Microbial  Treatment to Black Liquor

 

Bhaskar G. Gaikwad¹* and Yashita Thakur²

¹C.E.Division, National  Chemical  Laboratory, Pune-411008, India

²Department of  Biotechnology,  Dr. D.Y. Patil B.B.I., Tathawade, Pune

*Corresponding Author E-mail: bg.gaikwad@ncl.res.in

Black liquor is released from pulp and paper industry. It is hazardous in nature and harmful to environment. We have screened bacterial cultures for treatment of black liquor. 18 bacterial cultures were used for screening of biodegradation of black liquor (1:5 diluted). We have used two different pH i.e. 9.0 and 5.5 for study. Among bacterial cultures we attempted to use  Pseudomonas sp., Arthrobacter, Pseudomonas fluroscence and Pseudomonas pudita for screening. Baker’s yeast was also used for study at pH 9.0 and 5.5. Study of sugar utilization, phenol degradation and decolorization was carried out. Pseudomonas fluorescens NCIM 2174 utilized 47.5% sugar at pH 9. In case phenol degradation, 30.35% degradation was observed by Arthrobacter simplex NCIM 2449 at pH 9. 27.85% decolorization by done by Pseudomonas putida NCIM 2872 at pH 5.5. Pseudomonas fluorescens NCIM 2174 has shown  10.36% phenol degradation and 2.76% decolorization at pH 9. Arthrobacter simplex NCIM 2449 utilized 37.7% sugar and 8.23% decolorization observed at pH 9. 4.34% sugar utilization and 17.71% phenol degradation was observed with Pseudomonas putida NCIM 2872 at pH 5.5. Baker’s yeast has shown 10.94% sugar utilization and 15.59% decolorization at  pH 5.5. 14.27% phenol degradaton was obseved with Baker’s yeast at pH 9.

 

 

 

INTRODUCTION:

Paper and pulp industry releases black liquor which is rich in lignin, hemicellulosic material and it is highly colored. This effluent is hazardous in nature. Dense colour is harmful for aquatic organisms. It is essential to remove colour and toxic elements present in it. So many years efforts are being made but there is limited success. Here we have attempted different bacterial cultures and baker’s yeast to degrade, utilize the material present in the black liquor and reduce colour. Many researchers have attempted this problem to solve it. Study using bacterial culture is reported by Chandra and Abhishek (2011)¹, Chandra et al. (2011)², Chai et al. (2014)³ etc. Fungal cultures were used by Salony et al. (2007)⁴, Jin (2008)⁵, Zhao et al. (2008)⁶ etc.  In our study we have done screening of bacterial cultures to study treatment effect to black liquor (BL). We have used baker’s yeast also. In our study our main aim was to reduce load in black liquor and decolorize it which will lead to safe effluent.

 

MATERIALS AND METHODS:

Chemicals

Peptone, beef extract, glucose, agar were purchased from Hi-Media, Mumbai. NaCl, calcium chloride, sodium alginate etc. were purchased from SD Fine chemicals, Mumbai.

 

Cultures:

Bacterial cultures were brought from NCIM (National Collection of Industrial Microorganism), Pune. Bacterial cultures were maintained on Nutrient–agar medium (beef extract 10, NaCl 5, peptone 10 g/L, pH 7.0, agar 20g/L.) at 4⁰C. We have used commercial baker’s yeast for study.

 

Growth:

Bacterial cultures were grown on nutrient broth medium at 28⁰, 220rpm for 24h. Preinoculum was used. Cells were separated by centrifugation and used for study.

 

Biodegradation:

Black liquor (BL) was diluted by distilled water. 1:5 dilution was used. pH was adjusted as per experimental strategy. 4ml diluted black liquor was taken. 1 mL cell slurry was added. Cell concentration in reaction mixture was 2.5%. It was incubated at 220rpm and 28⁰C for 24h. Analysis of reducing sugar, phenol and colour was done.

 

Analysis:

Reducing sugar was analyzed by DNSA method [Miller (1959)]⁷. Non reducing were hydrolyzed by HCl and then DNSA method used. Phenols were analyzed by nitroprusside method [Rodenas-Torralba et al. (2005)]⁸. Colour of black liquor is estimated by spectrophotometer at 465nm [Chandra and Abhishek (2011)]¹.

 

Experimental:

18 Bacterial cultures and baker’s yeast were used for biodegradation of black liquor.1:5 diluted black liquor was used for study. pH was black liquor was adjusted to 9.0 and screening was done at 220rpm, 28⁰C. Results are shown in table 1. Similarly screening was done by adjusting pH at 5.5. Results are shown in table 2.

