Utilization of Waste Newspaper Using Oyster Mushroom Mycelium

Article pubs.acs.org/IECR

Utilization of Waste Newspaper Using Oyster Mushroom Mycelium Leonard Kopiński and Sylwia Kwiatkowska-Marks* Faculty of Technology and Chemical Engineering Science, University of Technology and Life Sciences, Bydgoszcz, Poland ABSTRACT: Research on the utilization of waste newspaper using a mycelium of the oyster mushroom Pleurotus ostreatus was conducted. The mycelium and fruiting bodies grown in growing substrates were cultured during the studies. The growing substrates were composed of waste newspaper or wheat straw, and mixtures thereof. Utilization of the components of the substrates was evaluated as the degree of utilization and performance of the fruiting bodies. The effectiveness of utilization depended on the amount of waste newspaper in the substrates. The maximum utilization was observed in the 3:1 mixture of waste newspaper and wheat straw. No fruiting bodies were formed in the substrate containing waste newspaper alone. The performance of the fruiting bodies was observed to increase as the concentration of waste newspaper in the substrates decreased. Heavy metals were present in the fruiting bodies grown in the substrates containing waste newspaper. Their concentrations were higher than acceptable by applicable standards, and the fruiting bodies were inedible. The substrates after cultivation were enriched in oyster protein.

1. INTRODUCTION Waste newspaper is printed paper which has lost its functional value because the information provided in it has become outdated. Waste paper is composed of organic matter (mainly lignocellulose) as well as printing inks, binding agents, and auxiliary materials (plasticizers, fillers, surfactants, preservatives). Printing inks are organic or inorganic pigments (with a content of heavy metals) which are dispersed in a binding agent which is in the form of a resin solution in mineral or vegetable oils.1−3 Although waste newspaper is not a hazardous waste, its specific composition may pose a degree of burden to the environment where it is stored or accumulated. Any chemicals or heavy metals contained in it as well as organic decomposition products may become eluted and penetrate to the soil and to surface waters, leading to their contamination. Owing to the phenomenon and applicable environmental laws, it is necessary to utilize this kind of waste with maximum efficiency. A considerable amount of waste newspaper is utilized by physical and chemical methods, using recycling or thermal treatment. Recycling is limited to decoloration, defibering, and processing the waste newspaper to obtain paper pulp for further processing to paper or cardboard. This helps reduce the consumption of wood and other raw materials in the pulp and paper industry, as well as energy required for their processing, to the advantage of the environment and the economics of paper production. Thermal treatment comprises combustion of waste newspaper or its processing to obtain activated carbons and building materials (such as ekofiber).4−6 At present, biotechnological methods based on the use of microorganisms are becoming more and more important in the utilization of waste newspaper. Less valuable material, which is not useful in recycling, is composted after being mixed with other lignocellulose waste or is subjected to biodegradation in anaerobic conditions to obtain methane.7−10 Cellulose which is present in the waste newspaper is hydrolyzed by means of enzymes. Any sugars present in the resulting hydrolyzate are © 2012 American Chemical Society

further fermented in anaerobic conditions to obtain ethanol.11,12 The lignocellulose substance contained in waste newspaper is biodegraded and bioconverted by means of various species of fungi.13 This results in the formation of a biomass which has a desirably different chemical composition, compared with the starting material. The resulting biomass has a content of mycoprotein and a much reduced amount of environmentally unfriendly substances. The enzymes which are synthesized by mushrooms (such as laccase) help decompose the pigments contained in printing inks as well as other not readily degradable substances, leading to a considerable decrease in their concentration and decoloration of waste paper. Moreover, its weight and volume are reduced, thus minimizing storage area. Specifically predisposed to decomposing the lignocellulose complex in all kinds of waste paper are higher fungi belonging to Basidiomycetes. Many of them are valued as edible mushrooms, such as oyster mushrooms of the genus Pleurotus. There are reports of attempts made to grow such mushrooms in substrates containing common waste paper among many lignocellulose materials.14−17 Such cultivation leads to the acquisition of a biomass composed of protein, enzymes, vitamins, and other organic compounds, and to the acquisition of the oyster mushroom fruiting bodies. The efficiency of its fruiting bodies was higher as the lower was the content of waste paper in the growing substrates. Common waste paper was not a good substrate for growing the oyster mushroom mycelium and cultivation of its fruiting bodies.14−17 Much more serious problems are encountered when an oyster mushroom growing substrate contains waste newspaper. Heavy metals and specific chemicals which are present in it may penetrate into the mycelium and the fruiting bodies, leading to Received: Revised: Accepted: Published: 4440

November 28, 2011 February 28, 2012 March 5, 2012 March 5, 2012 dx.doi.org/10.1021/ie202765b | Ind. Eng. Chem. Res. 2012, 51, 4440−4444

Industrial & Engineering Chemistry Research

Article

their accumulation above permissible levels according to applicable standards and laws. The resulting fruiting bodies may turn out to be inedible and may pose an environmental hazard. The research presented herein was intended to show the potential of the oyster mushroom mycelium in the utilization of waste newspaper by its biodegradation and bioconversion and to explain how high is the content of heavy metals in the fruiting bodies being developed.

