Lasisi etWaste Management and Bioresource Technology 1 (2011) 1-11 1 11 al / Waste Management and Bioresource Technology 1 (2011) Available

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Waste Management and Bioresource Technology

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Microwave Sterilization of Mushroom Production media

Abdul, A., Rozainee, M., Mutahharah, M. Department of Chemical Engineering, Fakulti of Chemical and Natural Resources Engineering, Universiti Teknologi Malaysia. A R T I C L E I N F O A B S T R A C T
Conventional sterilization process of mushroom growing media such as sawdust employing steam house is time consuming (7-8 hours) and energy intensive. Hence this study is aim at determining the kinetic parameters for bacteria and yeasts destruction in sawdust block under microwave exposure. Operating parameters such as microwave power density, exposure time and mass of sawdust block were also investigated. Thermal death time (TDT) and Arrhenius model were utilized in determining the destruction kinetic parameters. The decimal reduction times (D-values) at temperatures of 103, 101 and 99.4C from TDT model were 6, 7.8 and 11.3 min for bacteria and 5.1, 8.2 and 11.5 min for yeasts respectively. The temperature sensitivity values (z-values) were 13.3 and 10.2C for the corresponding microorganisms. While from the Arrhenius model, activation energy for bacteria and yeasts were 202.7 and 263.5 kJ/mol respectively. The study shows that the level of microbial destruction in sawdust block was susceptible to microwave power density, exposure period and mass of the sawdust block itself. However, optimum operating parameters varies depending upon applied combination of microwave power density and exposure period, since high power density requires shorter exposure period and vice versa. It could therefore be inferred that for 1000 g sawdust block, moulds could be totally inhibited at microwave exposure time of 20, 40 and 60 min at power density of 764.3, 395.3 and 105.4 W/kg respectively and for 600 g, an exposure time of 15, 25 and 30 min at power density of 1274, 660 and 175.7 W/kg respectively. 2011 woaj Ltd. All rights reserved

Article history: Accepted: Available online: 10 August 2011

Keywords: Sterilization, mushroom, kinetic parameters, microwave, activation energy.

1.

Introduction

Mushroom has long been recognized as an edible fungus which possess medicinal value dating back to 400 B.C. Depending on its characteristics and nutritional value there are various types of mushroom such as white cap (Agaricus bisporus), oysters (Pleurotus spp.) and shiitakes (Lentinus edodes). Unlike Plants which rely on sunlight to provide energy for their growth, mushrooms depend on its growth medium for *
Corresponding author. email address: abdulad2@yahoo.com

2011 woaj Ltd. All rights reserved

foods due to the inexistence of chlorophyll in mushrooms which prevents it from producing its own foods. The growth medium is commonly refers to as Mushroom growing media or scientifically as substrates. These substrates are composed of various lignocellulosic wastes such as grain, rice straw, wheat straw, cottonseed hulls, soybean meal and sawdust that act as source of nutrient for mushroom growth. Although, some types of mushroom can grow on a wide range of media like oyster while some like white cap grow well on composted media. Since mushroom growing media is made up of organic materials, microorganisms inherently exists in it. Microorganisms been ubiquitous in nature they can evolve even in extreme environment and they could be
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both helpful and harmful. Their existence in growing media helps to degrade the organic growing media thus releasing nutrient for mushroom growth but if microorganisms exist in a large quantity, they may compete to survive and indirectly this condition may retard the growth of mushroom. Hence, the need to kill microorganism in mushroom growing media is vital before cultivation can be made in order to ensure high mushroom yield. Various mushrooms growing techniques involving similar steps are as illustrated in Figure 1 below: i. Preparation of mushroom spawn Spawn is a media containing active hyphae and grain is the most common spawn media used by farmers. The grains provide nutrient for mushroom spores to develop into thread-like filament called hyphae. After a period of time, the spawn will start to produce mushroom. ii. Preparation of mushroom growing media Various organic materials can be used as mushroom growing media. Other than organic residues, logs are also preferably used to grow Shiitake mushroom. iii. Sterilization of mushroom growing media As mentioned before, this step aims to get rid of harmful microorganisms in growing media and to ensure better mushroom yield. iv. Inoculation The matured spawn is inoculated (i.e. placing the spawn) on sterilized mushroom growing media so that mushroom will grow on it. v. Incubation The inoculated media is incubated in damp and dark condition. Maintaining adequate moisture content of growing media is vital for mushroom growth. vi. Fruiting Fruiting bodies of mushroom start to emerge after the spawn colonize the growing media. The period for first fruiting varies with the spore strain. For instance, oyster mushroom takes about 8 weeks for the first flush but shiitake takes only 6 weeks. Figure 1: Various techniques to growing mushrooms (Stamets, 2000) Although many studies concerning sterilization using microwave energy had been carried out in food processing as in sliced beef sterilization (Tang et al. 2008); inactivation of microorganism in raw poultry (Pucciarelli and Benassi, 2005); in medical field as in sterilization of polyethylene catherers and bone allograft (Uchiyama et al. 2005); destruction of microorganisms in medium such as healthcare waste (Tonuci et al. 2008), and building materials (Gorny et al. 2007). To date, there are limited studies of microwave sterilization in agriculture industry. Hence, this work explores the usage of microwave to agricultural sector focusing on the improvement and sterilization process of mushroom growing media and the mechanisms involved. 2.0 Review on Process Condition for Application of Kinetic Destruction Model

