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Suppression of malodor from garbage during storage periods by yeast producing fragrances
Process Biochemistry 41 (2006) 1932–1939 www.elsevier.com/locate/procbio
Suppression of malodor from garbage during storage periods by yeast producing fragrances Kiyohiko Nakasaki *, Sung-Hwan Kwon, Hiroyuki Tanaka, Kumi Oyamada Department of Materials Science and Chemical Engineering, Shizuoka University, 3-5-1 Johoku, Hamamatsu 432-8561, Japan Received 30 October 2005; received in revised form 25 March 2006; accepted 4 April 2006
Abstract A strain of yeast (YT1) that is effective in suppressing malodor emission during the storage of garbage by producing fragrances and masking the malodors was isolated from garbage and identified as Candida krusei var. transitoria. The strain YT1 grew and formed pseudomycelium on the surface of the garbage and produced various fragrances, including alcohols and esters such as isoamylalcohol and ethyl ester. The quantities of the fragrances produced by strain YT1 were dependent on the various cultivation conditions, the temperature, pH, and kinds of substrates. The optimum conditions for fragrance production by strain YT1 were found to be a cultivation temperature of 40 8C, pH 4, and a glucose-rich substrate condition. # 2006 Elsevier Ltd. All rights reserved. Keywords: Suppression of malodor; Garbage storage; Microorganisms producing fragrances; Candida krusei var. transitoria
1. Introduction The treatment of municipal solid wastes (MSW) that are generated by humans poses a very serious problem in many countries. Even though MSW have mainly been treated by landfill or incineration, organic fractions like food wastes have been screened and then treated by biological methods such as composting [1–5] or biogas producing processes [6–8]. An energy recovery process to produce RDF (refuse derived fuel) from these organic fractions has also been considered [9,10]. However, whatever treatment methods were applied to treat these organic fractions, they were easily decomposed by microorganisms, and nuisance malodors were then being produced during the storage period. Generally, many microorganisms putrefy organic materials and emit malodors, but it is known that there are also some microorganisms that produce various fragrances. Willetts and Ugalde [11] reported that whey was metabolized into alcohols and esters by some genera of yeasts in the middle of the production process of single cell proteins (SCP). The production of fragrances is very important in the food and perfume industries [12–16]. In fact, some fragrances are produced by specified microorganisms, and almost all of these
* Corresponding author. Tel.: +81 53 478 1172; fax: +81 53 478 1172. E-mail address: [email protected] (K. Nakasaki). 1359-5113/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2006.04.003
microorganisms belong to different kinds of yeasts [17–21]. Murray et al. [22] reported that Candida utilis could produce selectively significant levels of ethyl acetate or acetaldehyde. Other methods using some enzymes instead of microorganisms have also been applied, and the production of fatty acids esters by these methods has recently attracted interest [12,23,24]. As mentioned above, many researchers have reported on the production of fragrances by some microorganisms or enzymes. However, there are few studies on the production of fragrances by some microorganisms to suppress malodors during the storage period of garbage, although it is empirically known that the fragrances could be emitted by chance when the garbage was laid aside. The objectives of this study were to identify the microorganisms related to the fragrances production from garbage, to analyze the composition of the fragrances, and to investigate the various environmental conditions influencing on the production of these fragrances. 2. Materials and methods 2.1. Raw material As a raw material, the remains of meals (called garbage throughout the rest of the paper) generated from the lunch-supply service industry were used. The garbage was collected in sufficient quantities to use in a series of experiments. It was first ground with a cooking grinder and mixed thoroughly, and then each
K. Nakasaki et al. / Process Biochemistry 41 (2006) 1932–1939 quantity for one experiment were packed separately into plastic bags and frozen once at 20 8C. The frozen raw materials were then thawed out and used when each experiment was begun. The carbon and nitrogen content of the raw material, as determined by element analysis, was 44.9%, and 3.8% on a dry weight basis, respectively. The carbon to nitrogen ratio was 11.8, somewhat smaller than that of the municipal solid waste excluding paper (C/N = 16–20) [25]. The pH and moisture content of the raw material were 5.6 and 77.0%, respectively.