 

Table 1: Microbial degradation of black liquor (1:5) at pH 9.0

Sr. no.	Microbial strains	NCIM No.	Sugar Utilization (%)	Phenol Degradation  (%)	Decolorization (%)
1	Arthrobacter simplex	2449	37.70	30.35	8.23
2	Arthrobacter nicotianae	2460	43.46	13.94	1.73
3	Arthrobacter sp.	2263	20.50	0	8.57
4	Pseudomonas sp.	2206	22.93	15.45	0
5	Pseudomonas sp.	2847	23.09	0	6.33
6	Pseudomonas sp.	2305	40.84	0	12.80
7	Pseudomonas sp.	2205	3.38	23.90	7.79
8	Pseudomonas sp.	2246	36.06	19.90	0
9	Pseudomonas sp.	2668	9.78	7.95	10.94
10	Pseudomonas fluorescence	2174	47.50	10.36	2.76
11	Pseudomonas putida	2176	46.39	19.73	0
12	Pseudomonas putida	2102	44.75	17.57	0
13	Pseudomonas putida	5088	41.47	20.08	0
14	Pseudomonas putida	2650	43.93	17.60	5.12
15	Pseudomonas putida	2152	44.26	16.22	0
16	Pseudomonas putida	5050	6.73	0	11.52
17	Pseudomonas putida	5087	17.91	0	6.33
18	Pseudomonas putida	2872	23.09	17.07	13.35
19	Baker’s yeast		5.00	14.27	10.35

 

Table 2: Microbial degradation of black liquor (1:5) at pH 5.5

Sr. no.	Microbial strains	NCIM No.	Sugar Utilization (%)	Phenol Degradation (%)	Decolorization(%)
1	Arthrobacter simplex	2449	2.80	24.12	7.07
2	Arthrobacter nicotianae	2460	6.24	27.98	10.82
3	Arthrobacter sp.	2263	19.04	11.16	14.35
4	Pseudomonas sp.	2206	35.10	8.25	22.33
5	Pseudomonas sp.	2847	9.52	4.60	6.72
6	Pseudomonas sp.	2305	20.21	4.60	5.27
7	Pseudomonas sp.	2205	14.28	3.04	4.39
8	Pseudomonas sp.	2246	13.56	26.77	12.92
9	Pseudomonas sp.	2668	0	14.41	12.59
10	Pseudomonas fluorescens	2174	9.58	8.82	0.46
11	Pseudomonas putida	2176	33.33	3.60	17.47
12	Pseudomonas putida	2102	3.44	1.05	17.86
13	Pseudomonas putida	5088	13.79	15.01	22.33
14	Pseudomonas putida	2650	20.68	5.40	24.66
15	Pseudomonas putida	2152	19.04	6.15	14.75
16	Pseudomonas putida	5050	7.69	13.06	20.68
17	Pseudomonas putida	5087	23.80	16.51	25.05
18	Pseudomonas putida	2872	4.34	17.71	27.85
19	Baker’s yeast		10.94	0	15.59

 

RESULTS AND DISCUSSION:

We have done screening at pH 9.0. Pseudomonas fluorescens NCIM 2174 has shown maximum sugar utilization 47.5%. Arthrobacter simplex NCIM 2449 has shown maximum phenol degradation i.e. 30.35%. Whereas Pseudomonas sp. NCIM 2305 has shown 12.8% decolorization.

 

Maximum sugar utilization at pH 5.5 was shown by Pseudomonas sp. NCIM 2206 35.10%. 27.98% phenol degradation was done by Arthrobacter nicotianae NCIM 2460.  Whereas Pseudomonas putida NCIM 2872 has done maximum 27.85% decolorization at pH 5.5. Baker’s yeast has not shown encouraging results at both pH. If we compare results of cultures showing maximum sugar utilization at pH 9.0 and 5.5, there is no particular trend w.r.t. pH variation and culture variation (figure 1). If we compare sugar utilization pattern of all culture, majority cultures have shown better sugar utilization at pH 9.0 than pH 5.5 except Pseudomonas sp. NCIM 2206, 2205; Pseudomonas putida NCIM 5050, 5087 and baker’s yeast. Results of maximum phenol degradation at pH 9.0 and 5.5 shows that there is no particular trend w.r.t. pH variation and culture variation (figure 2). If we compare phenol degradation  pattern of all culture, 9 cultures out of 18  have shown better phenol degradation at pH 5.5 than pH 9.0. Baker’s  yeast have phenol degradation at pH 9.0, while at pH 5.5 no degradation shown.

 

Figure 1:  Cultures showing maximum sugar utilization at pH 9.0 and 5.5

A-Pseudomonas fluorescens NCIM 2174;

B-Pseudomonas putida NCIM 2176; 

C-Pseudomonas putida NCIM 2102; 

D-Pseudomonas  sp. NCIM 2206;

E- Pseudomonas putida NCIM 5087

 

Figure 2: Cultures showing maximum phenol degradation at pH 9.0 and 5.5.

F- Arthrobacter simplex NCIM 2449;

G-Pseudomonas sp. NCIM 2205 ;                                             

H- Pseudomonas putida NCIM 5088;

I-Arthrobacter sp. NCIM 2460;                                                    

J- Pseudomonas sp. NCIM 2246

 

Comparison of results of cultures showing maximum decolorization at pH 9.0 and 5.5 was done. It seems at pH 5.5 more decolorization than pH 9.0 except Pseudomonas sp. NCIM 2305 (figure 3). If we compare decolorization pattern of all cultures, decolorization is more at pH 5.5 than pH 9.0 except with few exceptions e.g. Arthrobacter simplex NCIM 2449; Pseudomonas sp. NCIM 2305, 2205 and Pseudomonas fluorescence NCIM 2174. While adjusting pH precipitation might have removed hindering element while caused more decolorization.

