Notes on the physiology and morphology of Thamnostylum piriforme isolated for the first time in South Africa

The morphology and physiology of two fungal isolates. which were the first record of the species Thamnoslylum piriforme in South Africa, were s tud ied. In both isolates, which were morphologically typical representatives of their species, sporulation was stimulated by UV light. Optimum growth and sporulation in the dark was found to occur at 25 C. The isolates coul d utilize a wide variety of carbon sources aerobically. These carbon sources included pentoses, hexoses , disaccharides, trisaccharides, polysaccharides, gJycos ides , alcohols and organic acids. D-galactose, O-glucose and maltose could be fermented . The isolates we re able to grow in a medium without vitamins and were found to be sensitive to cyclohexamide at a concentration of 0.01 % w/v. The isola tes tested positive for urease activity on Christensen 's urea agar, while extracellu lar enzymatic activity was indicated by the abili ty to liquefy gelatin. The most biomass-containing high value dietetic oil was obtained with ace tic acid as carbon source. Consequently, it is possibile to utilize this fungus for the production of bioprotein and high va lue dietetic oil on industrial effluents containing acetic acid.


Introduction
Recen tl y j[ was discovered that some mucoral can fu ngi can grow and produce significant quantities of gamma-linol enic acid on petrochemical e fflue nts that contain high concentrations of ~H.:e ti c acid . [n a subsequent search [or add itional acetate to lerant l11ucoralean fu ngi, it was d isco vered that TJwmnosty/llm pinforme (Bain.) Arx & Upad hyay (Von Arx 1970 ). present in the leaf litter from the coastal forests and lhornveld of Northern KwaZulu-Natal (Acocks 1988). coul d utilize acetic acid as so le carbon source, while producing oi l containing gamm a-lin olen ic acid (Botha el af. 1995). This was the first report of this fungal species. able to prod uce a dieteti cally important oi l, in South Africa. Sin ce k nowledge of the morphology and physiology of a fungus is essential to assess its biotechnOlogical potential, these characteristics of the iso lates representing T piriforme wer~ investigated in this study.

Materials and Methods
Isolates used T. pirijorille isolates PPRI 5473 and PPRI 5534 from the National Collection ot" Fungi. Prdoria, were used. Both strains were isolated from lea f litter, obtained from the coasta l forest and thornve ld (Acocks 1988) of the tropical savannah region of northern KwaZulu-Natal , South Africa (Botha el al. 1995 ).

Morphology
Preparation of cultures. Pure cultures were grown on Mu cor-synthet ic medium (SMA) and Potato carrot agar (PCA) (Booth 197 1) at 21 ± 5°C in natu ral li ght on a window sill.
Optical microscopy. Material fo r optical microscopy was mounted in clear lactophenol and studied using bright tield illum ination.
Scanning eleclroll microscopy. Material for scanning electron microscopy was prepared acco rding to two reg imes: a) blocks of agar contain ing the frui ting structures were exposed o vernight to osm ium tetrox ide vapou r and then washed in 0.2 M Na cacodilate buffer, dried in a des iccat or and sputter-coated with gold; b) blocks of agar with fru iting structures were submerged in 0.075 M phosphate buffer. pH 7.4-7.6 fix ed in 0.25% osm ium tetrox ide and then dehydrated in acetone . The hlocks were mounted on SEM stubs and sputter-coated wi th gold.
Electron microgra phs were taken at 5 k V using a Jeol 840 scanning electron microscope.
{llflU ellCe of tempera/ure 011 growth alld sporulation . Petri dishes containing SMA were inoculated with mycelia of the isolates and incubated in the dark at 10°C, 15°C, 20°C, 25"C, 30°C and 37°C. After six days. the cult ures were exam ined for growth and sporulat ion.
Influence of UV fig/a 0/1 sporulation. Petri dishes containing SMA were inoculated in duplicate with mycelia of the isolates. One culture of each duplicate was incubated at 25°C under UV irrndiation. The other cul ture was incubated under identical conditions, except that it was incubated in the dark. G rowth and sporulati on were examined dail y for one wec:k .

