J. Basic Microbiol.

43 (2003) 3, 167 174

(Department of Agriculture Microbiology, National Research Centre Dokki, Cairo, Egypt)

Microflora involved in textile dye waste removal

WAFAA M. ABD EL-RAHIM*, HASSAN MOAWAD and M. KHALAFALLAH
(Received 04 July 2002/Accepted 17 October 2002)
Textile dyes are heavily used in factories for coloring different cloth materials. This work was
designed to identify microorganisms capable of removing textile dyes, either by biodegradation or by
biosorption.
We expected to isolate microorganisms adapted to high dye concentrations from sites near textile
industry complex. An experiment was conducted to study the efficiency of the isolates in removing
textile dyes. The tested dyes were used as carbon and nitrogen sources for isolation of soil and/or
water microorganisms capable of removing textile dyes wastes from factories effluent. The results
indicated the low efficiency of both bacteria and actinomycetes in clean-up the effluent from the waste
dyes in 10 21 days. On the other hand six fungal isolates were obtained by plating factory effluent on
MARTINs medium and media containing dyes as the sole source of carbon and nitrogen for growth.
These isolates fell in two genera, Aspergillus and Trichoderma. Results of these studies revealed
the potential capacity of these fungi to decolorize the tested dyes in comparatively short time
(2 24 hours) indicating strong efficiency of dye bioremediation by the fungal isolates. Since the
process involved is mostly fast interaction between the fungal mycelium and the dye in the media, the
possible mechanism could be based on a biosorption of such chemicals on the intact fungal biomass,
rather than direct biodegradation of the compounds.

The bioremediation of textile wastes is becoming an important issue in both developed and
developing countries (WAFAA 2000, DE MORAES et al. 2000, ONEILL et al. 2000, SARRIA
et al. 2001, KRULL and HEMPEL 2001, MCMULLAN et al. 2001, YESILADA et al. 2002). The
identification of microorganisms capable of assimilation or removal of textile dyes is one
approach to reduce environmental pollution by textile dyes. OGAWA and YATOME (1990)
found that azo dye assimilating bacteria isolated from drainage of a dyeing plant were identified as Pseudomonas cepacia. The acid azo dyes and p-aminoazobenzene, in model
wastewater, were effectively degraded by a continuous submerged culture of the bacteria.
HU (1994) also isolated a bacterium from sludge from a Taiwanese dye factory wastewater
treatment facility which removed the colour of four reactive azo dyes; Red G, RBB, RP2B
and V2RP. This bacterium was identified as P. luteola. After shaking incubation for 48 h,
P. luteola removed the colour from these dyes and in a further 2 days of static incubation,
the fraction of decolorization ranged between 37.493.2% of the original colour.
A microbial process was developed to treat wastewater effluent arising from a dyeindustry manufacturing methyl violet, rhodamine B, nigrosine and chrysoidine using a fixed
film bioreactor by PRADNYA-KANEKAR and SEEMA-SARNAIK (1995). A culture of Pseudomonas alcaligenes, isolated from cattle dung was applied. The microbial treatment resulted
in the reduction of 51% C.O.D. (chemical oxygen demand), 82% B.O.D. (Biological oxygen demand), 74% Total Organic Carbon (T.O.C.), and 75% phenol. The pH of the waste
effluent remained stable at 8.02 (PRADNYA-KANEKAR and SEEMA-SARNAIK 1995).
In this work we examined microbial resources of textile industrial sites to isolate microorganisms capable of bioremediating dye residues in Egypt.