2.2. Storage experiment of the garbage and isolation of microorganisms from the garbage Fig. 1 shows a schematic diagram of the storage bucket for the garbage. The volume of this cylindrical bucket was 15 L, and a perforated plate was set on the bottom of it. The inner cover of the bucket was equipped to drain the drip and delete the air space from the garbage. In this way, the anaerobic condition in the garbage was maintained because it was pressed by the inner cover from the top. The upper cover was made airtight to prohibit infiltration of the surrounding air. The drip was drawn out, if required, through a cock valve installed at the bottom of the bucket. The storage experiment was conducted for 10 d. The state of the garbage and malodor emission was checked once daily when the upper cover had been opened. The garbage was then well mixed and the sample withdrawn from the bucket in order to measure the pH, moisture content, and cell density of the microorganisms. Three grams of sample were suspended with 27 mL of sterilized water and then measured for the pH value using a pH probe. The moisture content was calculated from the weight loss after the sample was dried at 105 8C for 3 d. Both the population densities of the aerobic and anaerobic microorganisms were determined on an agar plate by dilution plating. The medium used was a Potato-dextrose (PD, Nissui) agar medium (potato-dextrose agar, 39 g; distilled water, 1 L), and the pH of the medium was adjusted at 4.0 by the addition of HCl solution. A GaspackTM anaerobic system (Becton-Dickinson Co. Ltd., USA) was used in the case of the anaerobic incubation. This apparatus can maintain an anaerobic condition with the addition of deoxygenate into the jar in which the agar plate was set. The incubation temperature was 30 8C, and the incubation period was 7 d. The population densities were expressed as a colony-forming unit per dry solid weight of the garbage (CFU/g-ds).
2.3. Experimental vessel and the test for fragrances production A kind of bacterium and two kinds of yeasts were isolated from the garbage, and were named for LT1, YT1, and YT2 in this paper, respectively. These microorganisms were purified and then inoculated into the sterilized garbage to demonstrate the contribution of each strain on the fragrances production from the garbage. The isolated microorganism was precultured in PD medium at 30 8C for 24 h. After the preculture, the microorganism was centrifuged at
Fig. 1. A schematic diagram of the garbage storage bucket.
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10,000 rpm for 10 min, washed twice by sterilized physiological saline, and then used as an inoculum. The tests for the fragrances production from the garbage were conducted using a modified sterilizing filtration apparatus (Sterifil1 Aseptic System, Millipore Co. Ltd., USA) to avoid microbial contaminations (see Fig. 2). The capacity of the apparatus was about 300 mL, and it was cylindrical and composed of two parts. A plastic screen was set on the bottom of the upper part to remove the drip from the garbage. Approximately 150 wet-g of garbage sterilized with g-irradiation at 10 kGy/ h for 3 h was packed into the upper part of the vessel. The combinations of all, two, and each single strain of the pure cultured microorganisms, LT1, YT1, and YT2 were inoculated into the sterilized garbage. For all seven combinations of microbial inoculation, the experiments were conducted under both aerobic and anaerobic conditions, as shown in Table 1. After the inoculation of the pure cultured strain(s), the surface of the garbage pile was compacted with a sterilized spatula in order to delete the air space in the pile. Four holes (4 mm in diameter) installed on the lid of the vessel were sealed with rubber caps. In the aerobic experiment, air was introduced into the vessel through one hole and then effused from another hole. The membrane filter of 0.45 mm-pore size (Cellulose nitrate A045047A, Toyo Filter Co. Ltd., Japan) was set on both holes in order to prevent microbial contamination. For the anaerobic experiment, however, the inner air of the vessel was exchanged by N2 gas once daily when the lid of the vessel was taken off for the mixing of the garbage, and then every hole on the vessel was sealed by rubber caps to prevent infiltration of the outer air. The vessel was placed into an incubator controlled at a temperature of 30 8C. The lid of the vessel was taken off every 24 h, and the materials in the vessel were manually mixed well in order to ensure uniformity. At adequate time intervals (once per 2 or 3 days), the samples were withdrawn and the population densities of microorganism in the samples were measured, as were their pH values. The operation was terminated when 9 d had elapsed from the start of the experimental run.
2.4. Qualitative analysis for the fragrances GC/MS analysis for the emitted gas in Run A-11, in which strain YT1 alone was inoculated under aerobic conditions (see Table 1), was conducted to specify the fragrances from the garbage after 1 d incubation and to compare these fragrances with the odorant emitted from the raw garbage itself. Emitted gas was sampled by suctioning the inner air with an air pump into a glass column packed with the absorbents (Tenax GC, Shimadzu Co. Ltd., Japan). The absorbed gas was introduced into the GC/MS (HP-5890, Hewlett-Packard Co. Ltd., USA; M2000A, Hitachi Co. Ltd., Japan) by heating at 250 8C. The capillary column contained BC-WAX (50 m 0.25 mm i.d., GL Science Inc., Japan), and the temperature was incrementally changed from 70 to 220 8C
Fig. 2. A schematic diagram of the modified sterilizing filtration apparatus.
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Table 1 Inoculation conditions of three strains into the garbage sterilized with girradiation and the fragrances production from the garbage Run no.
