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Utilisation of waste news paper hydrolysates in Growth and production system of Neurospora crassa.


Lignocellulosic biomass holds remarkable potential for conversion into commodity products presenting dual advantage of sustainable resource supply and environmental quality. Plant biomass as a useful and valuable resources of fermentable sugars in the biorefining industry has generate large interests for use in producing biofuels and chemicals[1]. Energy security, Petroleum depletion and global warming have been the main driving force for the development of renewable fuels that can replaces petroleum derived fuels such as gasoline and diesel. Bioethanol is currently the most commonly used renewable resources. Considerable research efforts have therefore focus on reducing the enzyme costs by producing more efficient enzymes from cheaper carbon source. Waste news paper hydrolysate is a cheap, abundantly available cellulosic secondary biomass. It can serve as an ideal substrate for microbial processes for the production of value added products .such as protein, enriched animal feed, enymes, amino acid and organic acids [2]. The goal of this work to evaluate the feasibility of using waste news paper hydrolysates as an inexpensive feedstocks for growth and production system of N.crassa.

Background Information

In this section we are dealing about the role of Neurospora crassa in environmental sectors along with the utilization of hydrolysates in the production of valuable products.

Role of Neurospora in environmental sectors: Neurospora crassa plays a very vital role in the environmental management process.

Role of Neurospora crassa in liquid waste management: Neurospora crassa a soft rot fungus also helpful in bioremediation process. Kiran et al reported the biosorptionof Pb(II) and Cu(II) aqueous solutions by pretreated biomass of Neurospora.crassa. Dried fungal cells exhibited the maximal sorption capacity (49.06 mg/g) for Pb (II) ions while detergent treated cells showed the maximum sorption capacity (12.28mg/g) for Cu(II) ions[3]. Luke and Burton(2001) reported a novel application of Neurospora Crassa for the bioremediation of Phenol and p-Cresol and compared the phenol degradation by N.Crassa in dynamic, static and in cappiliarry membrane immobilized reactor[4].

Application of Neurospora crassa in solid waste management: As the effective and efficient producers of cellulolytic enzymes, it becomes one of the major organisms for the lignocellulosic solid waste biomass utilization. Neurospora crassa is a true cellulolytic fungus and having the capability of synthesizing and secreting high level of cellulases.In addition this is well known producers of bioethanol. Utilizing wheat straw as solid substrate for cellulase production by Neurospora crassa have been investigated by Romero et al[5].

Role of hydrolysate in product formation: Hydrolysates, which are released after the hydrolysis of lignocellulosic biomass may contains inhibitory substances ,which hinders the microbial growth., this can be solved by using the following strategy such as large inoculum, diluted hydrolysate solution, tolerant strains or a combination of the three[6]. After treatment with acid various sugars have been released such as arabinose, glucose, xylose and acetic acid[7]. Only few literatures have been reported the utilization of hydrolysate for growth and production system of microbes. Damisha et al have been reported the efects of chemical pretreatment of some lignocellulosic wastes on the recovery of cellulose from Aspergillus niger AH3 mutants[1]. Utilizing waste paper hydrolysates could be a novel approach to reduce the cost of enzymes, through this process we can utilized all sugars generated from the hydrolysates. Biglow and Wyman have stated that few microbial strains were also able to consume or convert essentially all furans, aldehydes and acetic acid which released in the hydrolysates after hydrolysis process[8].

Materials and Methods

Pretreatment of waste news paper: The hydrolysates of waste news paper were prepared by acid digestion. Old news paper (Times of India) were collected form the local market. Before acid pretreatment, deinking process of the chopped and grounded waste news paper have been performed by using effective chemicals and surfactants(Tween-80). Deinked waste news papers were further used for the acid pretreatment. Acid treatment of WNP(Waste news paper)have been performed with 20-70% [H.sub.2]S[O.sub.4] (v/v)solution with maintaining the definite solid liquid ratio. Waste news paper were soaked in acid,at room temperature for one hour. These solution were cooled out, filtered and maked up the resulting solution pH 7.0 by dilute NaOH solution. The resulting hydrolyzates were used in the production medium.

Culture, Growth and Production

All the chemicals and reagents were used to perform experimental works are of Himedia and S.D.fine make.Neurospora crassa NCIM1021 strain was procured from National Chemical Laboratory (NCL), Pune. Fungal spores from a stock, kept at 4[degrees]C in 20% (v/v) glycerol. Neurospora cultures were grown on M2 slants at 28[degrees] C for 4-5 days. Slants were maintained at 4[degrees]C and subcultured about monthly intervals. For the study of growth and production rate separate sets of batch experiments have been performed. First set of experiment has been carried out(for getting culture solution) in a 250 ml erlenmeyer flasks containing 150 ml of [M.sub.2] broth in which 5 loopfull cultures of mycelial conidia were added and shaken at 180 rpm at 30[degrees]C in an incubator shaker for 3-4 days. While other sets of batch experiments were carried out in 250 ml Erlenmeyer flasks containing 100 ml of production media having components (g/l) Urea, 0.3; (N[H.sub.4]) SO4, 1.4; K[H.sub.2]P[O.sub.4], 2.0; Ca[Cl.sub.2]. 2[H.sub.2]O, 0.4; MgS[O.sub.4]. 7[H.sub.2]O, 0.3; Peptone, 1.0; Tween 80, 0.2; FeS[O.sub.4]. 7[H.sub.2]O, 0.005; MnS[O.sub.4]. 7[H.sub.2]O, 0.0016; ZnS[O.sub.4]. 7[H.sub.2]O, 0.0014; Co[Cl.sub.2]. 6[H.sub.2]O, 0.02, with acid pretreated waste news paper hydrolysates used as carbon source with the initial pH 5.0.Pretreated waste news paper hydrolysates containing production media were autoclaved and inoculated with ~ 8.0 ml of [M.sub.2] broth culture solution (specific cell dry weight) of Neurospora crassa. Growth and production experiments were performed in incubator shaker at 30[degrees]C and 180 rpm.