* Corresponding author: Dr. WAFAA M. ABD EL-RAHIM; e-mail: wafaa10m@hotmail.com

2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0233-111X/03/0306-0167 $ 17.50+.50/0

168

W. M. ABD EL-RAHIM et al.

Materials and methods

Isolation of microflora efficient in textile dye waste removal: Two soil and one water samples were
collected from the textile industry complex at El-Mahalla El-Kubra City, Gharbia governorate in the
Nile Delta, Egypt. One sample was collected from the soil adjacent to the drainage canal nearby the
complex, while the other soil sample was taken 200 meters away from this canal. The water sample
was collected from inside the drainage canal. All samples were transported at 4 C to the laboratory
and used immediately to do the work presented in this article. The rest of samples were stored into the
refrigerator at 7 C for possible further use. Serial dilution of the three samples was made in sterile
H2O. One ml inocula from the tenfold serial dilutions of soil and effluent samples were plated on the
surface of mineral salts agar medium containing (g/liter): 0.5 H2PO4, 0.2 MgSO4 7 H2O, 0.1 NaCl,
and 20 agar. The Direct yellow, Direct brown, Maxilon, Polar, Erio red, and Maxilon yellow dyes
were added in the amount of (0.3 g/l) as sole carbon and nitrogen source. PETRI dishes were incubated
at 28 C, and after two weeks colonies growth on the surface of agar plates were counted. Separate
colonies of the predominant types of microorganisms from appropriate petri dishes (30 100 colonies
density on the dish) were isolated on agar slants with MARTINs medium for fungi and nutrient agar
medium for bacteria and actinomycetes. The isolates were purified by re-streaking on agar plates of
the same media The purified isolates were examined microscopically to check their purity. The ability
of these isolates to grow on the dye containing media and/or to reduce the color intensity in the
medium containing dye was assessed.
Screening of isolated microorganisms for dye removal: Two representatives dyes, Direct yellow
and Erio red, were tested with the pure isolates of bacteria and actinomycetes isolated previously on
media containing the dyes (300 mg/l). The color removal efficiency of the isolates was measured in
liquid mineral medium the same as the one used for isolation (containing the aforementioned dyes at
concentrations of 1, 10, 100 mg/l). The concentrations used were selected to cover a range of possible
dye concentrations in the industrial effluent discharged into the environment
Removal of dyes by pure fungal isolates were also tested on the previous liquid mineral medium
using five dyes, Direct yellow, Direct brown, Maxilon red, Erio red and Maxilon yellow at
concentration of 100 mg/1. These dyes were obtained from Textile Industries Division, National
Research Center of Egypt. The experiment was done in 100 ml ERLENMEYER flasks with 40 ml media
in each flask. The flasks were inoculated with a loopful of growth from the slant and incubated on
shaker (150 rpm) at 28 C for 21 days for bacteria, and actinomycetes and 10 days for fungal isolates.
Inoculated flasks were examined at intervals for color changes and growth. The biosorption of dyes on
fungal biomass was also examined by using large inocula of fungal biomass from the start and
measuring decolorization after 2 and 24 hours.
Measuring the decolorization: The removal of dyes was judged by decolorization efficiency of the
solution, by measuring the optical density of the culture filtrates at intervals of 2, 10, 21 days at
different wavelength relevant to the studied dye (354 nm for Direct yellow; 423 nm for Direct brown;
515 nm for Maxilon red; 525 nm for Erio red; and 373 nm for Maxilon yellow). The values of
decolorization were estimated as percentage of changes of colour intensity (optical density reading) in
relation to control that contained the original dye concentration and no microbial inoculants.

Results
Isolation of bacteria and actinomycetes on dye agar medium
Suspension from soils and effluents collected from textile factories dumping sites were
plated each separately on mineral salt agar medium containing 0.3 g/l of six commonly used
dyes as sole source of carbon and nitrogen. Each dye was supplemented separately to the
agar medium. These dyes are Direct yellow, Direct brown, Maxilon red, Polar red, Erio red
and Maxilon yellow. The ppercentage of bacteria and actinomycetes on petri dishes with
50 100 colonies were calculated based on colony morphology. Table 1 shows the proportions of bacteria and actinomycetes isolated on different dyes. The results show that actinomycetes dominate on Erio red and Direct yellow dyes, being 74 and 100%, respectively.