The sterile growing substrates were inoculated with a liquid inoculum in the form of a suspension of a homogenized oyster mushroom mycelium. The inoculum was obtained by the submerged culture method and multiplication in the sterilized 4% aqueous suspension of dried breadcrumbs in shake-flasks. Erlenmeyer flasks (250 cm3) were filled with 100 cm3 each of the suspension and were shaken in a 358 S shaker with the speed of 120 rpm at 25 °C for 168 h (7 days). The resulting suspension of the mycelium beads (0.5−3.0 mm in diameter) was homogenized in the homogenizer 302 operating for 1 min at 10 000 rpm. The inoculum was introduced inside the growing substrates by injecting it with an automatic veterinary syringe. The syringe was equipped with a 35 cm long needle by means of which the substrate bags were pricked repeatedly to inject 1 cm3 doses of the inoculum each time. Each substrate bag was injected with 30 cm3 of the inoculum. The bags with the inoculated growing substrates were placed in a special incubation chamber made of organic glass to guarantee the required temperature and moisture of the air and light inside the chamber was maintained. The chamber was used for growing the oyster mushroom mycelium and cultivating its fruiting bodies according to the methodology used by the manufacturers of such mushrooms.22 Because of the commonly known22 decrease in performance of subsequent portions of the fruiting bodies cultivation, the growing and cultivation process was ended after harvesting the first portion of the fruiting bodies. The growing substrates which remained after the process were mixtures of the nondecomposed starting components of the growing substrates and hyphae. The amount of dry matter and total nitrogen (NK) were determined in each of the growing substrates.18,19 The content of dry matter (MO) and its concentration were determined in the harvested fresh fruiting bodies.18 The content of total nitrogen (NO) and heavy metals were determined in dry fruiting bodies.19,21 The effect of biodegradation and bioconversion of the components of the growing substrates was evaluated by the degree of utilization S, and performance of the fruiting bodies Y. Performance Y, denoted the relative proportion of dry matter of the fruiting bodies to that of the growing substrate and was defined as

2. THE MICROORGANISM USED AND THE METHODOLOGY The research on the microbiological utilization of waste newspaper was based on the use of the mycelium of the oyster mushroom (Pleurotus ostreatus) from the authors own collection. The research consisted in growing the mycelium in solid growing substrates and obtaining its fruiting bodies. Furthermore, the outcome of such growing processes was examined. The growing substrates consisted of waste newspaper (MG), which was the remainder of a number of issues of a popular Polish daily, or wheat straw (SP) collected from the fields near Bydgoszcz, or mixtures thereof. The straw was added to enrich the substrates in nutrients and improve their structure by enhancing oxygenation. The ratios of the components in the growing substrates are shown in Table 1. The qualitative and quantitative characteristics of the growing substrates are given in Table 2. Table 1. Growing Substrates substrate

composition of the substrate

1 2 3 4 5

MG MG + SP (7:1) MG + SP (3:1) MG + SP (1:1) SP

The concentration of dry matter, its organic content and total nitrogen (NP) and ash were found according to applicable standards.18−20 Its heavy metal concentrations were found by means of atomic absorption spectrophotometer (Buck Scientific 210 VGP) after mineralizing the substrate samples.21 The growing substrates were prepared by shredding the waste newspaper in a document shredder, cutting the straw to 1−3 cm long pieces, and mixing the material thoroughly in appropriate ratios (Table 1). The growing substrates were then packed in polypropylene bags (300 g dry MP each), wetted with distilled water to obtain desirable moisture, and pressed. The bags with the resulting substrates were then sealed and sterilized by the Koch method and cooled down to 25 °C.

