Chemical, biophysical and morphological changes of native ...calcium chloride were used as chemical...

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J. Rad. Res. Appl. Sci., Vol. 3, No. 4(B), pp. 1431 - 1446 (2010)

Chemical, biophysical and morphological changes of native and treated Phanerochaete chrysosporium pellets for dye biosorption application Ola M. Gomaa1 and Nabila S. Selim2 1 Microbiology Department, 2 Radiation Physics Department, National Center for Radiation Research and Technology (NCRRT), Atomic energy Authority, 3 Ahmad El Zomor St. 11371 Nasr City, Cairo-Egypt

E-mail: [email protected] Received: 07 /11 /2010. Accepted: 14/12/2010.

ABSTRACT The pretreatment of fungal biomass in general offers an increase in the biosorptive capacity according to the type of treatment used. Phosphoric acid and heat were the pretreatments chosen for their eminent removal of Brilliant Green (BG) from aqueous solution using waste Phanerocheate chrysosporium pellets. The results indicate that pretreatment with phosphoric acid resulted in increase in removal capacity by 92% in 30 minutes compared to 72.16 and 46.3% for heat treated and native pellets, respectively. The biosorptive capacities of phosphoric acid and heat treatment were 7.46 and 4.1, respectively compared to 2.63 mg dye/g biomass for native pellets. The dielectric properties of the fungal wall show that both the capacitance and conductance of phosphoric acid pretreated pellets are higher than those for heat treated or native pellets. Morphological changes and FT-IR reflect noticeable cell wall changes. Desorption experiments suggest that chemisorption could be considered as the primary mechanism for dye binding to phosphoric acid and heat pretreated pellets, while physiosorption could be the main mechanism for dye binding to native pellets. The study represents an in depth investigation of waste native and treated fungal pellets as possible low-cost biosorbents. Key Words: dye removal, biosorptive capacity, Phanerocheate chrysosporium,

biophysical changes, low-cost biosorption.

INTRODUCTION

The textile industry uses tons of water during the different processes involved in coloring fabrics, the use of such extensive water amounts leads to

Gomaa and Selim / J. Rad. Res. Appl. Sci., Vol. 3, No. 4(B) (2010)

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the release of large amounts of colored effluents (1). Colors pose a great aesthetic and environmental problem; therefore they have to be removed. The microbial decolorization being cost-effective, have received much attention in the past years for treatment of textile dyes (1-3). Although the use of live microbial cells is considered a safe alternative to conventional chemical and physical treatment methods, yet it has some drawbacks of requiring the appropriate conditions for growth, the need for prior adaptation, the extensive time needed for the dye removal or the toxicity of the resulting aromatic amines. To overcome such drawbacks, fungal biosorption was considered an interesting preference. Biosorption is considered an elegant option because it acts as an intermediate which brings about the benefits of the cell wall binding properties with the cost effectiveness (4). Dye biosorption depends mainly on the binding of the dye onto the biomass, dead biomass being favored for the lack of nutrients or cultivation conditions, it is also unaffected by the presence of toxic compounds in the media(5). Waste fungal biomass, which is a byproduct of industrial fermentations, can be used as a cheap source of biosorbent. The different groups present on the fungal cell wall are responsible for binding the dye molecules; the dye biosorption process could be accomplished quickly and often is completed in a matter of hours(6). Moreover, the biosorption sites could be chemically or physically modified to achieve a higher biosorptive capacity through exposure of latent binding sites(7-8). A variety of techniques are commonly used for detecting such modifications; usually through Infra Red Spectra (IR) and scanning electron microscopy (SEM). Whereas information gained from measuring the physical properties to detect changes is considered an additional tool which provides an insight to the modifications occurring on the fungal wall from these properties. The measurement of the cell dielectric properties could reflect important information about the cell’s physiology, particularly the properties of the membrane and the cell interior. Its value is strongly dependent on the level of folding of the cell membrane which could be microvilli or the blebs formed on the cell wall (9). Phanerocheate chrysosporium is considered a model fungus for dye decolorization from both the basic and applied stand points. It was used for removal of Brilliant Green (BG) with a limited decolorization in 4 days (30%) under in vivo conditions (10). Therefore, the aim of the present work is to study the biosorption of BG using in vitro incubation with Phanerocheate chrysosporium pellets and study the effect of some physical and chemical pretreatments on the fungal biosorption capacity, focusing on the changes occurring on the morphological and chemical levels as


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well as measuring the dielectric properties of the cell wall compared to those of the native pellets in an attempt to understand dye biosorption by used fungal pellets.