169

Microflora involved in textile dye waste removal

Table 1
Percentage of bacteria and actinomycetes colonies growing in different dyes as sole source of carbon
and nitrogen
Dye

Actinomycetes

Bacteria

Direct yellow
Direct brown
Maxilon red
Polar red
Erio red
Maxilon yellow

100%
34%
0%
0%
74%
38%

0%
67%
100%
100%
26%
63%

However, bacteria dominated the colonies on Polar red and Maxilon red dyes being 100%
on both dyes. The other two dyes had both bacteria and actinomycetes, in comparatively
higher proportions with bacteria having higher percentages than actinomycetes. The bacteria
recorded 67 and 63% of colonies, whereas actinomycetes recorded 34 and 38% on Direct
brown and Maxilon yellow dyes, respectively.
Efficiency of bacteria and actinomycetes in dye removal
Bacteria and actinomycetes representing the diversity of colonies on dye-containing agar
media were tested for their capacity to remove dyes from liquid media supplemented with 1,
10 and 100 mg/l dyes.
The results in Table 2 show that the percentage of decolorization by isolates of bacteria
and actinomycetes did not exceed 20 and 25%, respectively, of the original color of Direct
yellow and Erio red dyes (10 mg/l) after 21 days of incubation. It is likely that bacteria and
actinomycetes attack part of the dye, but they are not able to degrade the dye totally due to
the lack of proper enzymes and the synthetic nature of these chemical compounds.
Efficiencycy of fungal isolates in dye removal
The six promising fungal isolates from soil and water samples were identified to genera
using the scheme of BARNETT and HUNTER (1972) and GILMAN (1957). These six isolates
fell into two genera, five of them belonged to Aspergillus and one was identified as Trichoderma virdi.
The six fungal isolates from soil and water samples from El-Mahalla El-Kubra were
tested for removal of five commonly used dyes (Tables 3, 4 and Fig. 1). The application of
large starting inocula of fungi (30 mg fungal mycelia/40 ml dye) induced removal of the
dyes from liquid media containing 300 mg/l of dyes in comparatively short time between
2 24 hours, where the highest percentages of decolorization of dyes recorded with Direct
brown dye with most isolates after incubation for 24 hours. The decolorization percentages
ranged between 42 to 89% Table 3. This indicates that the removal takes place by means
other than degradation process and more likely this phenomenon takes place by biosorption.
It is expected that the synthetic dyes would require much longer time for complete degradation by water and soil microflora. The role of representative fungal isolates in biodegradation of those compounds was tested by inoculation of liquid media supplemented with
100 mg/l of dyes as sole source of carbon and nitrogen with standard fungal inocula (loopful
of fresh fungal growth). Then 10 days of incubation the decolorization was measured as
percentage. The results show small percentage of dye removal from the solution with the
exception of one dye namely Maxilon red, which was removed by comparatively large
proportion but not exceeding 30% (Table 4). This indicates that the tested dyes are not readily attacked by fungal isolates throughout the incubation period.

170

W. M. ABD EL-RAHIM et al.

Table 2
Decolorization percent of Direct yellow and Erio red dyes by bacterial and actinomycetes isolates
(21 days)
Isolate number

Direct yellow dye concentration (mg/l)

Erio red dye concentration (mg/l)

10

10

100

0
0
0
0
0
12
20
8
0
5
12
0
0
0
4
0
0
0
2
5
9
10
6
0
6
0
0

0
0
11
4
0
12
13
11
0
0
0
8
14
0
0
0
0
0
25
25
19
0
11
0
0
17
0

0
0
0
0
0

% Decolorization
1*
2*
3*
4*
5*
6*
7*
22*
23*
24
25
31
35
36
39
40
44
45
46
47
49
50
51
52
53
54
55

17
23
4
14
15
15
19
15
14
3
11
4
15
13
13
17
15
13
6
16
12
15
13
13
5
6
17

6
4
0
0
0
0
0
11
0
0
0
0
4
18
0
3
0
9
0
0
17
0

* Bacterial isolates, others are actinomycetes

Table 3
Biosorption of five textile dyes on biomass of six fungal strains
Strain number

Direct
yellow

Direct
brown

Maxilon
red dye

Erio red

Maxilon
yellow

25 (49)
23 (55)
25 (71)
64 (53)
34 (53)
30 (60)

14 (21)
28 (30)
9 (12)
10 (13)
10 (10)
6 (7)

0 (22)
9 (12)
15 (25)
36 (38)
7 (21)
0 (24)