Y=

MO MP

(1)

The metabolic activity of the mycelium led to decomposition of the lignocellulose substance and other components of the growing substrates. The process was accompanied, inter alia, by the emission of CO2 and H2O (as vapor) running off the culture medium. In consequence, the dry matter of the growing substrates was reduced and its nitrogen concentrations NK were increased pro rata of the present weight of the mycelium. The phenomenon was additionally intensified by the loss of

Table 2. Characteristics of Growing Substrates content in dry matter substrate (%)

(mg/kg)

substrate

dry matter (%)

NP

organic substance

ash

Zn

Cu

Ni

Cd

Co

Pb

1 2 3 4 5

38.0 31.55 33.67 33.85 28.06

0.21 0.27 0.34 0.45 0.70

90.66 91.43 92.2 93.73 96.80

9.34 8.57 7.8 6.27 3.20

98.0 88.8 79.5 61.0 24.0

37.0 33.0 29.0 21.1 5.1

35.0 31.5 28.0 21.0 7.0

9.0 7.9 6.7 4.5 trace

6.0 5.6 5.3 4.5 3.0

199.0 174.5 149.9 100.8 2.6

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dx.doi.org/10.1021/ie202765b | Ind. Eng. Chem. Res. 2012, 51, 4440−4444


Article

fruiting bodies at all. The low metabolic activity of the mycelium resulted probably from the too high levels of toxic and inhibiting substances, a great number of which were present in the substrate. Moreover, there were not many nutrients in the substrate and the structure of the moist waste paper appeared to have an added negative impact on the growth of the mycelium. The shredded fragments of the waste paper would easily stick together, considerably affecting the porosity of the substrate. In consequence, its oxygenation was reduced by higher air diffusion resistance. In the other substrates (substrates 2−5), higher contents of straw were accompanied by higher densities of compaction of the hyphae and the presence of fruiting bodies being developed. These substrates were covered by a dense mycelium entirely. After adding some shredded wheat straw, the growing substrates were enriched in nutrients and their structure became more porous. At the same time, the concentration of toxic and inhibiting substances was reduced by diluting them. This led to an improved metabolic activity, more intense utilization of the components of the growing substrates, and development of the fruiting bodies. The characteristics of all of the postculture substrates are shown in Table 3.

total nitrogen and the components of dry matter of the substrates, penetrating into the fruiting bodies being eveloped.

Table 3. Characteristics of Substrates after Cultivation

Figure 1. Fruiting bodies oyster mushrooms grown on substrate 3.

The degree of utilization S, denoted a relative reduction in the dry matter of the postculture substrates (MK), compared with that of the starting growing substrates (MP). It was defined as MK MP The value of S was calculated from the equation S=

S = A(1 − BY )

(2)

(3)

NP (4) NK while B characterized a relative change in the concentration of total nitrogen in the dry matter of the fruiting bodies and the growing substrates and is defined as A=

NO NP

dry matter (%)

concentration of nitrogen in the growing substrates (%)

1 2 3 4 5

42 45 43 42 39

35.25 31.13 32.42 31.08 29.04

0.29 0.40 0.67 0.78 1.10

Those substrates produced the flavor of vanilla and mushrooms, which is typical of the oyster mushroom mycelium. Their nitrogen content NK grew higher as their content of waste paper decreased. The duration of the culturing experiments were comparable in the range 39−45 days. In terms of morphology, the harvested fruiting bodies were only slightly different from those obtained by manufacturers.22 They were slightly funnel-shaped, 5−10 cm in diameter, grayyellow in color. Their properties are shown in Table 4. The dry matter MO, of the fruiting bodies tended to increase as the content of waste paper in the substrates from which they were harvested decreased. The level of nitrogen NO in the dry fruiting bodies was quite high, ranging from 3.60 to 4.36%. Heavy metals penetrated from the growing substrates to the fruiting bodies. The higher was the content of waste paper in the substrates, the higher were the concentrations of some of the heavy metals in the dry matter of the fruiting bodies. Their concentrations tended to exceed the permissible limits for the fruiting bodies to be edible.23,24 In the fruiting bodies harvested from substrate 5 alone the concentrations of heavy metals were lower than the permissible upper limit. These limits are 2 mg/ kg for Pb and 1 mg/kg for Cd.24 Also the performance of the fruiting bodies, Y, depended on the composition of the growing substrates. The less waste paper was contained in those substrates, the higher were the values of Y. In substrate 5 the performance Y, reached 0.064 which is the maximum value, comparable to that obtained by the manufacturers of the fruiting bodies.22 All values of perform-

where A is a coefficient denoting a relative change in the concentration of total nitrogen in the dry matter of the substrates in the process of growing the mycelium and the fruiting bodies. Coefficient A is expressed as

B=

substrates

duration of the culturing experiments (days)

(5)

3. RESULTS AND DISCUSSION The present research work has shown a considerable impact of the composition of the growing substrates (Table 1) on the growth of the mycelium and the formation of the oyster mushroom fruiting bodies. The mycelium, growing in the substrates, was in the form of fluffy white hyphae. In substrate 1, not all of the waste paper area was covered by the hyphae. On the contrary, some areas were entirely devoid of the mycelium. The mycelium in that substrate failed to produce any 4442