Physiological properlies
Preparation of inOClIla . Cultures of T pinforme PPRI 5473 and PPRI 5534 were incubated for two week s at 25°C under UV light on malt extract agar (Difco). A sterile , wet inocu lating loop was used to transfer the sporangi ospores from the hyphal growth of each culture to.5 ml sterile distilled wate r. The resu lting suspension s (approx. 1.3 x 10 7 spores ml-I) were used as inocula.
Carbo" assimilation l esl.{ . The abili ty of the iso lates to assim ilate a series of carbon sources in symhetic media aerobically was determined according to the standard methods for yeast identification as described by Van der Walt & Yarrow (1984). Test tubes, each containing 7.6 g 1-1 Yeast nitrogen base (YNB Difco) and a different carbon source (Table I) were inoculated wi th 40 J.lI of spore suspension per tube. On ly 150 x 12-mm test tubes were used throughout th is study. and the carbon sources, obtained from Sigma. Merck and Fluka. were of analytical reagent grade and used at a concentration of 2 g carbon I-J . Assimilation was visuall y determined by comparing the fungal growth of the inoculated cultures to a blank without carbon after six, eight and [Q days of incubation at 25°C on a rollordrum rotating at 100 rph. The biomass was also harvested by filtration (What man GFI A), freeze-dried and weighed after each period of incubation.
Fermentation of carbohydrates. The standard method was used to test the ability of a yeast to ferment a series of carbohydrates (Van def Walt & Yarrow 1984). Six milliliters of liquid medium, consisting of 0.5% w/v yeast extract (Difco) and 2.0% w/v of a different fermentab le carbohydrate (Table 1), was inoculated with 40 ~l spore suspension. The inoculated cultures in test tubes were incubated in an upright position at 25°C and monitored for gas production (i.e. fermentation) over a period of 14 days. Gas production was observed as bubbles accumulating in a Durham tube submerged in the medium Growth ill vitamin~free medium. The ability of the fungal spores to germinate and grow in a synthetic medium without vitamins was analysed according to the method described by Van der Walt & Yarrow (1984). The spore suspension (40 } .. tI) was used to inoculate 5 ml liquid medium consisting of 16.7 g 1-1 Bacto-vitamin~free yeast base (Oifco) in a test tube. The culture was incubated at 25°C on a rollordrum rotating at 100 rph. Growth was visually determined by comparing it with the fungal growth of a blank without a carbon source after six , eight and 10 days of incubation. In addition. the bjo~ mass was harvested by filtration (Whatman GF/A), freeze~dried and weighed after each period of incubation.
Cycloheximide resistallce. The influence of 0,0 I % w/v and 0.10% w/v cyclohex imide on fungal growth was examined according to the method described by Van der Walt & Yarrow (1984). The spore sus~ pension (40 I . . ll) was used to inoculate 5 mlliquid medium consisting of 6.7 g I -I Yeast nitrogen base (Oifco), 5.0 g I-I glucose and 0,1 g I-l or 1.0 g I-I cycloheximide in test tubes. The inoculated media were incubated at 25°C on a rollordrum rotating at 100 rph. Growth was monitored after six, eight and 10 days of incubation.
Gelatinliquefactioll. The ability of the fungal isolates to liquefy gel~ atin was analysed according to the method described by Van def Walt & Yarrow (1984). The surface of a solidified medium consist~ ing of 100 g I -I gelatin, 5 g 1-1 glucose and 11.7 g I -I Yeast carbon base (Difco) was inoculated with 40 J-ll of the spore suspension. The inoculated culture was incubated in an upright test tube at 25°C for seven days, whereafter the depth of the liquid layer was measured.
Splitting of arbutill , A slant consisting of 5 g 1-1 arbutin. 5 g I-I yeast extract (Oi1'co). 12 mg I-I ferric ammonium citrate and 20 g I-I agar was inoculated with 40 J.ll of the spore suspension and incubated at 25°C (Van der Walt & Yarrow 1984). The slope was examined after seven days for the presence of a dark brown colour, characteristic of the complexes formed between ferric salts and hydroxyquinonc due to the splitting of arbutin by p-glucosidase.
Urease activity, The ability of the fungal isolates to produce ammonia from urea, indicating the presence of urease, was tested as described by Van der Walt & Yarrow (1984). A slope of Chris~ tensen 's urea agar was inoculated with 40)11 of the spore suspension and incubated at 25°C. After seven days, the slope was examined for the presence of a deep pink colour, as the phenol red indicated a rise in the pH due to ammonia in the medium.
Lipase activity. The ability to produce lipases was determined as described by Kouker & Jaeger (l987). A loopful of the fungal growth was streaked out on a plate containing nutrient agar supple~ mented with 2.5% (w/v) olive oil and 0.001 % (w/v) of the nuores~ cent dye, rhodamine B. The plate was incubated at 2S"C for seven days, after which it was examined under UV irradiation for an orange fluorescent halo around the fungal colony -indicating lipase activity Results Morphology s. Mr. J. Bot. 1997. 63(2) At 21 ± 5°C in natural light. T. pirt/orme produced only sporangioles on SMA after 7 days, but eventually, and especially on PCA, terminal sporangia also formed ( Figure 1). Some sporangiophores produced only terminal sporangia. Sporangia deliquesced before the sporangioles ripened (Figure 1): the outer membrane, which consisted of cells bearing spinules. disinte~ grated and released the sporangiospores. A basal frill remained at the apex of the apophysis. Lateral clusters of sporangioles of varying sizes occurred along the axis of the sporangiophore (Figure 2), The sporangioles developed characteristic circinate stipes (Figure 3) similar to those in members of the genus CircineLJa, The largest groups of sporangioles were usually terminal ( Figure   4) and arranged all a verticillateiy branched structure ( Figure 5).
To ensure correct identification, cultures must. therefore. be grown under conditions that encourage the development of both states of this fungus. As in Circillella, the sporangioles did not deliquesce: the spores were released by disintegration of either the sporangioles themselves or by the severing of their stipes, Each individual, smooth sporangiole ( Figure 6) contained a small number of spores, All sporangioles were black in colour when mature, but they did not develop at the same rate. They measured 15 ~m in diameter. The columellae were ovoid and small spinules on the outer surface were lost at maturity,