% Decolorization
Aspergillus 1
Aspergillus 2
Aspergillus 3
Aspergillus 5
Aspergillus 11
Trichoderma virdi

29 (72)
38 (66)
11 (67)
19 (62)
30 (82)
0 (2)

80 (85)
81 (82)
47 (70)
55 (88)
81 (89)
0 (42)

* Numbers outside and inside parenthesis are the % of decolorization after 2 and 24 hours, respectively

171

Microflora involved in textile dye waste removal

Table 4
Bioremoval of five textile dyes by the growth of six fungal strains after 10 days
Strain number

Direct
yellow

Direct
brown

Maxilon
red dye

Erio red

Maxilon
yellow

29
30
13
22
15
0

2
5
2
2
1
0

23
19
15
16
15
19

% Decolorization
Aspergillus 1
Aspergillus 2
Aspergillus 3
Aspergillus 5
Aspergillus 11
Trichoderma virdi

1
2
0
0
0
0

17
16
9
9
20
0

* Numbers in table are the % of decolorization after 10 days

Therefore in this study we found it more practical to concentrate on the removal of dyes
by biosorption by fungal biomass, as this seems to be the reliable approach to remove dye
residues in short time. The other biodegradation approach will require extremely longer
time and effort (Tables 3 and 4).
Biodegradation (10 days)

Biosorption (24h)

100

% Decolorization

80
60
40
20

Dye

Y B R

Strain

Y B R

Y B R

Y B R

Y B R

Y B R

11

Tv

Fig. 1
Bio-removal of three textile dyes (Y, Direct yellow, B, Direct brown, R, Maxilon red) by six fungal
strains

Discussion
Several authors (ITOH et al. 1996, SARNAIK and KANEKAR 1995, VYAS and MOLITORIS
1995, HU 1994, LIU and YANG 1989) reported the active bioremediation of toxic and hazardous dyes by microorganisms. This bioremediation can be achieved either by biodegradation of certain dyes such as disperse Yellow 3 dye (Spadaro and Renganathan 1994) or by
biosorption of these toxins on strains of microorganisms such as bacteria and fungi (PRADNYA-KANEKAR and SEEMA-SARNAIK 1995 and COOKSON 1995).

172

W. M. ABD EL-RAHIM et al.

In this study an attempt was made to isolate microorganisms efficient in decolorization of