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Table 4. Characteristics of Harvested Fruit Bodies content in dry matter substrate (mg/kg) substrate

dry matter (%)

MO (g)

NO (%)

Zn

Cu

Ni

Cd

Co

Pb

1 2 3 4 5

30.6 20.7 32.25 21.15

9.9 12.8 16.8 19.2

3.6 3.59 3.89 4.36

88.9 70.5 45.6 25.2

91.8 68.5 31.8 14.2

1.7 trace trace trace



9.3 7.8 4.0 1.6

substrates 4 and 5 the values of S started to rise moderately, up to 0.298 and 0.382, respectively. The values of degree of utilization S tended to increase probably because more fruiting bodies developed. Performance Y of those substrates was 0.056 and 0.064, respectively. The substances which penetrated into the growing fruiting bodies consumed more nutrients and mycelium growth stimulants from the growing substrates. The hypothesis has been verified by the fact that heavy metals penetrated from the substrates to the fruiting bodies. In the fruiting bodies harvested from the substrates having a content of waste paper (substrates 2−4), the level of some of the heavy metals was higher than permissible in edible products. In particular, the level of lead was very high, several times as high as the permissible limit (2 mg Pb/kg24). In the fruiting bodies harvested from substrate 5 the permissible limits of the heavy metals determined were not exceeded. Postculture substrates, which were mixtures of a nondecomposed lignocellulose (waste paper and straw) and other components as well as mycelium hyphae were enriched in fungal biomass. At the same time, their weight and volume were considerably reduced, leading to higher levels of heavy metals and other harmful substances. The phenomenon limited the possibilities of utilization of the substrates. The postculture substrates with a content of waste newspaper may not be used as a high-protein additive to animal foods, as is frequently the case in the commercial growing of fruiting bodies.22

ance Y obtained in the growing substrates were shown in Figure 2.

Figure 2. Dependence of Y on the type substrate.

The degree of utilization S varied in a typical way in the respective growing substrates, as illustrated in Figure 3.

4. CONCLUSIONS The efficiency of utilization of the components of the growing substrates by the oyster mushroom was highly various. It was measured by means of the degree of utilization S and performance Y. Its performance depended on the proportion of waste newspaper to the growing substrates. In substrate 1, containing waste paper alone, the efficiency of utilization was the lowest and no fruiting bodies were formed by the mycelium. This may lead to the conclusion that waste newspaper was not a suitable kind of substrate for growing the oyster mushroom mycelium and fruiting bodies. The degree of utilization S decreased with decreasing amount of waste newspaper in substrate. The maximum efficiency of utilization was observed in substrate 3, which was composed with an addition of 25% straw. After harvesting the fruiting bodies from substrate 3, its dry weight MK was 3.61 times as low as the initial weight MP. However, further addition of straw to the growing substrates (substrates 4 and 5) tended to result in lower efficiency of utilization of their components, probably because more fruiting bodies developed. In the fruiting bodies harvested from substrate 2−4, the level of some of the heavy metals was higher than permissible in edible products.24 In the fruiting bodies from substrate 5

Figure 3. Dependence of S on the type substrate.

In substrate 1 the value of S was 0.724, which means that dry matter MK, was as much as 72.4% of its starting dry matter MP. In substrate 2 the value of S was 0.378. The substrate had a low (12.5%) content of straw, which enhanced considerably the metabolic activity of the mycelium. Consequently, the dry matter MK, of the postculture substrate was as low as 37.8% of its starting matter MP. The most considerable reduction in MK was observed in substrate 3, where S was the lowest, at 0.277. In 4443