Inoculum
Condition
Result
A-1 A-2 A-3 A-4 A-5 A-6 A-7 A-8 A-9 A-10 A-11 A-12 A-13 A-14
LT1, YT1, YT2 LT1, YT1, YT2 LT1, YT1 LT1, YT1 LT1, YT2 LT1, YT2 YT1, YT2 YT1, YT2 LT1 LT1 YT1 YT1 YT2 YT2
Aerobic Anaerobic Aerobic Anaerobic Aerobic Anaerobic Aerobic Anaerobic Aerobic Anaerobic Aerobic Anaerobic Aerobic Anaerobic
: produced; : not produced. Fig. 3. A schematic diagram of Jar-fermentor used in this study. at a rate of 4 8C/min. The temperature of the injector was set at 250 8C. Helium (1.5 mL/min) was used as a carrier gas.
2.5. Effects of the cultivation conditions on the fragrances production by strain YT1 The pure cultured strain YT1 was investigated for its fragrances production capability under various cultivation conditions of pH, temperature, and kinds of medium. Details on the experiment conditions are summarized in Table 2. The pH was controlled at 2, 3, 4, and 6 in the PD medium under incubation temperature at 30 8C. In addition, the incubation temperature was changed at 20, 25, 30 and 40 8C with the PD medium under pH 4.0 in order to investigate the effect of cultivation temperature. Finally, the effect of substrates was examined under three different kinds of media, PD, Trypticase-soy, and ISP No. 2 medium, respectively, under pH 4.0 and a cultivation temperature of 30 8C. Strain YT1 was precultured in the PD medium using a shaker (TA-12R, Takasaki Scientific Instruments Co. Ltd., Japan) under 180 rpm and an incubation temperature of 30 8C. The cultivation tests for strain YT1 were conducted using a Jar-fermentor (Fig. 3). One-thousand five-hundred milliliters of the liquid medium was put into the Jar-fermentor and incubated under mixing at 200 rpm and with air supplied at 1 L/min from the bottom of the Jar-fermentor. The temperature of the medium was maintained by a temperature controller. The pH of the medium was continuously measured by a pH sensor set at the Jar-fermentor and then adjusted at the constant value throughout the cultivation by automatically dropping 1N H2SO4 or 5N NaOH solution according to the signal from a pH controller. The inoculation size of strain YT1 at the start of the experiment
was adjusted at around OD570 = 0.05. The incubation period was 20 h, and the OD570 value of the liquid medium was measured every 2 h. The emitted gas was collected every 4 h with a Tedlar bagTM from the exhaust gas outlet of the Jarfermentor and then analyzed for fragrances by a GC-FID (G-3000, Hitachi Co. Ltd., Japan). A capillary column (BC-WAX, 30 m 0.53 mm i.d., GL Science Inc., Japan) was used, and the oven temperature was first maintained at 40 8C for 4 min then increased at 170 8C at a rate of 5 8C/12 min. The temperature of the injector and detector were also set at 250 8C. Nitrogen (8 mL/min) was used as a carrier gas. 1 mL of the collected gas was directly introduced into the GC using a syringe.
3. Results and discussion 3.1. Changes in physicochemical and microbial properties during the storage experiment of the garbage The time courses of the pH and moisture content of the garbage during the storage period are shown in Fig. 4. The pH value of the garbage was decreased until the 4th day from the start of the storage experiment, then leveled off at around 3.9 thereafter. It is deduced that this pH decrease may be due to the
Table 2 Cultivation conditions of pure cultured strain YT1 under various cultivation conditions of pH, temperature, and kinds of medium for the fragrances production Run no.
Medium
pH ( )
Temperature (8C)
B-1 B-2 B-3 B-4 B-5 B-6 B-7 B-8 B-9
PD a PD a PD a PD a PD a PD a PD a Tripticase-soy ISP No. 2
2 3 4 6 4 4 4 4 4
30 30 30 30 20 25 40 30 30
a
Potato-dextrose liquid medium.
Fig. 4. Time courses of pH and moisture content of garbage during the storage period.