textile dyes from the wastes from industrial textile factories and the surrounding environment. It was expected that the heavily polluted sites near textile industries harbor microorganisms capable to co-exist with such high levels of pollution. These microorganisms adapt
to the new environment and likely that they can contribute in modifying this environment
through their growth, and function. SARNAIK and KANEKAR (1995) isolated Pseudomonas
alcaligenes, P. mendocina, P. putida biovar B, and P. stutzeri from cattle dung enrichments
and soil samples in the premises of a factory manufacturing methyl violet in India. All four
species had the capability to remove phenol and methyl violet dyes.
In our studies suspensions from soils and effluents collected from sites nearby a textile
factory were plated on a medium supplemented with the six commonly used dyes as sole
sources of carbon and nitrogen. Our results showed that actinomycetes dominated the colonies on Erio red and Direct yellow dyes as they represented 74 and 100% of all colonies.
Whereas bacteria dominated the colonies on Polar red and Maxilon red dyes as they represented 100% of the colonies. The other two dyes had both bacteria and actinomycetes. The
high capacity of bacteria in dye removal was stated by OGAWA and YATOME (1990) who
isolated and identified azo dye assimilating bacteria from effluent from a dyeing plant. HU
(1994) isolated P. luteola strain from a Taiwanese dye factory wastewater treatment sludge,
which removed the color of reactive azo dyes. KIRBY et al. (2000) found that Phlebia
tremellosa decolorized eight synthetic textile dyes by greater than 96% within 14 days under stationary incubation conditions. PRADNYA and SEEMA (1995) found that P. alcaligenes
isolated from cattle dung and immobilized on rock media packed in a rectangular steel tank
increased the efficiency of treatment of waste effluent arising from a dye manufacturing of
methyl violet, rhodamine B, nigrosine and chrysoidine dyes. KRULL and HEMPEL (2001)
while investigating a newly developed sequencing batch process for the purification of
residual water containing concentrated azo dye, found that the split flow can destructively
purify 90% of the pollutants. The decolorization took place by 98%.
In this study, the percent of decolorization by both bacteria and actinomycetes isolates did
not exceed 25% of the original dye. It is likely that the bacteria and actinomycetes were not
able to attack the dye and they were not able to degrade the dye completely due to the lack
of the proper metabolic pathways. PASTI-GRIGSBY et al. (1992) found that some actinomycetes strains Streptomyces rochei A10, S. chromofuscus A11, S. diastaticus A12, S. diastaticus A13, and S. rochei A14 were able to degrade the commercial Acid Yellow 9 or monosulfonated mono azo dye derivatives of azobenzene. ZHOU and ZIMMERMANN (1993)
isolated and identified actinomycete strains capable of decolorizing effluents containing
different types of reactive dyes. Adsorption of anthraquinone, phthalocyanine and azo dyes
on the cells of some of the strains resulted in the decolorization of the effluents, but no degradation of the dyes was observed. In contrast, effluents containing an azo-copper complex
and a formazan-copper complex dye were almost completely decolorized by several strains
without adsorption to the cells. The observed changes in the visible spectra indicated the
degradation during incubation with the strains.
Fungi are known as active agents in removing toxins from the environment. In our study,
six fungal isolates were obtained by plating factory effluent on MARTINs medium and media containing dyes as the sole source of carbon and nitrogen for growth. Based on the
capacity of the fungi to remove dye from solution, these isolates were further studied and
identified according to the scheme BARNETT and HUNTER (1972) and GILMAN (1957). These
isolates belonged to two genera: Aspergillus and Trichoderma. The role of fungi as bioremediating agent was reported by SPADARO and RENGANATHAN (1994) who stated that the
whole cultures of white rot basidiomycete Phanerochaete chrysosporium were able to degrade the disperse Yellow dye [2-(4-acetamidophhenylazo)-4-methylphenol] (DY3) (I) used
in industry and previously reported as a carcinogen. YESILADA et al. (2002) investigated the
effects of dye concentrations and amount of pellet on decolorizing activity by Funalia

Microflora involved in textile dye waste removal

173

trogii. They found that decolorization of the Astrazon Red dye involved adsorption of the
dye compound by fungal pellets. These pellets produced 55% decolorization in 24 hours.
Screening of these isolates in relation to six dyes most commonly used in textile industry,
revealed that these fungal isolates were capable of removing dyes in a relatively short period
of time. This indicates that the process involved is mostly fast interaction between the fungal mycelium and the dye in the media. Consequently, the possible mechanism could be
based on a biosorption of such chemicals on the intact fungal biomass. This is in consistent
with LIU and YANG (1989) and HU (1994) who found fast removal of azo dyes by the biomass of the bacterium Pseudomonas, and with SANI et al. (1998) who found the rate of
decolorization of several dyes (Red HE-8B, Malachite Green, Navy Blue HE-2R, Magenta,
Crystal Violet) in the presence of P. chrysosporium was dependent on biomass concentration in the media. ONEILL et al. (2000) reported that decolorization of azo dyes during
biological treatment can involve both adsorption to cell biomass and degradation by azobond reduction during anaerobic digestion. The active bioremediating power of the fungi we
identified in bioremoval of three dyes through biosorption (after 24 hours), as compared
with slow degrading power with the same fungi (after 10 days of inoculation), is clearly
shown from Fig. 1. It seems that the growth of fungal biomass in dye containing media is
limited by readily assimilated carbon and nitrogen sources.
In conclusion the biosorption looks very promising for removal of dyes from effluents, as
the fungi can be easily cultivated in waste materials to harvest large amounts of biomass for
use in bioremediation programs.

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Mailing address: Dr. WAFAA M. ABD EL-RAHIM, Department of Agriculture Microbiology,
National Research Centre Dokki, Cairo, Egypt
e-mail: wafaa10m@hotmail.com