Lipid accumulation and membrane fluidity influence mycelial stability and riboflavin production by the riboflavinogenic fungus Eremothecium ashbyii
V S., S R, T Chandra, M A.K.
dph, eremothecium ashbyii, fluorescence anisotropy, hemiascomycete, membrane fluidity, riboflavin overproduction, unsaturated fatty acids
V S., S R, T Chandra, M A.K.. Lipid accumulation and membrane fluidity influence mycelial stability and riboflavin production by the riboflavinogenic fungus Eremothecium ashbyii. The Internet Journal of Microbiology. 2009 Volume 8 Number 1.
The plant pathogenic filamentous hemiascomycete fungus
One previously reported interesting feature of riboflavin over production by
The present study was undertaken as a first step to understanding the accumulation of lipids in the hyphae, the influence of growth substrate on composition of stored lipid and correlating the composition of the membrane phospholipids, with the membrane fluidity and changes in mycelial morphology of
Materials and methods
Organism and culture conditions
Determination of mycelial dry weight and residual substrate
For determination of the mycelial dry weight, 100ml of the pooled culture broth was filtered through a preweighed Whatman filter paper No 1. The supernatent was used for the estimation of the residual substrate, lipase activity and extracellular riboflavin . The harvested mycelia were dried overnight on the preweighed Whatman filter paper No 1 at 60 C till a constant weight was obtained.
Residual glucose in the culture supernatent was determined by the DNS method (Miller, 1959). Residual oil was determined by the gravimetric estimation of lipid extracted from the cell-free culture supernatant using hexane (Plummer, 1978).
Riboflavin was estimated fluorimetrically using the ISI standard procedure (IS 1374, 1979). From the cell free culture supernatent, 10 ml of a suitable dilution was taken in two tubes marked A and B, 1 ml of the riboflavin standard (1 g/ml) was added to tube A and 1 ml of distilled water was added to tube B. The solutions were then acidified using 1 ml of glacial acetic acid followed by the addition of 0.5 ml of 4 % KMnO4 to each tube in order to oxidize the impurities. After 2 minutes 0.5 ml of 3 % H2O2 was added to both the tubes in order to oxidize the residual KMnO4. The fluorescence of the solutions was measured using an ELICO Fluorometer Model CL-53 to give readings A (Standard + Sample) and B (Sample alone). Into tube B, 20 mg of Sodium dithionite was added and the fluorescence measured within 10 seconds (reading C). The riboflavin concentration in the original sample was calculated using the formula :
Care was taken to ensure that the ratio
Microscopic observation of lipid accumulation
The intracellular lipids were observed by fluorescence microscopy using a Leica fluorescence microscope after mixing 1ml of Nile blue solution (1mg/ml in Methanol) with 100 l of the culture broth. Blue light was used for excitation.
Gravimetric estimation of intracellular lipid
Mycelial lipid was extracted using Hexane-Isopropanol mixture (3:2) (Hara and Radin, 1978). The mycelia were pooled and harvested as described above at intervals of 24, 48, 72, 96 and 120 h. The mycelia were given a quick wash with Hexane-Isopropanol mixture to remove adhering lipid and crushed in a pestle and mortar alongwith acid washed glass beads to obtain a homogenate. The total mycelial lipid was extracted by adding 10 ml Hexane-Isopropanol thrice to the homogenate. The pooled lipid extracts were filtered through cotton wool into a preweighed round bottom flask and the solvent was rotary evaporated. The residue was weighed to obtain the gravimetric weight of intracellular lipid. Membrane fluidity studies were done for the 48 h and 96 h old mycelia only. For this mycelia were harvested from 8 Erlenmeyer flasks each containing 100 ml of the riboflavin production medium was used for each growth substrate at each time point, a known weight of the harvested mycelium was used for gravimetric estimation of lipid and the remaining was used for membrane fluidity studies. The gravimetric estimation was done in triplicate as a time course for each medium and the standard deviation values were calculated.