Thermal Death Time (TDT) and Arrhenius are modeled to describe a process occurring at constant temperature (isothermal condition). However, most of the thermal destruction of microorganisms processes occurs at non-isothermal condition as in microwave processing, autoclaving, water bath, etc. Literature on kinetic studies shows that the kinetic analysis of nonisothermal process can utilize the TDT and Arrhenius isothermal models. Earlier work by Fujikawa et al., (1992), use Arrhenius law to describe the kinetics of E.coli destruction by batch microwave irradiation at 100, 200 and 300 W, obtaining survival plot with three linear parts for each microwave power. Rate constant (k) was considered constant during the exposure period corresponding to
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each linear part. In determining (k) the following equations were used (Fujikawa et al., 1992):

process, by using different sizes of test tubes (i.e. to obtain different D-value) containing E.coli culture immersed in water bath circulator set at 53C carried out at different runs. Residence time was the dependent variable, and longer time was required for the treated liquid to reach 53C with increasing tube diameter. In all, Arrhenius law was adjusted to express mean value of the rate constant (k) over time and the defined equation was used to derive the effective temperature (Teff) formula as shown below (Farid and Alkhafaji, 2009).

Tonuci et al., (2008), in his study on microwave sterilization of healthcare waste also used Arrhenius model to describe the kinetic of disinfection process. He obtained microwave heating profile in the form of increasing temperature field; by defining k-value at constant power per mass of (60, 80 and 100 W/kg) unit instead of constant temperature. The Arrhenius preexponential factors were assumed as constant since the only reagent was microorganism and the activation energies were determined using the least square method. In the study, equations derived to calculate the rate constant allows the estimation of the required time for a desired level of inactivation at known temperature (i.e. temperature in this case was determined from timetemperature profile fitted with polynomial second degree function). In a study of the inactivation of microorganisms in apple juice during continuous flow microwave heating by Tajchakavit et al., (1998), the TDT model was applied in explaining the process mechanism, where the flow rate of the apple juice was varied to obtain a given exit temperature taken as (Tref). Then the corrected heating time was obtained by applying effective comeup time (CUT) and come-down time (CDT) added to the isothermal hold-time (heating time). A survival plot as a function of corrected heating time was found shorter than the uncorrected heating time. Koutchma et al., (2001), employed similar concept in their work of E.coli destruction in continuous flow microwave. Other than finding the effective time to obtain kinetic parameters for non-isothermal condition, Farid and Alkhafaji, (2009), used the concept of effective treatment temperature to minimize error in a non-isothermal

3.0 MATERIALS AND EQUIPMENT A. Materials/Procedure 3.1 Mushroom Growing Media Mushroom growing media was obtained from an organic mushroom farm in Sungai Tiram, Johor, and was made up of a mixture of rubber tree sawdust, rice bran and lime in ratio 100: 10: 1. Rice bran acts as nitrogen additive while lime functions to adjust the pH of the mixture. The mixture was adjusted to a moisture content of 60 % (i.e. optimum moisture for mushroom growth used by mushroom cultivator). The prepared mixture was filled into heat resistance polyethylene bag in form of a sawdust block representing the mushroom growing media for 1000 and 600 gram (Figure 2). To avoid fermentation reactions which may render the sawdust block unsuitable, direct sterilization after makeup is highly desirable Stamets, (2000).