MATERIALS AND METHODS

Dye

Brilliant Green (BG) belongs to the triarylmethane dye group (CI name: Basic green 1; its formula is C27H34O4N2S) was kindly provided from the dye lab, chemistry department at the National Center for Radiation Research and Technology (NCRRT). This dye is used for coloring silk and wool and was chosen for its frequent use in different factories in Egypt.

Cultivation and preparation of Phanerochaete chrysosporium pellets

The white rot fungus Phanerochaete chrysosporium ATCC 34541 was cultivated in liquid medium using the shake flask method under laccase enzyme production conditions. The growth medium consisted of malt extract (20 g/l); glucose (20 g/l); peptone (1 g/l). Once inoculated, flasks were incubated on an orbital shaker at 150 rpm and 30oC for 2 days for the pellets to be formed.

Chemical and physical treatment

After incubation, the pellets were separated from the culture fluid by filtration, rinsed with distilled water twice, 1 ml of 1N CaCl2, H3PO4, H2CO3, NaOH were added separately to each flask and incubated under the same previously mentioned conditions for 15 minutes as chemical treatment. For physical treatment, another set of flasks containing pellets were killed using heat (100oC for 30 minutes) and another by autoclaving (at 121oC for 20 min), after which the dye was added to each flask so that the final dye concentration would be 250 mg/l; the flasks were incubated further for 30 minutes. All treatments were performed in triplicates, the concentrations of the added chemical treatments, the time of incubation with the dye under study and the time chosen for heat treatment were all chosen according to a set of prior experiments. The pellets were harvested by filtration and the culture filtrate was used for residual dye measurements using UV-Vis spectrophotometer (Schimadzu UV 2100 spectrophotometer). The decolorizaion was calculated from the following equation:

Decolorization (%) = (Ao-A/Ao) 100


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Where Ao is the initial dye absorption on the day of inoculation at 620 nm; A is the final dye absorption after incubation.

Dielectric studies

The harvested pellets were suspended in 50 ml distilled water and dielectric measurements were performed for samples representing native, chemical and physical treated pellets. The dielectric measurements were carried out using LCR meter type HIOKI 3531, manufactured in Japan. The measuring cell is a parallel plate conductivity cell with platinum electrodes, coated with a platinum black layer to reduce electrode polarization (11) which covers an area of 4 cm2 and a separating distance of 2 cm. The measured parameters were the capacitance C (F) at 1 kHz, and conductance G (S) at 10 kHz.

Biosorption studies

The biosorptive capacity calculated from the following equation:

q= (Co-C )V/M

where q is the amount of dye biosorbed onto the unit amount of the biomass (mg/g); Co and C are the concentrations of each dye in the initial solution (mg/l) and after biosorption respectively, V the volume of the aqueous phase (l) and M is the amount of biomass (g).

Characterization of the fungal biomass

Fourier Transfer Infra Red spectroscopy (FT-IR):

The IR spectra of native and treated pellets of Phanerochaete chrysosporium were obtained using FT-IR spectrophotometer (Mattson 1000 FT-IR, England), individual pellets were dropped on a clean glass slide cover, excess water was removed using filter paper, samples were left to dry at room temperature. The FT-IR spectra were recorded in the range from 400 to 4000 nm using refractive measurements and plotted as wave number Vs transmittance using WinFirst software.

Scanning Electron Microscopy (SEM):

Scanning electron micrographs of the nature of the pellets was carried out using a JOEL JMS 5600 scanning electron microscope; the pellets were collected after treatment with phosphoric acid and heat and dried in oven at 50oC. The pellets were then glued separately onto brass stubs using a double sided adhesive tape and were coated with a thin layer of gold under reduced


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pressure. The images were captured at magnifications of 25,000 X using an electron beam high voltage of 30 KV.

Light Microscopy:

Native and treated pellets were studied under using Labomed Microscope CX RIII connected to Benq 510 E Digital camera. The pellets were placed on glass slides using a clean forceps, the pellets were air dried and excess liquid from media was removed using a filter paper. The pellets were examined using a 40X lens and photographed at once.