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(10) Fox, M.; Noike, T. Wet oxidation pretreatment for the increase in anaerobic biodegradability of newspaper waste. Bioresour. Technol. 2004, 91, 273. (11) Xin, Y.; Geng, A.; Chen, M. L.; Gum, M. J. M. Enzymatic hydrolysis of sodium dodecyl sulfate (SDS)-pretreated newspaper for cellulosic ethanol production by Saccharomyces cerevisiae and Pichia stipitis. Appl. Biochem. Biotechnol. 2009, 158, 186. (12) Sun, Y.; Cheng, J. Hydrolysis of lignocellulosic materials for ethanol production: A review. Bioresour. Technol. 2002, 83, 1. (13) Sanchez, C. Lignocellulosic residues: Biodegradation and bioconversion by fungi. Biotechnol. Advances 2009, 27, 185. (14) Yildiz, S.; Yildiz, U. C.; Gezer, E. D.; Temiz, A. Some lignocellulosic wastes used as raw material in cultivation of the Pleurotus ostreatus culture mushroom. Process Biochem. 2002, 38, 301. (15) Baysal, E.; Peker, H.; Yalinkilic, M. K.; Temiz, A. Cultivation of oyster mushroom on waste paper with some added supplementary materials. Bioresour. Technol. 2003, 89, 95. (16) Mandeel, Q. A.; Al-Laith, A. A.; Mohamed, S. A. Cultivation of oyster mushroom (Pleurotus spp.) on various lignocellulosic wastes. World J. Microbiol. Biotechnol. 2005, 21, 601. (17) Baysel, E.; Peker, H. An alternate to waste paper recycling: Mushroom cultivation. Teknoloji 2001, 3−4, 9. (18) Polish Standard PN-75/C-04616/01/, Determination of dry weight, organic and mineral substances in sludge (19) Polish Standard PN-75/C-04576/17/, Determination of total nitrogen in sludge (20) Polish Standard PN-80/G-04512, Determination of ash content by the weight method (21) Łoginow, W., Cwojdziński, W., Andrzejewski, J. Agricultural Chemistry; WU ATR: Bydgoszcz, Poland, 1996. (22) Gapiński, M., Woźniak, W., Ziombra, M. Oyster Mushroom; PWRiL: Poznań, Poland, 1992. (23) Polish Standard PN-89A-78510, Mushroom preserves. Dried Mushrooms (24) Journal of Laws No. 37, Item 326, Regulation of the Minister of Health of 13 January 2003 in the matter of maximum permissible levels of chemical and biological contaminants in foods, food components, permissible additives, processing adjuvants or on food surfaces.

(containing wheat straw alone), the permissible limits of the heavy metals determined were not exceeded. The results indicate that waste newspaper ought not to be used as a component of substrates used for the commercial growing of the oyster mushroom fruiting bodies.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +48 52 3749005. Tel: +48 52 3749022. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS

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REFERENCES

A = coefficient denoting a relative change in the concentration of total nitrogen in the dry matter of the substrates in the process of growing the mycelium and the fruiting bodies B = coefficient denoting a relative change in the concentration of total nitrogen in the dry matter of the fruiting bodies and the growing substrates MG = waste newspaper MK = dry matter of the postculture substrates (g) MO = dry matter of the fruiting bodies (g) MP = dry matter of the starting growing substrates (g) NK = concentration of nitrogen in the growing substrates (%) NO = content of total nitrogen in dry fruiting bodies (%) NP = concentration of nitrogen in the dry matter of the substrates (%) S = degree of utilization SP = wheat straw Y = performance of the fruiting bodies (relative proportion of dry matter of the fruiting bodies to that of the growing substrate)

(1) Przybysz, K. Paper Technology, Part 2; WSiP: Warszawa, Poland, 1997. (2) Biedermann, M.; Grob, K. Is recycled newspaper suitable for food contact materials? Technical grade mineral oils from printing inks. Eur. Food Res. Technol. 2010, 230, 785. (3) Shin, H. J.; Kim, Ch. J.; Kim, S. B. Kinetic study of recycled newspaper liquefaction in polyol solvent. Biotechnol. Bioprocess Eng. 2009, 14, 349. (4) Katsumata, H.; Kaneco, S.; Suzuki, T.; Ohta, K. Effect of metal nitrates on the formation of PCDD/Fs during newspaper combustion. Bull. Environ. Contam. Toxical. 2004, 73, 479. (5) Okada, K.; Yamamoto, N.; Kameshima, Y.; Yasumori, A. Adsorption properties of activated carbon from waste newspaper prepared by chemical and physical activation. J. Colloid Interface Sci. 2003, 262, 194. (6) Grigoriou, A. H. Waste paper-wood composites bonded with isocyanate. Wood Sci. Technol. 2003, 37, 79. (7) Ball, A. S.; Shah, D.; Wheatley, C. F. Assessment of the potential of a novel newspaper/horse manure-based compost. Bioresour. Technol. 2000, 73, 163. (8) Alvarez, J. V. L; Larrucea, M. A.; Bermudez, P. A.; Chicote, B. L. Biodegradation of paper waste under controlled composting conditions. Waste Manage. 2009, 29, 1514. (9) Xiao, W.; Clarkson, W. W. Acid solubilization of lignin and bioconversion of treated newsprint to methane. Biodegradation 1997, 8, 61. 4444


Utilization of Waste Newspaper Using Oyster Mushroom Mycelium

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