Influence of temperature on growth and sporulation. The results
obtained are depicted in Figure 7. Maximum growth and sporulation took place at 25°c'  Figure 7, The colony diameter and vegetative reproductive structures produced by T piriforme PPRI 5473 and PPRI 5534 on Mucorsynthetic agar after six days of incubation in the dark at various tem~ peratures, The key to the different structures is given on the right side of the graph. a, Aerial hyphae: b, sporangiophore bearing restricted clusters of sporangioles: c. sporangiophore bearing well~ developed clusters of sporangioles; d, sporangiophore bearing well~ developed clusters of sporangioles and a terminal sporangium: e, sporangiophore bearing a terminal sporangium, S. Afr. J. !lot. 1997.63(2) /njiuclIl:e of UV fight Oil sporulation. At 25°C on SMA, UV irradiatio n ~nhancl!s sporangioie fo rmation and ~s pecia ll y sporangium formation (not illustrated).

Physiological properties
T he results or t he physio iogicailL!sts arc sum marized in Table 1.
Spo rangiospores o f T. piriforme PPRI 5473 ao d PPRI 5534 ge r minat~ and grow in aerobic conditions on a wide diversity of carbon sources. When comparing the penrose and he xose assim ilation, as summarized in Table 1, it is interesting to note that the assimi lahlc aldoses (L-arabinosc. D-xylosc, D-galaclosc and D-glucose) all show the same configuration at the asymmetric carbon alOms next to the carbony l cnrbon atom (carbon atoms numbers two and three), The ketose, L-sorbose, could not be assim ilated .
The disaccha ride:.; consis ting of two D-glucopyranosyl moieties (i.e. cellobiose. ma lto:.;e and trehalose), or a D·glucopyranosyl and a D.galactopyranosy l moiety bou nd in an a( J ---). 6) li nkage (i .e. melibiose) were assimi lated ( Table I ) Dn ly (wo of the nine alcoho ls tested as :.;ale: carbon sou rce cou ld support grow th: the po l yo Is D· manni tol and sorbito l (Tab le I). Regard ing the assi mi lali o n of organic acids, short-chain fatl y ac ids wilh an even num hcr of carhon alOms (i.e. acelic ac id and butanoic ac id) werl! uti li zed. Short-chai n fatt y aci ds with an od d numbe r of carbo n atoms (fo rmic acid and pro pa noic :.Jcid), however. were not util ized. One of the complex organ ic acids (i.c. lactic acid) was utilized , wh il e two (i.e. citric acid and g lu con ic aci d) were not. It is in ten!sting to nole (hal in the stationary phase unde r aerobic cond itions (Figure 8   -. negati ve resu lt of bio mass was obtained with acetic acid as carbon 50Un.;C th an from th e ot her ca rbon sources.
Ot her tests showed th at o nly D -g lucose, D-galactose and maltose coul d be fer mented, the spora ngiospores could germ inate and grow in a sy nthetic med ium devoid of vitamins. and that the fungus was sens itive to the anti fungal drug cyclohex imide at a concentration of 0.01 % w/v.