Desorption studies:

The study was carried out using the adsorbate-adsorbent obtained at the end of the biosorption experiment. The pellets for native, phosphoric acid and heat treated pellets were incubated for 30 minutes in 1N NaOH. The resulting color released was measured spectrophotometrically in the media, after removal of fungal cells. The dye shows a major absorption band at 620 nm at pH 2.2, but as the pH increases its color changes from green to bluish green. The different treatments applied in this study had different pH values, which resulted in changes at 660 nm. Therefore, the changes in dye concentrations were monitored for both peaks. The visible spectrum from 500 to 700 nm of the dye was recorded.

RESULTS AND DISCUSSION

Decolorization by chemically and physically treated pellets as compared to native pellets

For preliminary selection of chemical and physical pretreatment for waste fungal pellets, sodium hydroxide, bicarbonate, phosphoric acid and calcium chloride were used as chemical pretreatments, while heat for 30 min and autoclaving were used as physical pretreatments. Figure (1, a) represents the visible spectrum for the decolorization for native and chemical pretreatments of fungal pellets shown two peaks at 620 and 660 nm for both green and bluish green color respectively. The comparison of the sum of the two characteristic peaks of the dye color was as follows: calcium chloride> phosphoric acid> bicarbonate> native> sodium hydroxide (fig. 1, b). Visual observation of the chemically treated pellets after decolorization showed that the pellets treated with calcium chloride were light green while the rest of the treatments resulted in the normal green color of the BG, this indicates the possible involvement of another mode for dye removal other than external biosorption, therefore, the


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second choice was the phosphoric acid which was employed in the following experiments. NaOH was employed to increase the biosorptive capacity of Rhizopus stolonifer (5), while in the present study, the least biosorption was obtained from treating the pellets with NaOH, dissimilarly, and the addition of calcium chloride and phosphoric acid provided the best decolorization percentages as observed from the UV-Vis spectrum.

Fig. (1): a) Visible spectrum for BG decolorization and b) Comparison of the absorbance of both 620 and 660 nm peaks for BG decolorization after chemical pretreatment of fungal pellets.

The study of the dielectric properties is represented by the capacitance and conductance which are the major parameters representing the biophysical changes occurring on the cell wall. The membrane capacitance Cm determines the amount of charges that can be stored across a membrane when a cell is exposed to an electric field while conductance reflects membrane permeability (9). The results represented in Fig. 2 a&b show that the stored charges and the permeability of the cell wall respectively are at their highest levels when the pellets were pretreated with phosphoric acid, slightly lower after incubation with the dye. Treatment with alkaline salts (calcium chloride), weak alkali (sodium bicarbonate) and strong alkali (sodium hydroxide) showed moderately higher capacitance and conductance than native pellets.

It was shown that chemical pretreatments led to the rearrangement of the cell wall components against the treated chemicals, although acid treatment is known to give rise to fragmentation of the cell wall differently from alkali treatment to raise cross-linking reactions (12). These effects could be the reason behind the decrease of the capacitance and conductance of the alkali treated cells compared to those pretreated with the acid. The type of pretreatment of

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Fig. (2): Capacitance (a) and conductance (b) for chemical pretreated fungal pellets in the presence and absence of dye.

fungal biomass determines how the fungal cell wall will be affected, therefore controlling the extent of biosorption and/or the type of adsorbed dye. For example: the pretreatment of R. arrihzus with NaOH led to an increase in the biosorptive capacity, the reason was attributed to the removal of proteins and glucans from the cell wall which resulted in changes in the chitin/chitosan ratio generating anionic sites. The use of autoclaving results in changes of the fibrous nature of the biomass which was caked as a result of exposure to high temperature (12). Aksu (7) explained that autoclaving is the major cause for cell wall disruption, thus leading to an increase in biosorption which might also be due to the increase in surface area and opening of more latent sites.

Fig. (3): a)Visible spectrum for RBG decolorization and b) Comparison of the absorbance of both 620 and 660 nm peaks for BG decolorization after physical pretreatment of fungal pellets.

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The physical pretreatment indicates that the decolorization ability is represented by the same decolorizing spectrum for the dye in cases of autoclaving and heat treatment which were both higher than that for native pellets (Fig 3). Therefore, heat treatment was chosen for its ease and speed.

