Journal of Environmental Biology � July, 2010 �

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* Corresponding author: shweta_kulshreshtha@rediffmail.com

Bioremediation of industrial waste through mushroom cultivation

Shweta Kulshreshtha*1, Nupur Mathur2, Pradeep Bhatnagar3 and B.L. Jain4

1Department of Zoology, University of Rajasthan, Jaipur - 302 015, India2 Centre for Converging Technology, University of Rajasthan, Jaipur - 302 004, India

3International College for Girls, Mansarovar, Jaipur, India4Department of Botany, University of Rajasthan, Jaipur - 302 015, India

(Received: March 13, 2009; Revised received: September 05, 2009; Accepted: September 25, 2009)

Abstract: Handmade paper and cardboard industries are involved in processing of cellulosic and ligno-cellulosic substances for making

paper by hand or simple machinery. In the present study, solid sludge and effluent of both cardboard and handmade paper industries was

collected for developing a mushroom cultivation technique to achieve zero waste discharges. Findings of present research work reveals that

when 50% paper industries waste is used by mixing with 50% (w/w) wheat straw, significant increase (96.38%) in biological efficiency over

control of wheat straw was observed. Further, cultivated basidiocarps showed normal morphology of stipe and pileus. Cross section of

lamellae did not show any abnormality in the attachment of basidiospores, hymenal trama and basidium. No toxicity was found when fruiting

bodies were tested chemically.

Key words: Mushroom cultivation, Pollution abatement, Handmade paper, Pulp and cardboard industries

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Introduction

Handmade paper industries (HMPI) are involved in

processing of cellulosic and ligno-cellulosic substances for making

paper by hand or simple machinery and thought to be non-polluting

whereas cardboard industries (CI) are also involved in processing

of similar raw material as well as recycling of waste paper and

boards having shortened fibres, and thought to be polluting industries.

The pulp, paper and cardboard industries are also utilizing huge

amount of water which is discharged as untreated effluent to natural

water bodies and pollutes it.

Presence of dyes and chemicals, in handmade paper and

pulp industries effluent reveals by its colour, low pH and high COD

(Kulshreshtha et al., 2008). Secondly, problem also arises due to

the repeated recycling of paper. In the last stage of recycling, cellulosic

fibres become too short to be recycled and utilized for making

cardboards. Paper can be recycled to 4-5 times, after that non-

recycled pulp residues accumulated in the front of cardboard industries

as solid sludge and produce enormous odour in the area due to its

biologically degraded compounds. These industries are not only

present in India but also in many other European, American and

Asian countries. Hence, the problem is of global concern.

The present work is based on effluent and sludge utilization

of handmade pulp, paper and cardboard industries situated around

Amani Shah Drainage in Sanganer near Jaipur. These industries

are utilizing cellulosic hosiery rags, cotton rags; mill broke, and ligno-

cellulosic waste paper and cardboard for making paper and

cardboards respectively.

Further, after making paper and boards, effluent is

discharged to the Amani Shah Drainage which is then utilized for

irrigating nearby agricultural fields. Solid sludge of non-recycled

pulp residues is also accumulated in the front of these industries at

the rate of 10 Kg day-1.

The present research work was planned to protect

environment by utilizing the pulp residues separated from effluent

and solid sludge of both industries for mushroom cultivation because

it is not only capable of bioremediation of waste but also provides a

highly proteinaceous food (Mane et al., 2007; Kuforiji and Fasidi,

2009).

Cultivated fruiting bodies were collected for further analysis

of toxicity, morphological and anatomical studies.

Materials and Methods

Sampling site and collection of sample: Sanganeri handmade

pulp, paper and cardboard industries were selected for collection of

solid sludge and effluent. Effluent and solid sludge of these industries

were collected separately, in a clean plastic container, transferred to

laboratory and stored at 4oC until use for analysis.

Physico-chemical analysis of effluent: Effluent samples were

analyzed for physicochemical parameters such as pH, temperature,

salinity, conductivity and total dissolved solids (TDS) by water

analyzer kit. Biological oxygen demand (BOD) was analyzed by

membrane electrode method and chemical oxygen demand (COD)

was analyzed by closed reflux method as given in American Public

Health Association (APHA) manual (1995). All these parameters

were analyzed within 24 hrs. in each of three replicates.

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Kulshreshtha et al.

Mushroom culture and cultivation on waste: Mushroom

(Pleurotus florida) culture and prepared spawn were obtained from

National Research and Mushroom Center, Solan, Shimla, India.

Strain was subcultured on Potato dextrose agar and maintained

at 4oC.

The effluent was mixed with solid sludge. Due to mixing,

thick slurry of composite waste was obtained; therefore no presoaking

is required like wheat straw. 1.25 ml l-1 of 40% formalin solution (v/v)

and 0.75 g l-1 (w/v) of anti-fungal substance bavistin were added to

the slurry. After 18 hrs, slurry was filtered by a clean cloth and

collected pulp fiber spread on a surface clean previously by formalin,

to remove excessive water and maintain 80% humidity. Waste was

then sterilized by steam for 1 hr. In control, wheat straw (WS) was

soaked in water containing formalin and bavistin, for 18 hrs and

sterilized before using it as a substrate. Pulp fibres, alone as well as

in combination of wheat straw (1:1) was tested for mushroom

cultivation.