Fractionation of intracellular lipids into different classes
The fatty acid content of the triglycerides and phospholipids was analysed for glucose, olive oil and sunflower oil grown mycelia at 48 h and 96 h only, since these points denoted growth (maximum lipid accumulation) and riboflavin overproduction respectively. A known weight of lipid obtained on the three different media at 48 h and 96 h of growth were reconstituted in 10 ml. of Methanol-Dichloromethane (1:1) mixture and fractionated into polar lipids (phospholipids), glycolipids and triglycerides (non-polar lipids) using the Solid Phase Extraction (SPE) procedure as follows (Abraham,
The total lipid extract was dried on a hot plate (45oC) under a gentle stream of nitrogen and re-suspended in 10 ml Dichloromethane (DCM). The sample (5 ml sample volume x 2) was applied to a pre-equilibrated Solid Phase Extraction glass column (SPE column) with a silica based sorbent (Insolute silica 1g/6ml column reservoir, average pore size = 54 Å; specific surface area 521 m2 g-1; surface pH = 7). Samples and solvents were passed through the SPE column by vacuum pressure. Non-polar lipids (triglycerides) were eluted with 20 ml DCM and the fractions stored in stoppered vials, glycolipid fraction was eluted with 20 ml acetone from the column and stored. Polar lipids (phospholipids) were eluted with 20 ml methanol and collected in stoppered vials. The solvent in the different lipid fractions was rotary evaporated and weight of each fraction was determined by gravimetry. The triglycerides and phospholipids were reconstituted using 10 ml of Methanol-Dichloromethane (1:1) mixture and subjected to methylation as detailed in the following section.
Preparation of methyl esters for GC-MS analysis
The extract prepared as above for the triglycerides and phospholipids was dried under a stream of pure N2 gas on a hot plate at 45 C and saponified overnight at 4 C by adding 0.05 ml of dichloromethane (DCM), 2 ml of methanol and 0.5 ml of 1M KOH. Next morning, 1 ml of DCM, 1 ml 0.1M Phosphate buffer (pH 7.0) and 0.18 ml 6N HCl was added to effect phase separation. The lower DCM phase containing the lipids was collected in a 4 ml sample vial. The extraction with DCM was repeated twice with 0.5 ml DCM. The DCM extracts were pooled together and dried under a stream of pure N2 gas on a hot plate at 45 C, 1.5 ml of the methylating reagent (Methanol:Dichloromethane:33%HCl::10:1:1) was added to the residue and incubated in a hot air oven at 105 C for 1.5 h. For phase separation 1 ml of phosphate buffer pH 7.0 and 1 ml of DCM was added and vortexed for 20 seconds. The lower DCM phase was transferred to a clean GC vial with teflon liner. The procedure was repeated and the DCM extract containing the fatty acid methyl esters (FAME) was dried under a stream of pure N2 gas at 45 C. The sample of FAME was stored in 0.5 ml of Octane and kept frozen until GC analysis.
GC analysis of fatty acid methyl esters
The fatty acid methyl esters were separated and identified by running a GC MS analysis using 6890 N system for GC (Agilent Technologies) and 5973 N MSD fitted with a 30 m x 25 µm BPX5 capillary column. Helium gas at 16 lbs pressure was used as carrier at a flow rate of 1 ml/min. A temperature programme of 80 C to 280 C and a split ratio of 1:10 was used. Individual fatty acid methyl esters were identified by comparison with standard FAMEs obtained from Promochem, GmBH and Sigma Chemical Company, U.S.A. The individual fatty acid concentration in each sample was computed by area normalization of peaks using the Chemstation software (Agilent Technologies) according to the formula:
% free fatty acids =
Sum of total peak areas
The concentration of each fatty acid so obtained was multiplied by the dilution factor and then divided by the weight of lipid to obtain the relative proportion of each fatty acid in the triglyceride and phospholipids fractions for each growth substrate. Samples were analyzed in duplicates and averages calculated.
Screening and assay for lipase activity
Lipase activity was screened using a plate assay as well as by TLC of the products of Triolein utilization.