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Figure 3: Radiation cycles of various power levels of SHARP Model: R-958A microwave matic diagram of the thermocouples arrangement in the microwave cavity is shown in Figure 5. To prevent electromagnetic effect of overheating, part of the thermocouple probe inside the chamber was covered by a inch OD copper tubing. The copper with low resistivity (1.7 cm) can tolerate the current flow without significant overheating and also by grounding the copper tubing at one end, the problem of induced voltage was minimized Ramaswamy et al,. (1991, 1998).

Figure 2: Comparison of (A) wet and dry and (B) 1000 and 600 g sawdust block

3.2

Microwave

Domestic microwave oven (SHARP Model R958A) with maximum power output of 900 W at 2450 MHz was used in this study. The irradiation cycle of various power levels of the microwave is shown in Figure 3. One complete irradiation phase for every power mode is 30 s and the total time for irradiation to occur is 30 s for high, 20 s for medium high, 15 s for medium, 10 s for medium low and 5 s for low mode. 3.3 Temperature Measuring

Experimental Set-up

Three shielded type-K thermocouples with a 1 mm diameter probe were employed to measure the temperature variation inside sawdust block. Each thermocouple was inserted into the oven through a 2 mm hole from the top, while the other ends of the thermocouples were connected to data logger (TC08, Pico Technology Limited, United Kingdom). The length of each thermocouple in the microwave cavity was varied to measure temperature at different depth of sawdust block. Sche4

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Figure 4: Schematic diagram of experimental set up

Figure 5: Thermocouples arrangement (A) 1000 g block (18 x 8 x 7 cm) (B) 600 g block (12 x 8 x 7 cm) (C 1 denotes Channel 1)

Microwave Power Calibration

In order to obtain the actual microwave power (Diprose, 2001), calibration was conducted for low, medium, medium high and high power mode in which seven hundred grams (700 g) of de-ionized water was placed inside the cavity in a central position. Then, the desired power level was set and the temperature of the water was recorded for 5 minutes. The real dissipated
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power was calculated by the principle of energy conservation, using equation (5).

4.1

Effect of Microwave Power Density

The calculated microwave power outputs for each power level are tabulated in Table 1. C Sterilization Procedures

The microwave oven was set to operate at low, medium, medium high and high power mode. For each microwave power, sawdust block was exposed to microwave at least 4 times intervals to determine the effect of exposure time to microbial content. Using the pour plate dilution method (Liao, 1993), at each time interval, 10 g sawdust was taken from the centre of sawdust block and kept in sealed sterile Stomacher bag. The samples were stored at 4C before been subjected to microbe test to avoid new microbial growth after microwave sterilization process as shown in Figure 6.

Figure 7 shows the heating profile of 1000 g sawdust block as a function of time for microwave power level of high, medium high, medium and low for 30 min exposure period. The corresponding power densities (power output per mass) are 764.3, 401.4, 395.3 and 105.4 W/kg. There was slight difference in comeup time (i.e. the time to reach constant temperature) with duration of 8, 9 and 10 minutes respectively for power densities of 764.3, 401.4 and 395.3 W/kg. More than 30 minutes heating time was required to reach the constant temperature for 105.4 W/kg power density.

Figure 7: Temperature measured at the centre of sawdust block (Moisture content: 60%, Mass: 1000 g) Figure 6: Sampling point for microbial testing (10g sawdust) 4.0 Results and Discussion The recorded heating trend could be explained by radiation cycles of different microwave power as shown in Figure 2. For high mode, microwave is continuously being irradiated in the cavity during 30s irradiation phase causing high power density and temperature increase. However, for medium high, medium and low modes, microwave is irradiated intermittently for
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Table 1: Moisture losses in 1000 g sawdust block upon 30 min microwave exposure at different power densities Microwave power density (W/kg) 764.3 401.4 395.3 105.4 20, 15 and 5 seconds respectively resulting in descending power density order with temperature increase. Apart from different heating trend, the moisture content of sawdust block also varies with respect to microwave power density. The moisture losses measured after 30 min microwave exposure at different power densities are shown in Table 1. It could therefore be inferred from the Table that, increasing microwave power density results in greater moisture loss in sawdust block equivalent to its weight loss. It was also observed that microwave sterilization of sawdust block with moisture content of 60%, mass of 1000g, at power density of 764.3 W/kg for 30 minutes greatly reduce the weight of sawdust block to almost half of its initial mass. A microbe test on 10 g sawdust sample shows that it have adequate moisture content for mushroom growth because ideal moisture content is about 60%. Hence, microwave sterilization should be conducted taking into consideration the effect of moisture loss anticipated to influencing mushroom growth. Moisture loss (%) 44 25 16 7

Figure 8: Heating profile of 1000 and 600 g sawdust block at high microwave power

The heating profile of low microwave power for sawdust block having a mass of 1000 and 600 g is shown in Figure 9. Heating of 600 g sawdust block resulted in considerable reduction in come-up time to 30 min (i.e. half of come-up time for 1000 g), suggesting that low microwave power will be more suitable to sterilizing smaller load.