K. Nakasaki et al. / Process Biochemistry 41 (2006) 1932–1939
production of organic acids in the garbage [2,5,26]. The moisture content of the garbage was kept in the range of 74% and 77%, and no significant change was observed throughout the experiment. The fragrances were not detected from the garbage when the lid of the vessel was taken off on the 2nd day, but were detected after the 4th day of the experiment. Since the malodor comprised of organic acids was detectable after the mixing of the garbage, even after the 4th day, it was thought that the fragrances were produced only near the surface of the garbage, and that the fragrances masking the malodor associated with the anaerobic degradation of the garbage occurred deep inside the vessel. Fig. 5 shows a comparison of changes in the population densities of the aerobic and anaerobic microorganisms in the garbage with time. In the case of the aerobic microorganisms, two distinctive microbial groups of bacteria and yeasts were detected, whereas in the case of anaerobic microorganisms, the yeasts could not be distinguished from the bacteria on the agar plate, since the colonies of both groups of microorganisms were quite small. Therefore, the total number of colonies on the agar plate was compared for both the aerobic and anaerobic microorganisms. Both the aerobic and anaerobic microorganisms grew rapidly, increasing two orders of magnitude by the 4th day, then finally leveling off around 108 CFU/g-ds. In addition, no significant difference was observed between the aerobic and anaerobic microorganisms. As mentioned above, bacteria and yeasts were observed in the aerobic microorganisms. Among these, it was confirmed from the colony appearance on the agar plate that three strains of microorganisms, a kind of bacterium, LT1, and two kinds of yeasts, YT1, and YT2, predominated (Fig. 6). These microorganisms showed good growth on the PD agar medium adjusted at pH 7 with lime, and the strain LT1 creates a halo related to the acid production around the colony. The other two strains were yeasts with clear differences in the colony appearance and microscopic observation. In addition, these characteristic colony appearances made it possible to distinguish and separately count each microorganism on the agar plate. The time courses of the cell densities of strains LT1, YT1, and YT2, determined by the dilution plating under an aerobic incubation, i.e., the composition of aerobic microorganisms as
Fig. 5. Population densities of aerobic and anaerobic microorganisms in the garbage withdrawn from the garbage storage bucket during the storage period.
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Fig. 6. A photograph of the colony appearances of microorganisms in the garbage withdrawn from the garbage storage bucket that were cultivated aerobically on the potato-dextrose agar medium adjusted at pH 7 with the addition of lime.
already seen in Fig. 5, are shown in Fig. 7. The strain LT1 grew rapidly with the progression of time, and the cell density was up to 108 CFU/g-ds by the 4th day. The cell density of strain YT1 increased at around 107.5 CFU/g-ds by the 6th day, and the values held up. Strain YT2 was attained at 107.5 CFU/g-ds by the 4th day, but had decreased gradually by one order of magnitude by the 8th day. And it was observed that strain YT1 formed pseudomycelium on the surface of the garbage when the fragrances were produced from the garbage. 3.2. Microorganisms responsible for the fragrances production The fragrances production was observed in the experiments inoculated with strain YT1 alone, and the combination with YT1 and LT1 as well as YT2, and also all three strains, irrespective of aerobic or anaerobic conditions (Table 1). In all of the experiments which produced the fragrances, strain YT1 was involved. Therefore, strain YT1 can be considered the main contributor to the fragrances production from garbage. In addition, it was ascertained that the fragrances were produced
Fig. 7. Time courses of cell densities of the strains, LT1, YT1 and YT2 in the garbage determined by dilution plating under an aerobic condition during the storage period.
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whether oxygen was present or not. The morphological and physiological characteristics of strain YT1 are summarized in Table 3. Based on those characteristics, strain YT1 was identified as Candida krusei var. transitoria, by Deutche Sammlung von Mikroorganismen und Zellkuturen (DSMZ) GmbH, Germany. It was known that some kinds of yeast strains including the genus Candida could produce flavors via bioconversion called specific conversions of appropriate precursor-compounds, and denovo process, which means by fermentation staring from simple nutrients such as sugars and amino acids [17,19,21]. It was however, characteristic that the strain YT1 isolated in this study could produce fragrances from garbage rather than the selected substrates such as sugars and amino acids. The time courses of the cell densities for strain YT1 during Run A-11 are shown in Fig. 8. The strain YT1 increased immediately after the inoculation on the garbage. The cell density was about 109.5 CFU/g-ds by the 5th day, and was maintained thereafter. Although the cell density of strain YT1 was not measured on the 1st day when the fragrances began to be produced, it could be assumed that YT1 had grown at around 107 CFU/g-ds from the curve of the YT1 cell density. As mentioned above, the fragrances were detected at the 4th day in the experiment using the unsterilized garbage, when strain YT1 grew at around 106.5 CFU/g-ds level (see Fig. 7). It was thus considered that the ranges of the cell density for strain YT1 to initiate the fragrances production were similar for both sterilized and unsterilized garbage. These results seem to indicate that the threshold cell density of strain YT1 for the fragrances production is at around 107 CFU/g-ds. Table 3 Morphological and physiological characteristics of the strain YT1 Morphology Colony on potato-dextrose agar butyrous, smooth, creme colored, fringed with mycelium, blastospores ellipsoidal; pseudomycelium present; no true mycelium; no sexual reproduction detected; dry pellicle in liquid culture Condition
Sources
Growth
Sources
Fig. 8. Time course of cell density of the strain YT1 during Run A-11, where the pure cultured YT1 was inoculated into the garbage sterilized with girradiation and cultivated at 30 8C for 9 d under an aerobic condition.