After cooling, 1 Kg of wet waste alone and mixing with 50%

(1:1, w/w) wheat straw was inoculated with spawn and packed in

polythene bags possess holes at 3 cm distance. After inoculation,

polythene bags were incubated at 22oC±1oC. When Pleurotus

mycelium fully colonized the substrate, polythene bags were removed

and substrate bundles were hanged. To maintain humidity bundles

were sprayed with water twice a day. Six bundles of each combination

of substrate were prepared. Each substrate bundle was observed

daily and time was recorded for pinhead formation, immature fruiting

body and mature fruiting bodies formation.

One bag of each substrate and its combination was dried to

calculate the actual dry weight of the substrate. Biological efficiency of

mushrooms were calculated by dividing weight of fresh mushroom

yield (in Kg) by weight of air dried substrate (in Kg) and multiply by

100 (Banik and Nandi, 2004).

Experiment was repeated thrice. The data was pooled and

ANOVA was used to test the significance of biological efficiency of

mushroom.

Chemical toxicity test of mushroom: A drop of juice was pressed

out of the fresh fruit body on a piece of newspaper. After the spot had

dried, hydrochloric acid was dropped on it. A blue spot indicated the

presence of toxins (Svrcek, 1998).

Anatomical studies: Anatomical structures were analyzed in cross-

section of lamellae which was stained with lactophenol cotton blue

staining technique and then observed under light microscope at

10x10x and 10x45x magnification.

Results and Discussion

Physicochemical characteristics: The pulp, paper and

cardboard industries are major industrial sector which are polluting

natural water bodies by its discharges (Malviya and Rathore, 2007).

The generation of waste water and characteristics of pulp, paper

and cardboard industries effluent depends upon the type of

manufacturing process adopted and the extent of reuse of water

employed in plant (Singh and Thakur, 2006) and it should be checked

before it is used for the cultivation of mushrooms (Gray, 2000).

In this context, the physico-chemical characteristics of waste

effluent of Sanganeri handmade paper and cardboard industries

were carried out and results are shown in Table 1.

Fig. 2: Spore print of fruiting body

Fig. 1: Fruiting bodies appeared on the coloured pulp residues of handmade

paper industries when mixed with 50% wheat straw

Table - 1: Analysis of different physico-chemical parameters of handmade

paper industries (HMPI) and cardboard industries (CI) effluent

Parameters HMPI effluent CI effluent

Temperature (oC) 30.4 ± 4.98 31.3 ± 3.76

pH 4.37 ± 0.26 6.56 ± 0.1

Salinity(in ppt) 2.22 ± 0.63 2.16 ± 0.18

Conductivity (mS cm-1) 4.7 ± 2.01 4.35 ± 1.77

TDS (ppt) 2.03 ± 0.44 1.87 ± 0.47

Colour (Pt-Co units) 0-3242 8.1-9200

Turbidity (NTU) 0-77 0.38-21

BOD (mg l-1) 30.7 ± 4.27 18.76 ± 9.03

COD (mg l-1) 6263.33 ± 805 3213.66 ± 170.67

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Bioremediation of industrial waste through mushroom cultivation

Salinity, conductivity, and total dissolved solids were not found

to be very high. Temperature and BOD of effluent of both industries

is in acceptable range but pH and COD were found to be above the

limits described by Central Pollution Control Board (MINAS, 2000).

Low pH was also observed by Saakshi (2005). The reason of low

BOD and high COD is the presence of high molecular weight

compounds such as cellulose and lignin. This finding was evidenced

by the work of Raj et al. (2007).

Handmade paper industries are using different dyes for

making coloured papers revealed by the presence of colour in the

effluent whereas brown or black colour of cardboard industries effluent

is due to ink, dyes or chemicals released during pulp making process.

Hence, effluent is not suitable for discharge in untreated form.

Growth characteristics and biological efficiency: A

number of research reports have been published for biological

treatment of kraft pulp and paper industries (Ragunathan and

Swaminathan, 2004; Singhal et al., 2005; Bishnoi et al., 2006;

Singh, 2007; Kousar and Puttaiah, 2009; Satyawali et al.,

2009). However, no work has been done on treatment of

solid as well as effluent wastes of handmade paper and

cardboard industries.

Although effluent having low pH, high COD and colour yet it

can be utilized for the production of mushroom because mushrooms

have ability to degrade dyes (Espíndola et al., 2007), xenobiotics

(Eggan, 1999), lignin and cellulose containing sawdust and

wastepaper (Chang and Chiu, 1992; Baysal et al., 2003).