For the plate assay CaCl2-Tween 80 plates were used (Sierra, 1957). The products of Triolein utilisation were visualised by TLC of the culture supernatent using precoated silica gel plates (Merck, Darmstadt, Germany). The solvent system used as the mobile phase consisted of Petroleum ether: Diethyl ether: Glacial acetic acid in the ratio of 16: 3: 1 and the reagent for development of plates consisted of Ceric sulphate / Ceric ammonium sulphate (1g) plus Ammonium molybdate (21g) dissolved in 31 ml concentrated H2SO4 in a total volume of 500 ml.
Lipase was assayed colorimetrically (Lakshmi, 2000). The reaction mixture consisting of 500 l substrate emulsion (1mM p-nitrophenyl stearate in undecane), 500 l culture supernatant and 500 l Phosphate buffer (pH 7.2) was incubated at 30 °C in a gyratory shaker at 200 rpm for 20 minutes. From the aqueous layer 500 l was transferred to 500 l borate buffer (pH 10.6) and O.D. was measured at 410 nm. A standard graph of p-nitrophenol in the range 10 nmole to 100 nmole in Borate buffer was used.
One unit of Lipase activity is defined as the amount of enzyme necessary to produce 1nmole of p-nitrophenol/min under the assay conditions using p-nitrophenyl stearate as substrate.
Preparation of cell membranes from protoplasts of
The cell wall of
Purification of protoplasts and membrane preparation for fluorescence anisotropy
The protoplasts formed were sedimented by centrifugation at 2,500 rpm and 4°C at the end of the incubation period (2 h). The pellet obtained was suspended in 5ml Tris-HCl buffer (15 mM, pH 7.4) and allowed to lyse by incubation for 1 h at 4 °C. The mixture was centrifuged at 15,000 rpm for 30 minutes at 4 oC and the supernatant containing the intracellular organelles as well as solubles was decanted. Sediments of ghosts were re-suspended in buffer of same strength, washed thrice with Tris buffer and the supernatant fluid discarded. The membrane pellet was suspended in 15 ml of 0.1 M Tris-buffer pH 7.4 and used for anisotropy studies after measuring the optical density of the membrane suspensions at 540 nm.
Fluorescence anisotropy studies using DPH as membrane probe
The absorption and emission spectra of DPH (10-5 M) in THF (Tetrahydrofuran) and emission spectrum of DPH in membrane were recorded. The absorption spectra of DPH revealed the absorption maxima to be 356 nm. Taking this to be the excitation wavelength the emission spectrum was scanned and maximum was found to be 458 nm. Hence anisotropy measurements were carried out at 458 nm. For the probe DPH to go and position itself into the membrane and thereby give reasonable fluorescence intensity in the range 60-100 during the anisotropy experiment, a molar ratio of DPH to lipid of 1:100 was maintained in the present study.
The weight of the membrane preparation was determined as follows: 1ml of the membrane suspension was dried overnight at 60C in an oven and the total weight of the membrane obtained thereby was calculated. Amount of protein present in that volume of the membrane suspension was determined and hence amount of lipid present was calculated by subtracting the weight of protein from the total weight. From the GC-MS analysis, the fatty acid components in the phospholipid of membranes obtained in each medium was determined and since the molecular weight of these lipids were known, the approximate molarity of the membrane suspension in terms of the lipids present was determined. A molar ratio of DPH to lipid as 1:100 was maintained accordingly for the three different media. Since the anisotropic measurements were done for membrane preparations from 48 h and 96 h old mycelia obtained on each medium, samples having equal weights of membranes and equal O.D. at 540 nm were taken for the two time points for each medium. The final reaction mixture consisted of 3ml of membrane preparation and 0.3 ml of DPH in THF such that the final concentration of DPH was 10-6 M. In order to deproteinise the membranes 0.1mg/ml proteinase (pepsin) was added and incubated for 1 hour. Fluorescence polarization was measured in an F-4500 Hitachi spectrofluorimeter at 356 nm excitation and 458 nm emission (slit width 5/5). Fluorescence anisotropy was measured over the temperature range 15 °C - 60 °C. All the experiments were done in triplicates using membrane preparations from mycelia grown on glucose, olive oil and sunflower oil media at both time points (48 h and 96 h) and sd values calculated. For calculating the anisotropy, both the horizontal and vertical polarizations were measured to obtain the correction factor G and the I and I respectively. Fluorescence intensities were measured for the following positions of the polarizer (90, 90), (90, 0), (0, 0) and (0, 90). The first two values are for the horizontal polarization and the latter two are for the vertical polarization. Extent of fluorophore rotation can be quantified .by anisotropy (r)
G is the correction factor for transmission efficiency.