Discussion
Morphology, growth and sporulation T he isolates of Tlwmllostylum piriforme produced morpho logically simi lar spows from sporangia and sporangioics. These spores, however. were released in different ways. The sporangia deliquesced whereas the sporangioles disintegrated. Ultra-vio let li ght stimul ated sporang iale formation and especially promoted spo rangium formation. Optimum grow th and sporu lati on in the dark was found to occur al 25°C.

Physiology
The two st rains o f T. piriforme ass imil ated sim il ar carbon sources to those ass im il ated by another mucoralean stra in , represe ntin g Mucor circillelloides , tested under ide nti cal conditions (Bolha el (I f. 1996). However, in contrast to the strain representing M. circine/Joides , th e strains of T. piriforme cou ld not ass imilate D-ribose, ethanol, ribitol or gluconic acid. On th e other hand , the strain s represe nting T. piriforme were able to utili ze me li biose, while the strain representing M. circinelloides did not utilize this disaccharide. Be fore carbon assimilation patterns can be used in the taxo no my of these fungi , as is evident in the yeast domain (Van der Walt & Yarrow 1984), many more strains should be te sted under id entical conditions. It was fo und [hat the strains representing T. piriforme produce the most biomass with acet ic acid as carbon source. This is no t surpri sing, since these strains were originall y iso lated on a medium containing thi s organi c acid as carbo n source (Bo tha er (II. 1995). The resu lts therefore ind icate that as in GeOlricJllIUI Cl lfldidulII (Both a & Kock 1993), the funga l strains in this study sho uld a lso be inves ti ga ted for the production of biopro te in from an aceti c ac id -rich pe troche mical effl uent. In addit ion, ga mma-lino lenic acid can simultaneous ly be produced by the strain s of r piriforme during the same process. The ability of the strains to grow in a medium devoid of vitamins, may also be of bi otechnological val ue, since th e addition of these growth factors is expensive and may render a process uneconomicaL The isolates we re ab le to fe rment D-galactose, D -glucose and maltose, T he results of the carbon assimilation tes ts indicate the production of certain glycosidases by the fungus. The ability to assi milate the P-D-glucopyranosides arbutin , cell obiose and salicin is ind ica ti ve of p-glucosidase activity (Barrett 1976). Further, the ability to ass imi late maltose and trehalose m ay indicate the presence o f a-D-gl ucosidase (Barrett 1976). Similarly. o ne can assu me that the presence of a-D-glucosidase may fac ilit ate the assi mil atio n of the tri sacc haride me li zitose, whi le the ability to assi milate mdibiose may be indicati ve of the presence o f a -D-galactos id ase.
S. Afr. J. Bot. 1997, 63(2 ) The fungus tested positi ve for urease ac ti vity o n C hri ste nsen's urea agar. while e xtracell ul ar e nzy mati c ac ti vity was indi cated by the abi li ty of t he fungal strains to lique fy gelatin. 1n contrast to find ings rega rdi ng representatives of the genus Mucor (Rat ledge 1989). no lipase activ ity was detectccJ wit h the strains re presenting T. pin/orme in thi s study.

Concluding remarks
T he fungal strains representing T. pin!orme in this study we re able to grow in a medium devoid of vi tam ins. Th is ability, together with the fact that the st rain s produced the most biomass on acetic acid as carbon source, indi cate that the fungus should be further investigated for economi c bioprotein production fro m an acetic acid-rich petrochem ical effluent.
Before planning a bi otechnolog ical process utilizing a fungus, as many characteristics as possible thereof must be known. Therefore, knowledge of the mode o f spo rulation , optimum physiolog ical conditions and the utilization of carbon sou rces are cri ti ca lly im portant in uti li zi ng fung i in these processes. Consequen tl y, it is impo rtant to investigate the abi lity of fu ngi to assi milate aerobicall y a nd fe rme nt different carbo n sources, produce extrace llular enzy mes, grow and spo ru late under various conditions. Other mu coralcan fu ngi of pot ential biotechnological importance are currently being invest igated in thi s m anner.