In the present study, composite waste (effluent + solid sludge)

of handmade paper industries was found to be good support in the

formation of fruiting bodies of P. florida (ANOVA, p<0.5) when mixed

with 50% wheat straw (Table 2). The results are consistence with

Table - 2: Biological efficiency, weight and average yield of substrates

Waste substrate usedWeight of each substrate Average yield in three flushes Biological efficiency

(in kgs) (in kgs) (in %age)

100% WS 0.18 ± 0.08 0.09 ± 0.02 59.3

100% HMPI 0.47 ± 0.05 0.013 ± 0.02 2.88

50% HMPI + 50% WS 0.16 ± 0.01 0.15 ± 0.03 96.38

100% CI 0.27 ± 0.09 0.045 ± 0.03 17.02

50% CI + 50% WS 0.31 ± 0.05 0.17 ± 0.04 56.54

WS = Wheat straw, HMPI = Hand made paper industry, CI = Cardboard industry

Table - 3: Time periods (mean ± standard deviation) of the different phases of P.florida mushroom cultivation (in days)

Waste substrate for Spawn running time Pinhead formation Complete fruiting body

cultivation of Pleurotus (in days) (in days) formation (in days)

100% WS (control) 9.4 ± 0. 55 33 ± 2.4 36.6 ± 2.2

100% HMPI 8.8 ± 0.8 48.8 ± 5.4 51.8 ± 5.4

50% HMPI+ 50%WS 7.2 ± 0.44 26.2 ± 1.8 29.2 ± 1.7

100% CI 10.2 ± 0.4 51.4 ± 3.78 54.6 ± 4.0

50% CI + 50% WS 8.0 ± 1 26.6 ± 3.2 30.4 ± 2.96

WS = Wheat straw, HMPI = Hand made paper industry, CI = Cardboard industry

Fig. 3: Cross section of lamellae of fruiting bodies cultivated on 50%

handmade paper industries waste mixed with 50% wheat straw. It shows

hymenophoral trama, and hyaline basidiospores containing basidium on it

Table - 4: Measurement of pileus, stipe and spores of Pleurotus florida

cultivated on industrial waste as mentioned above

Substrate usedStipe Pileus Spores

(in cm) (in cm) (in cm)

100% WS 1.26×2.26 11.2×8.2 7.2×3.1

50% WS+ 50% HMPI 1.3×2.4 11.2×8.23 7.7×3.36

100% HMPI 1.4×2.46 11.0×8.1 6.76×2.76

50% CI + 50% WS 1.3×2.3 11.1×8.1 7.2×3.06

100% CI 1.1×2.26 11.1×8.0 6.86×2.93

WS = Wheat straw, HMPI = Hand made paper industry, CI = Cardboard

industries

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Baysal et al. (2003) who reported increase yield of P. ostreatus on

mixing paper waste with rice straw. Biological efficiency of P. florida

is not decreasing significantly over control in case of combination of

cardboard industrial waste with wheat straw.

When waste was used alone significant decrease in

biological efficiency of P. florida was observed. Spawn running

time of P. florida was found to be highly co-related with 50%

HMPI+50% WS, followed by 50%CI+50%WS, 100%HMPI and

then control (Table 3). This may be due to the higher CO2

concentration in the center of substrate bundles which might have

resulted from the growth of mycelium.

Due to recycling, fibres of cardboard industry pulp residues

are finer than that of paper industry, resulted in over compaction of

substrate bundles. This resulted in lower yield due to high time

requirement for complete fruiting body formation. This reasoning is

evidenced by the work of Zhang et al. (2002).

Morphological studies of Pleurotus: Fruiting bodies of P. florida

appeared in groups from a same point on each substrate, were

normal with white colour. Content of fruiting bodies were found to be

fleshy and odour fungoid. Oyster shape pileus was found to be

spatulate when young, convex (Fig. 1) then later becoming concave

on maturity, have smooth surface and white colour.

Lamellae were long-decurrent, crowd, white coloured and

having smooth edge when young, later become undulating. Stipe

was short and having solid context, longitudinally striate surface,

and white in colour. Presence of white coloured spores confirms by

spore print (Fig. 2).

Hence, presence of chemicals in the effluent did not found to be

inducing any morphological dissimilarity as no significant differences

were observed in measurement of mature fruiting bodies (Table 4).

Anatomical study of Pleurotus: Cross-section of Pleurotus

lamellae shows no abnormality (Fig. 3). Unstained preparation of

gills, stalk and pileus mycelium of P. florida reveals no absorption of

dyes on it. Spores were cylindrical, smooth and hyaline. Four

spored, slenderly clavate basidia were found to be attached to

irregular, hymenophoral trama and showed normal morphology.

Chemical toxicity test: P. florida cultivated on each of above

substrate, showed no blue spots on reacting with HCl on lignin

containing paper, revealing the presence of non-toxic compounds.

The present study deals with an approach that integrates

biotechnology with waste management to achieve zero waste and zero

discharges from handmade paper and cardboard industries. Cultivated

mushrooms were not only found to be normal but also non-toxic.

Acknowledgments

Authors are thankful to Department of Science and

Technology, Jaipur for providing financial assistance and Dr. R.C.

Upadhyay (Principal Scientist), National Research Centre for

Mushroom, Solan for providing the culture of P. florida. Thanks are

also due to Prof. Ishan Patro, Department of Zoology, Jiwaji University,

Gwalior for providing photographic facility.

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