 

Figure 3: Cultures showing maximum decolorization at pH 9.0 and 5.5

K- Pseudomonas putida NCIM 2872;

L- Pseudomonas sp. NCIM 2305; 

M- Pseudomonas putida  NCIM 5050;

N- Pseudomonas putida NCIM 5087;

O- Pseudomonas putida NCIM 2650

 

Chandra and Abhishek (2011)¹ studied decolorization of black liquor using mixed bacterial culture and found 79% decolorization in 144h. Chandra et al. (2011)² reported 85% decolorization using Serratia marcescens (GU193982), Citrobacter sp. (HQ873619) and Klebsiella pneumoniae (GU193983). Zheng et al. (2013)⁹ observed 50.5% decolorization of BL using mixed microorganisms. Chai et al. (2014)³ have reported 54% decolorization of BL in 7 days using Comamonas sp. B-​9. Wang et al. (2009)¹⁰ found 73.74% decolorization with Coriolus sp. S1. Aspergillus fumigatus JX-5 decolorized BL to 93.15% [Jin (2008)]⁵. Zhao et al. (2008)⁶ found 76% decolorization of black liquor using Pleurotus ostreatus B1. 60-80% decolorization of diluted BL was observed by Salony et al. (2007)⁴ in 3-4 days using Cyathus bulleri. Removal of colour from diluted black liquor was reported by Marton et al. (1969)¹¹ using Polyporus versicolor.

 

CONCLUSION:

Baker’s yeast has shown lower results. Maximum sugar utilization was observed at pH 9.0 i.e. 47.5% using Pseudomonas fluorescens NCIM 2174. Maximum phenol degradation was observed at pH 9.0 i.e. 30.35% by Arthrobacter simplex NCIM 2449. At pH 5.5, we observed 27.85% decolorization by Pseudomonas putida NCIM 2872. It is possible to degrade BL  with more efficiency provided better culture than this gets.

 

REFERENCES:

1.        Chandra R and Abhishek A. Bacterial decolorization of black liquor in axenic and mixed condition and characterization of metabolites. Biodegradation. 22;  2011: 603–611.\

2.        Chandra R, Abhishek A and Sankhwar M. Bacterial decolorization and detoxification of black liquor from rayon grade pulp manufacturing paper industry and detection of their metabolic products. Bioresource Technology. 102(11); 2011: 6429-6436.

3.        Chai  LY, Chen YH, Tang  CJ, Yang ZH, Zheng Y and Shi Y. Depolymerization and decolorization of kraft lignin by bacterium Comamonas sp. B-​9. Applied Microbiology and Biotechnology.  98(4); 2014: 1907-1912.

4.        Salony, Mishra S and Bisaria VS. Decolorization and detoxification of textile dyes and black liquor by laccase of Cyathus bulleri. Journal of Scientific and Industrial Research. 66(8); 2007: 84-688.

5.        Jin XC. Research on treating black liquor of straw pulp with strain JX-​5. Jiangxi Shifan Daxue Xuebao Ziran Kexueban. 32(5); 2008: 555-558 (Chinese) [CAN 151:455069].

6.        Zhao LH, Zhou JT, Lv H, Zheng CL, Yang YS, Sun HJ and Zhang XH.  Decolorization of cotton pulp black liquor by Pleurotus ostreatus in a bubble-column reactor. Bulletin of Environmental Contamination and Toxicology. 80 (1); 2008: 44-48.

7.        Miller  GL. Use  of  dinitrosalicylic  acid  reagent  for  determination  of  reducing  sugar. Analytical Chemistry. 31(3); 1959: 426-428.

8.        Rodenas-Torralba E, Morales-Rubio A, and Guardia Miguel de la.  Determination of phenols in waters using micro-pumped multicommutation and spectrophotometric detection: an automated alternative to the standard procedure. Analytical and Bioanalytical Chemistry. 383; 2005: 138–144.

9.        Zheng Y, Chai  LY, Yang ZH, Tang  CJ,  Chen  YH and  Shi Y. Enhanced remediation of black liquor by activated sludge  bioaugmented with a novel exogenous microorganism culture. Applied Microbiology Biotechnology.  97; 2013: 6525–6535.

10.     Wang HX, Zhang ZM, Mao YB and Zhang XY. Continuous treatment of pulp black liquor by acid-​producing white rot fungus in open system. Huanjing Kexue Yu Jishu 32(4); 2009: 156-158 (Chinese) [CAN152:103800].

11.     Marton J, Stern AM and Marton T. Decolorization of kraft black liquor with  Polyporus versicolor, a white rot fungus. Tappi. 52(10); 1969: 1975-1981.

 

 

Received on 04.01.2015         Modified on 15.01.2015

Accepted on 20.01.2015         © AJRC All right reserved