Results and Discussions

Separate sets of batch experiments have been carried out for the growth and production study of Neurospora crassa using acid treated waste news paper hydrolysates as carbon source. From the above experimental observation we have observed that higher dosages of acid concentration was not suitable for the growth and production system of N.crassa. Pretreatment of waste news paper with medium acid dosage was found better for the growth and production system, in comparison to lower and higher acid dosages.

It is evident from Table 1 and Fig1, that 40% acid treated hydrolysates was found much more suitable for the growth and production system of N.crassa. Distinctly large lag phase ~ 24hr were observed in the cell growth in hydrolysate system, presumably because of the presence of the hydrolysis by products. The microbes also utilized the sugares present in the hydrolysates for their growth as well as cellulase production.


Maximum cell concentration were approximately in hydrolysate system, indicating the easier assimilation of hydrolysates than less insoluble cellulose. Hydrolysates with lower than 20% of oligomers (H5 and H6) supported active cellulase production. Xylose is also known to induce cellulase synthesis, although not as effectively as cellulose. The nonglucose sugars present in the hydrolysates also facilitated the induction for cellulase synthesis[9-11].


In conclusion, despite the potentially, inhibitory effects, the hydrolysates supported cell growth and induced active cellulase biosynthesis. 40% [H.sub.2]S[O.sub.4] treatedwaste news paper hydrolysate is more favourable for growth and production of Neurospora crassa. Utilisation of waste news paper hydrolyzates for growth and production is the novel approach for the complete utilization of waste raw material, as the waste news paper used as secondary fiber for waste paper recycling, whereas on the other hand hydrolyzates released after treatment can also be used for product formation in SmF system, which revealed the complete utilization of the waste secondary cellulosic biomass.


[1] Damisha, D.,Ameh, J.B.,Umeh, V.J .,2008,Efects of chemical pretreatment of some lignocellulosic wastes on the recovery of cellulose from Aspergillus niger AH3 mutants,Afr J Biotechnol,17 ,2444-2450.

[2] Singh, P.,Nigam, Nee.,Pandey, A.,2009,Sugarcane Bagasse ,Biotechnology for agroindustrial utilization ,239-252,ed-1,

[3] Kiran, I.,Akar, T.,Tunali, S.,2005,Biosorption of Pb(II)and Cu(II) from aqueous solution by pretreated biomass of N.Crassa.Process Biochem,;40:3550-3558.

[4] Luke, A.K.,Burton, S.G.,2001,.A novel application for Neurospora crassa:Progress for batch culture to a membrane bioreactor for the bioremediation of phenols.Enzyme Microbial Technol ,29:348-356.

[5] Romero,M.D.,Aquado,J.Gonjalez,L,.Ladero,M.,1999,Cellulase production by Neurospora crassa on wheat straw,Enzyme Microbial Technol,25,244-250.

[6] Yu, J.,and Kieko, S.,2008,Microbial utilization and biopolyester synthesis of bagasse hydrolysates ,Bioresour Technol,99,8047-8048.

[7] Bernhar, D.H.H.W.,and Prudfoot, S.,1990, Proceddings of the south African sugar technologists association,Dilute acid hydrolysis of bagasse ad fermentation of hydrolysates to produce single cell proteins.

[8] Biglow, M., and Wyman, C.E.,2002,Cellulase production on bagasse pretreated with hot water ,Appl Biochem Biotechnol, 98-100.

[9] Lo, C.M.,Zhang, Q.,Lu,P.,Ju,L.K.,2005,Cellulase production by T.reesei using saw dust hydrolysate.Appl Biochem Biotechnol,121-124,561-573.

[10] Sternberg,D.,and Mandels,G.R.,1979,Induction of cellulolytic enzymes in T.reesei by sophrose.Journal of Bacteriology,139,761-769

Nitin Verma *, M.C. Bansal and Vivek Kumar

Department of Paper Technology, Indian Institute of Technology, Roorkee, Saharanpur Campus, Saharanpur, U.P., 247001, India Corresponding Author E-mail:
Table 1: Growth of Neurospora crassa in various waste news paper
hydrolysates containing production medium.

S.No Raw Acid treatment Hydrolysate colour
 material dosages after hydrolysis

 1. Without Whitish colour
 2. Waste news 20% Whitish colour
 3. papers 30% Whitish colour
 4. 40% Slight brown colour
 5. 50% Dark brown colour
 6. 70% Deep brown colour

S.No Growth of
 Neurospora crassa

 1. ++

 2. +++
 3. ++
 4. ++++++
 5. +++
 6. Not suitable

* + indicates the growth percentage of Neurospora in waste news paper
hydrolysates medium after48 h incubation.
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Author:Verma, Nitin; Bansal, M.C.; Kumar, Vivek
Publication:International Journal of Applied Environmental Sciences
Date:Jul 1, 2010
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