Relationship between polarization (P) and anisotropy (r) can be given by the following expression (Lakowicz, 1983).
Protein was estimated by the method of Lowry et al. (Lowry et al., 1951) using the Phenol reagent.
1,6 - Diphenyl - 1,3,5 - hexatriene (DPH) was obtained from Sigma Chemical Company, St.Louis, USA. All other chemicals used were of analytical grade from local sources.
Visualisation of intracellular lipids and mycelial stability
The stability of the hyphae, in terms of non-production of bulbous forms and resistance to lysis was greater on olive oil than on glucose and sunflower oil. No bulbous forms were observed even on the 5th day (Fig 1 a-c).
Sporulation was not evident even on the 6th day. In the case of mycelia grown on sunflower oil medium, filamentous nature was observed up to the 4th day, sporulation and bulbous mycelial forms were seen on the 4th day with onset of lysis by the 5th day (Fig 1 d-f).
The growth and maturation of mycelia was faster on glucose. Sporulation was observed by the 2nd day, formation of bulbous forms of mycelia with intracellular riboflavin accumulation was seen on the 3rd day, onset of lysis was observed on the 4 th day and complete lysis was evident by the 5th day (Fig 1 g-i ).
The microscopic observations corresponded well with the pattern of biomass changes. The biomass on glucose medium declined after 72 h of growth, on sunflower oil the decline in biomass started after 96 h of growth while the mycelial growth on olive oil was most stable and did not show any decline even after 96 h of growth (Fig 2).
Lipid accumulation and riboflavin production
On all three media the maximum lipid accumulation was observed at 48 h of growth (Table 1) and the riboflavin production started increasing after 48 h of growth (Figs 3-5). Lipids constituted 46.1 % of the weight of olive oil-grown mycelia, 15.3 % of the weight of sunflower oil-grown mycelia and 10.2 % of that of glucose-grown mycelia at the end of 48 h (Table 1) (Figs3-5). Maximum riboflavin was obtained on olive oil medium (282.03 g/ml) followed by glucose (216.96 g/ml) and sunflower oil (108.64 g/ml) (Figs3-5).
Fatty acid profile of the triglycerides and membrane phospholipids
Irrespective of the growth substrate used, the 48 h and 96 h old mycelia obtained on glucose, olive oil and sunflower oil medium showed the presence of a large proportion of unsaturated fatty acids in the triglycerides and phospholipids (Tables 2 and 3). An observation was the decrease in the Octadecadienoic acid (C18:2; Linoleic acid) content in the triglycerides of glucose and olive oil grown mycelia at 96 h during riboflavin production (Table 2). In the case of glucose and olive oil-grown mycelia the decrease was very significant, and was about ten-fold or more while it was minimally reduced in sunflower oil-grown mycelia (Table 2) (Fig 6).
Interestingly, Octadecenoic acid (C 18:1) was found to be present in the highest percentage in the phospholipids of the membrane preparations obtained on all the three media during growth (48 h) and riboflavin over production (96 h) (Table 3). In the case of membrane preparations from glucose and sunflower oil grown mycelia, a decrease in the content of Octadecenoic acid was observed during riboflavin over production (96 h) while in the case of membrane preparations from olive oil-grown mycelia, the Octadecenoic acid content increased in the 96 h old membrane preparations (Table 3).
Fluorescence anisotropy of the membrane preparations
In general, the highest fluidity (low anisotropy) was observed in the case of membrane preparations obtained from olive oil grown mycelia both during riboflavin over production (96 h) and exponential growth (48 h) (Figs 7 and 8).
During growth the membrane preparations obtained from sunflower oil grown mycelia and those from mycelia obtained on glucose medium showed similar fluidity characteristics. While during riboflavin over production, membranes of sunflower oil grown mycelia showed intermediate fluidity and least fluidity was observed in the case of membrane preparations of glucose grown mycelia (Figs 7 and 8).