4.2 Effect of Mass of Sawdust Block to Microwave Heating The mass of the sterilized glassware essentially determines the length of the come-up time (Jeng et al., 1987). Similar trends were observed as in Figure 8, where lower mass of sawdust block exhibits shorter come-up time and higher heating rate compared to higher mass exposed to similar microwave power. This could be due to the fact that lower mass of sawdust block results in higher microwave power density (i.e. power per mass).

Figure 9: Heating profile of 1000 and 600 g sawdust block at low microwave power destruction kinetic parameters Enumeration of bacteria and yeast survival in microwave sterilized sawdust at different operating condition is shown in Table 4.3 and Table 4.4 respectively. Microbial population in untreated sawdust was 5 x 107 CFU/g bacteria and 1 x 108 CFU/g yeast.

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Table 2: Experimental results of bacteria survival in microwave sterilized sawdust at different microwave power and exposure time High (764.3 W/kg) (T = 103C) Time (min) 6 10 14 18 CFU/g 8.2 x 106 1.27 x 106 1.7 x 105 1.3 x 105 Medium high (401.4 W/kg) (T = 101C) Time (min) 10 20 25 30 CFU/g 4.8 x 106 1.54 x 105 1.45 x 105 9.8 x 104 Medium (395.3 W/kg) (T = 99.4C) Time (min) 5 10 20 25 CFU/g 5 x 105 9.4 x 106 3.3 x 105 2.3 x 104

Table 3: Experimental results of yeast survival in microwave sterilized sawdust at different microwave power and exposure time

High (764.3 W/kg) (T = 103C) Time (min) 6 10 14 18 CFU/g 3 x 106 9.4 x 104 1.4 x 104 1.2 x 104

Medium high (401.4 W/kg) (T = 101C) Time (min) 10 20 25 30 CFU/g 5.1 x 105 9.4 x 104 1.73 x 104 1.03 x 104

Medium (395.3 W/kg) (T = 99.4C) Time (min) 5 10 20 25 CFU/g 3.8 x 107 6.6 x 105 2.5 x 104 2.3 x 104

Linear regression on log CFU/g (for bacteria and yeast) versus exposure time gives D-value (i.e. negative reciprocal of slope) as shown in Table 4 and Table 5 respectively. Although, the correlation coefficient, R2 is near unity representing good fit for the original data, the D-values obtained are not in accordance with the theory of kinetic parameter, which shows D-value increasing with decreasing temperature. Table 4: Regression equation and D-values for bacteria D-value (min) 6.4 11.8 6.2 Liner regression equation y = -0.156x + 7.718 y = -0.085 + 7.314 y = -0.162 + 8.576

Microwave power High, 764.3 W/ kg Medium high, 401.4 W/kg Medium (395.3 W/kg)

T C 103 101 99.4

R2 0.93 0.85 0.99

Table 5: Regression equation and D-values for yeast D-value (min) 5 11.2 6.4 Linear regression equation y = -0.200x + 7.326 y = -0.089 + 6.623 y = -0.157 + 7.898

Microwave power High, 764.3 W/ kg Medium high, 401.4 W/kg Medium (395.3 W/kg)

T C 103 101 99.4

R2 0.86 0.97 0.89

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One of the probable reasons why appropriate D-values cannot be obtained from the linear regression survival curve might be due to the composition of the various microorganisms in sawdust block. Microorganisms in sawdust block are Gram-negative bacteria, fungal, coli-

401.4 and 395.3 W/kg and rate constant, k calculated are shown in Table 6 and Table 7. The temperature sensitivity curves for bacteria and yeast based on TDT and Arrhenius models are illus-

Table 6: Calculated D and k values for bacteria at time intervals D-value (min) 6 7.8 11.3

Microwave power High, 764.3 W/kg Medium high, 401.4 W/kg Medium, 395.3 W/kg

T C 103 101 99.4

k 0.38 0.30 0.20

Table 7: Calculated D and k values for yeast at time intervals

Microwave power High, 764.3 W/kg Medium high, 401.4 W/kg Medium, 395.3 W/kg forms (i.e. E.coli, Klebsiella spp) and streptococci based on identification test and literature review. Hence, it could be stressed that, the reduction of microorganism represents the destruction of mixed microorganism population in sawdust block. It was anticipated that during microwave sterilization of sawdust block, the mixed composition of microorganism was inactivated at different temperature where the least heat resistance microorganisms were killed at lower temperature and vice versa. Karel and Lund, (2003), suggested that those spores or cells with the greatest resistance are believed to contain proteins with the greatest stability.