Autoclaved biomass is likely to be caked by heat and pressure which weakens the physical strength of the hyphae of the fungus (12). This physical change can be the cause beyond the decrease in capacitance and conductance of the heat treated and autoclaved cells compared to the native pellets (Fig 4 a&b) in the absence of the dye while there were no differences in the presence of the dye.

Fig. (4): Capacitance (a) and conductance (b) for physical pretreated fungal pellets in the presence and absence of dye. When physically treated cells where exposed to the dye, the biosorption

of the dye on the cell surface showed an increase in its surface charge thus increasing its capacitance and providing ion movement around the cells, consequently increasing their conductance values. Therefore, heat treatment was chosen for pretreating Phanerocheate. chrysosporium pellets. It is also known to be the chosen treatment to increase the biososrptive capacity of Lentinus sjor-caju (13) and for Trametes versicolor (8).

Biosorptive calculations and in depth comparison of phosphoric acid and heat pretreatments on fungal pellets

The biosorption calculations indicate that the pretreatment with phosphoric acid led to a 92% decolorization as compared to 72.16% for heat pretreatment and 46.3% for native pellets. The biosorptive capacity increased also in the same respect along with the calculated biosorbed dye (Table 1). The results indicate that the pretreatment affects the decolorization values.

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Table (1): Biosorbed and decolorization for native, heat treated and phosphoric acid treated pellets of Phanerocheate chrysosporium.

Native pellets Heat treated pellets

Phosphoric acid treated pellets

Decolorization (%) 46.3 72.16 92 Residual dye (mg/l) 134.23 69.57 20.27 Biosorbed dye (mg/l) 115.711 180.42 229.724 Biosorptive capacity (mg/g) 2.63 4.1 7.46

This is also represented as the change in the optical density of the BG after incubation with native, heat treated and phosphoric acid treated pellets (Fig. 5 a&b).

Fig. (5): a) Visible spectrum representing decolorization of RBG and b) Comparison of the absorbance of both 620 and 660 nm peaks for native, heat-treated and phosphoric acid treated pellets.

Fig. (6): Capacitance (a) and conductance (b) for native, heat-treated and phosphoric acid treated pellets, in the presence and absence of dye.

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The dielectric properties of the fungal wall show that the capacitance and conductance of the phosphoric acid treated pellets are much higher than those of native and treated cells (Fig. 6 a&b). The positively charged dye attached to the cell wall may increase its surface charge as can be seen from the increase in the capacitance recorded at 1 kHz compared to native pellets, while no change in the conductivity was recorded after the addition of the dye to native pellets.

The use of pretreatments resulted in a number of changes on the fungal cell wall. The morphological changes represented in SEM photos (Fig. 7) which show that the native fungal pellets have rough and porous surface of interwoven mycelia which provide the surface area for adsorption; this is in accordance with fungal SEM micrographs presented by Bayramoğlu and Arica (8).

Fig. (7): Scanning electron micrographs for native, heat-treated, phosphoric acid treated pellets in the absence of dye (a, b and c, respectively), and in the presence of dye (d, e and f, respectively).

The surface texture and porosity of both heat treated and phosphoric acid treated pellets show that the loose networking has disappeared. The pellets were caked by heat treatment; this in turn is expected to cause weakness of the fungal hyphae. The amorphous homo and hetero-polysaccharides, often in association with proteins, play the role of cementing substances (14). While for


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phosphoric acid treatment, the partial dissolution of the wall by the imposed treatment may lead to rearrangement and displacement of the remaining wall constituents, therefore leading to change in the obtained SEM micrographs (12). The light microscopy (Fig. 8) shows clearly the loose hyphal network of native pellets as compared to the tightly woven and agglomerated structure of the pretreated pellets, but it was observed that the phosphoric acid pretreated pellets had the dye entrapped inside, a dark green color was observed in the core of the pellets as compared to a faint green color on the outside.

