

5.5

3.5

8.5

6.5

9.0

4.5

7.5

5.5

4.5

3.5

6.5

9.0

8.5

Medium

CB

AF

DE

CF

AD

BE

DC

BF

AE

See

captionto

Table

1fordetails

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fungal growth and bioactivity might be slightly differentfrom our results when multiple factors were consideredtogether. Our results suggest that the fungus might producemore defensive compounds (such as antibacterial or otherbioactive compounds) under stress condition. The additionof spent culture medium of some bacteria into the fungalculture increased the fungal growth and its bioactivity,which indicated that certain bacterial metabolites in thespent culture medium could promote the fungal growth andantibacterial compounds production. These findings willfacilitate further studies to gain a better understanding ofactive metabolite production in this fungus, which is veryhelpful for the optimization of culture conditions in masscultivation as well as the biotechnological mass productionof active compounds in the future.

Acknowledgements The authors would like to thank Dr. SCK Lauand all the lab mates for their constructive comments in revising themanuscript, and Dr. Virginia Unkefer for proofreading the manu-script. This work was supported by a CAG grant (CA04/05.Sc01) ofthe Research Grant Council of HKSAR and a CAS-CroucherFoundation grant (CAS-CF03/04.SC01) to PY Qian.

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