T C 103 101 99.4

D-value (min) 5.1 8.2 11.5

K 0.45 0.28 0.20

4.3

Destruction Kinetic Parameters

A close look at the experimental results in Table 3 shows that at exposure time of 10 minutes, bacteria survival count was the lowest at power density of 764.3 W/kg, followed by 401.4 and 395.3 W/kg. Similar trend was observed in time intervals of 18 minutes for 764.3 W/kg and 20 minutes for 401.4 and 395.3 W/kg. Similar trend was observed for yeast (Table 3) in the first 10 minutes exposure time. Further yeast reduction was observed in 18 minutes exposure for 764.3 W/kg and 25 minutes for the other two power densities. Based on the observation, D-values re-calculated at 8, 10 and 20 min time interval with respect to power density of 764.3,

Figure 10: Temperature sensitivity curve of bacteria and yeast based on TDT model
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pH and buffer component, ionic environment, water activity, composition of the medium) (Stumbo, 1973). According to Stratford, (2006), yeasts are relatively sensitive to heat. This is true since z-value for yeasts is 10.2C, slightly lower than bacteria (z = 13.3C). Conclusions Comparisons of the sterilization period show that variations in microwave powers results in different destruction trend because the application of high power require much shorter exposure time than low power, to achieve the same microbial destruction. This is due to difference in the length of come up time exhibited by both microwave powers. Similarly, it could be seen that, reducing the mass of sawdust block could reduce the come up time, although this is beneficial to low power that shows considerable decline in come up time with higher microbial destruction when using 600 g sawdust block in place of 1000 g. The only observed similarity was in the application of microwave powers; higher microbial destruction occurred at early sterilization period but became slower towards the end of the sterilization period. Generally, moisture content seems to have no effect on absorption of microwave energy, because, even though high moisture loss was observed after 30 min high microwave exposure, the temperature of sawdust block kept increasing during the exposure period. Microwave power density did influence the destruction of microorganism, because higher microwave power density results in higher destruction of microorganism. A long exposure time result in lowest survival but of note is that the maximum microbial destruction occurred at shorter exposure time. Smaller mass of sawdust block did result in higher microbial destruction which might be due to the high absorption of microwave energy as indicated by high microwave power density. Finally, the optimum operating parameters could not be ascertained because microwave power density and exposure time were interrelated, since, using high power density required shorter exposure time and vice versa. References 1. The results of temperature sensitivity curves show that yeasts were easier to inactivate due to lower z -value of 10.2C when compared to bacteria (13.3C), suggesting that bacteria were more heat resistance. This might be due to such factors such as temperature, ionic environment, organic compound, lipids, age or phase for growth and condition present during heat treatment (i.e. Diprose, M.F ( 2001).Some Considerations when using a Microwave Oven as a Laboratory Research Tool.Plant and Soil.229:271-280. Farid, M. and Alkhafaji, S (2009).Determination of an Effective Treatment Temperature of Chemical and Biological Reactions.Food and Bioprocess Technology. Fujikawa, H., Ushioda, H., and Kudo, Y
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Figure 11: Temperature sensitivity of bacteria and yeast based on Arrhenius model

4.4 Destruction Kinetic Parameters of Bacteria and Yeasts The TDT kinetic parameters for bacteria (initial population of 5 x 107 CFU/g) determined in this study were D103, D101, D99.4 of 6, 7.7, 11.3 min and z-value of 13.3C. This illustrates that, for D103 at high microwave power (764.3 W/kg) 1 log reduction of bacteria was achieved in 6 min exposure time and so on, while, z -value indicates increase in temperature of 13.3C which reduce D-value by 1 log cycle. Tonuci et al., (2008), reported that 1 log destruction of E.coli inoculated in healthcare waste was achieved in 13.3, 20.5 and 30.5 min exposure time for microwave power of 100, 80 and 60 W/kg at initial population of 5 x 105 CFU/g, suggesting that faster bacteria destruction occurred in sawdust block due to application of higher microwave power density. D-value assumed a consistent reduction over time (Spinks et al., 2006).

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