Evidence of lipase activity was seen as a white precipitate of Ca-oleate on CaCl2-Tween 80 plates upon addition of culture supernatants of olive oil, sunflower oil and glucose media (Fig 9).
TLC of the cell free culture supernatant of Triolein medium showed the appearance of Octadecenoic acid as one of the degradation products indicating an extracellular cleavage of this substrate via extracellular lipase activity (data not shown).
The lipase activity was quantified by a colorimetric assay. Maximum lipase production was observed at 48 h of growth on all the three media (Figs 3-5), which corresponded to the accumulation of maximum lipid prior to riboflavin over synthesis. Maximum lipase was produced on olive oil medium (5 U/ml) (Fig 3) followed by sunflower oil (4.25 U/ml) (Fig 4) and glucose medium (2.5 U/ml) (Fig 5). The increase in extracellular lipase activity corresponded to an increase in biomass and decrease in the carbon source in the culture supernatant (Figs 3-5).
A 1.5 fold increase in the lipase activity (7.5 U/ml, 6.5 U/ml and 4.2 U/ml on olive oil, sunflower oil and glucose respectively) was observed in all the three cases upon addition of Tween 80 to the medium. Tween 80 alone as sole carbon source did not support growth of
In the riboflavin over producer
Lipid accumulation in
In this study lipid accumulation during riboflavin over production by
Our study shows for the first time that lipid accumulation and riboflavin over production in
Fatty acid profile of triglycerides and riboflavin production
A GC-MS analysis of the triglycerides of
Mycelial stability is influenced by growth substrate and membrane fatty acids
In general, morphology and stability of
It has been reported that microorganisms respond to stresses such as changes in osmolarity, pH and presence of toxic chemicals by altering the permeability and fluidity of the membranes. In particular, alterations in the fatty acid composition of the membrane phospholipids have been reported. Fatty acid analyses of the plasma membrane of the salt tolerant yeast
The appearance of Octadecenoic acid as one of the products of Triolein degradation as well as the appearance of a white precipitate of Ca-oleate upon addition of the cell free culture supernatant to CaCl2 Tween 80 plates, confirms the activity of an extracellular lipase which is involved in cleaving the lipid substrates. The observation of maximum lipase production at 48 h of growth on all the three media which corresponded to the accumulation of maximum lipid prior to riboflavin oversynthesis needs to be studied further for a probable role of the lipase in providing precursors for flavinogenesis (Figs 3-5).
Tweens, like triacylglycerol, contain an ester group that can be hydrolysed by lipase. This behaviour of Tweens as a lipase substrate could explain the inductive effect of Tween 80 on the production and release of lipase by
The production of lipase is mostly inducer-dependent, and in many cases, oils act as good inducers of the enzyme (Sharma
In the present study we have shown intracellular lipid accumulation and changes in membrane fluidity during growth and flavinogenesis by
Mycelial stability was the greatest on olive oil medium followed by sunflower oil, then glucose-grown mycelia. Membrane fluidity was correlated with the fatty acid composition of the membrane phospholipids and our observations on the mycelial stability in terms of non production of bulbous forms and resistance to lysis correlated with the Octadecenoic acid content of the membrane phospholipids Maximum fluidity was observed in the case of membrane preparations of olive oil grown mycelia due to the above reason followed by those obtained on sunflower oil and the membranes obtained from mycelia grown on glucose exhibited least fluidity. Hence we postulate that the content of Octadecenoic acid in the membrane phospholipids may play a role in stabilising the mycelia by influencing the membrane fluidity.
Based on our results we postulate that the decrease in Octdecadienoic acid content in the triglycerides and the content of Octadecenoic acid in the phospholipids of
We thank Prof. Dr. J. Zeyer of the Institute of Terrestrial Ecology, ETH, Switzerland and the Postdoctoral fellow in his group, Dr. O.Pelz for initiating us into techniques of lipid fractionation and lipid analysis. We also thank M/s. SGS Lab house India, Ltd., for the GC-MS analysis.