In order to confirm the existence of functional groups which are responsible for biosorption, the FT-IR spectra of the native pellets and pretreated

Fig. (8): Light microscopy for native, heat-treated, phosphoric acid treated pellets in the absence of dye (a, b and c, respectively), and in the presence of dye (d, e and f, respectively).

pellets were compared. The obtained spectra of the native pellets show intense peaks at a frequency level of 3500-3200 and 1540 cm-1 representing amino groups stretching vibrations, and 3500-3300 cm-1 representing hydroxyl group. There are strong peaks around 1650, 1400 and 1240 cm-1 which are caused by the C=O stretching band of carbonyl groups. The phosphate groups show some characteristic adsorption peaks around 1150 and 1078 cm-1 representing P=O and P-OH stretching, respectively. The band between 610 and 535 cm-1 for the fungal pellets represent C-N-C scissoring which is only found in protein structure. Some


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changes could be observed comparing native fungal pellets to those pretreated with phosphoric acid or heat (Fig 9a), an evident shift could be observed after pellets were incubated with dye for native and treated pellets (Fig. 9b).

Fungal cell wall are known to contain amine, carboxyl and phosphonate groups (15), these groups could bind BG which has a net positive charge. The phosphoric acid pretreatment is expected to increase the protonation. The binding between the dye and the fungal functional groups is known to take place via the formation of Van der Waals interaction and also by ion exchange with other groups (12).

Fig. (9): IR spectrum for native, heat and phosphoric acid treated pellets in the absence (a) and in the presence of dye (b).


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Desorption data reflect the mechanism of adsorption. The results show that the binding of the dye to phosphoric acid pretreatment is very strong, this is followed by heat and the least dye binding occur for native pellets (Fig. 10), this indicates that the pellets underwent changes in surface charges that is normal in native pellets but irreversibly changed in both heat and phosphoric acid pretreatments. This also indicates a strong bondage between the adsorbate and the adsorbent and the possibility of chemisorption, moreover, only the physiosorbed dyes could have desorbed from the fungal surface, and since it was of low values, it indicates the involvement of both chemisorption and physiosorption for pretreated pellets while physiosorption is the main mechanism for native pellets.

Fig. (10): a) Visible spectrum representing desorption of RBG and b) Comparison of the absorbance of both 620 and 660 nm peaks for native, heat-treated and phosphoric acid treated pellets,.

CONCLUSION

Since the type of treatment affects the fungal cell wall-dye interaction through morphological, chemical and biophysical changes, therefore, it is easy to manipulate this interaction towards higher removal levels according to the chemical nature of the adsorbate and the adsorbent employed. The use of native and modified waste fungal pellets is not only effective because of the potential increase in the biosorptive capacity, but also because it encourages the economical use of cheap cast-off biomass as the adsorbent. The fate of the adsorbate, being removed from aqueous media for re-use or for mere disposal, dictates the pretreatment to be employed.

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3. Yeh, M-S and Chang, J-S. (2004): Bacterial decolorization of an azo dye with a natural isolate of Pseudomonas luteola and genetically modified Escherichia coli. Journal of Chemical Technology and Biotechnology 79, 1354-1360.

4. Nigam, P; Armour, G; Banat, IM; Singh, D; and Marchant, R. (2000): Physical removal of textile dyes from effluents and solid-state fermentation of dye-adsorbed agricultural residues. Bioresoure Technology 72, 219-216.

5. Zeroual Y; Kim, BS; Blaghen, M; Lee, KM. (2006): Biosorption of bromophenol blue from aqueous solutions by Rhizopus Stolonifer biomass. Water, Air and Soil Pollution 177, 135-146.

6. Kaushik, P and Malik, A. (2009): Fungal dye decolourization: Recent advances and future potential. Environment International 35, 127-141.

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8. Bayramoğlu, G and Arica, MY. (2007): Biosorption of benzidine based textile dyes "Direct Blue 1 and Direct Red 128" using native and heat-treated biomass of Trametes versicolor. Journal of Hazardous Materials 143, 135-143.

9. Patel, P and Markx, GH. (2008): Dielectric measurements of cell death. Enzyme and Microbial Technology 43, 463-470.

10. Gomaa, OM; Linz, JE; Reddy, CA. (2008)" Decolorization of Victoria blue by the white rot fungus, Phanerochaete chrysosporium .World Journal of Microbiology and Biotechnology 24, 2349-2356.


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11. Iwamoto S and Kumagai H. (1998): Analysis of the dielectric relaxation of a gelatin solution. Bioscience Biotechnology Biochemistry 62, 1381-1387.

12. Binupriya, AR; Sathishkumar, M; Swaminathan K; Ku CS; and Yun SE. (2008): Comparative studies on removal of congo red by native and modified mycelial pellets of Trametes versicolor in various reactor modes. Bioresoure Technology 99, 1080-1088.

13. Arica, MY and Bayramoğlu,G. (2007): Biosorption of Reactive Red-120 dye from aqueous solution by native and modified fungus biomass preparations of Lentinus sajor-caju. Journal of Hazardous Materials 149, 499-507.

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اإلشعاعیةاإلشعاعیةبحوث بحوث مجلة المجلة ال لتطبیقیةلتطبیقیةوالعلوم اوالعلوم ا

)2010( 1446 – 1431 ص ص )ب(4 دعد 3 مجلد

لكریات فطر الفینوركیتى كرایسوسبوریوم ) المورفولوجیة(التغیرات الكیمیائیة والبیوفیزیائیة والشكلیة المعالج و الغیر معالج بھدف استخدامھا فى مجال االدمصاص الحیوى

2نبیلة سید سلیم 1ةعال محمد جمع ھیئة الطاقة الذریةـ المركز القومى لبحوث وتكنولوجیا اإلشعاع قسم الفیزیاء اإلشعاعیة، 2قسم المیكروبیولوجى، 1

مما ینتج كمیات كبیرة من األنسجةعملیة صباغة أثناءمن الماء أطنانتستھلك صناعة النسیج ى وتعتبر . النفایات السائلة المحملة بالصبغات ة الت اكل البیئی ن المش بغات م ذه الص ن ھ تخلص م عملیة ال

، ومن الحلول التى أثبتت كفاءة عالیة فى ھذا المجال إستخدام أكتسبت اھتماما كبیرا فى السنوات األخیرة . خاصیة اإلدمصاص الحیوى لبعض أنواع من الفطریات فى عملیة إزالة الصبغات

ف " الفینوركیتى كرایسوسبوریوم"إستخدمت ھذه الدراسة فطر ر مكل درا غی والذى یعتبر مصوتعتمد ھذه العملیة على خواص اإلدمصاص لجدار خالیا الفطر، . للتطبیق فى مجال اإلدمصاص الحیوى

ت ومن ثم تھدف ھذه الدراسة الى زیادة قدرة الفطر المستخدم على االدمصاص الحیوى باستخدام معامالوئیة ات الض ف التقنی تخدام مختل امالت باس ل المع أثیر أفض ة ت ة و دراس ة مختلف ة وفیزیائی ؛ كیمیائی

ح ى الماس وئى واإلكترون كوب الض ة المیكرس ات الفیزیائی ة ، والتقنی یلیة الكھربی عة والتوص ، ؛ الس .والخواص الطیفیة فى المدى من األشعة تحت الحمراء والمرئیة وفوق البنفسجیة

یوم أما د الكالس ى كلوری ت تعریض الفطر ال ذه الدراسة فكان المعامالت التى استخدمت فى ھن ا م ا لھ ة لم امالت كیمیائی فوریك كمع ض الفوس ودیوم وحم ید الص ودیوم وھیدروكس وبیكاربونات الصتأثیر على الجدار الخلوى كما تم تعریض مجموعة أخرى الى الحرارة لمدة ست ساعات وساعة ونصف

.درجة مئویة والمعالجة بالحرارة والضغط العالى كمعامالت فیزیائیة 100عند الساعةى ادة ملحوظة ف ى زی فوریك أدى ال ض الفوس ى أن معالجة الفطر بحم ة وتشیر النتائج ال إزال

ى ٪92 إلىاللون وصلت ل ٪46و ٪72المقارنة بالمعالجة بالحرارة والتى وصلت ال ر معام للفطر الغیون ة فى خالل ثالث رات مورفولوجی ى تغی ة الفطر أدت ال ائج ان معامل الل النت ن خ ث ظھر م ة، حی دقیق

كما أدت الى ) زیادة فى السعة الكھربیة(وكیمیائیة فى جدار المیسیلیوم أدت الى زیادة الشحنات المختزنة . زیادة السطح المعرض للصبغة مما أدى الى زیادة القدرة على االدمصاص

Chemical, biophysical and morphological changes of native ...calcium chloride were used as chemical...

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Transcript of Chemical, biophysical and morphological changes of native ...calcium chloride were used as chemical...