3.3. Analytical results of the fragrance components Fig. 9 shows the comparisons of the GC/MS analytical results of the emitted gas from the raw garbage and the 1st day garbage of Run A-11. By comparing the two chromatograms, it was confirmed that the concentrations of some chemical components, the esters ethyl acetate, prophyl acetate, isobutyl acetate, and isoamyl acetate and the alcohols isopentanol, isobutanol, and isoamylalcohol, were apparently different each other and were higher in the 1st day garbage than in the raw garbage. The fragrances could be smelled in the gas collected from the 1st day garbage. Therefore, it was considered that the fragrances were composed of these chemicals. It was demonstrated that these chemicals were produced by yeast strains [20,21]. In addition, Buzzini et al. [20] reported that isoamylalcohol and isoamyl acetate were main compounds of VOC produced and accounted for the banana smell, which was detected during the manipulation of some yeast culture. In this study, the smell of the fragrances was similar to that of isoamlyalcohol. This may be because the peak of isoamylalcohol was the largest among all fragrance components.
Growth
Utilization of carbon and nitrogen sources Anaerobic Glucose + Aerobic
Additional test Growth at 40 8C
Glucose Galactose Sorbose Rhamnose Dulcit Inositol Mannitol Sorbitol Glycerol Erythritol D-Arabinose L-Arabinose Ribose D-Xylose L-Xylose Adonitol Nitrate
+
+
+
a-Methylglycoside Salicin Cellobiose Maltose Lactose Melibiose Sucrose Trehalose Inulin Melezitose Raffinose Starch Xylitol Gluconate 2-Keto-gluconate 5-Keto-gluconate Acetate production
Fig. 9. Gas chromatograms of the emitted gas from the raw garbage and the 1st day garbage of the storage experiment of the garbage in Run A-11, where the pure cultured YT1 was inoculated into the garbage sterilized with g-irradiation and cultivated at 30 8C under an aerobic condition.
K. Nakasaki et al. / Process Biochemistry 41 (2006) 1932–1939
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3.4. Effect of cultivation conditions for strain YT1 on fragrance production The effects of cultivation conditions such as pH, temperature, and kinds of medium for the fragrance production by strain YT1 were investigated using the Jar-fermentor (see Table 2). The time courses of the OD570 for Runs B-1–B-4 are compared in Fig. 10. In Runs B-3 and B-4, the values of OD570 began to increase at almost the same time from the start of the cultivation experiment. The values then became nearly constant after 10 h. Even though the growth rate of strain YT1 in Run B-2 was a little late, the OD570 for Run B-2 reached similar values for both Runs B-3 and B-4 after about 18 h. However, the OD570 in Run B-1, in which the pH was controlled at 2, increased very slowly, and finally reached the lowest value. These results suggest that the low pH was not suitable for the growth of strain YT1. The maximum specific growth rates of strain YT1 for Runs B-1–B-4 were 0.148, 0.464, 0.592, and 0.567 h 1, respectively. Fig. 11 shows the time courses of the relative concentrations of the fragrance components emitted during Runs B-1–B-4. Four kinds of fragrance components: ethyl acetate, ethanol, isobutanol, and isoamylalcohol were analyzed in these experiments, and the relative concentrations of each fragrance component were calculated with dividing by those emitted in Run B-3. The relative concentrations of every component were the highest in Run B-3 among all experimental runs. In Runs B2–B-4, all of the fragrance components began to be detected after 12 h and then their concentrations increased gradually, whereas these fragrances were not detected in Run B-1 throughout the cultivation period. Fig. 12 summarizes the pH dependence of the fragrances production for Runs B-1–B-4. The relative concentrations of each fragrance component at 20 h of cultivation time are shown here. The value for every fragrance component was the largest in the cultivation condition of pH 4, and the values decreased distinctly in the conditions of pH 6 or 2. Therefore, it was ascertained that the quantities of the fragrances produced by strain YT1 depended on the pH conditions, and that pH 4 was the optimum for the production of the fragrances. The effects of the cultivation temperature and kinds of medium used, together with the results of the pH dependence of
Fig. 10. Time courses of OD570 during the cultivation experiments of strain YT1 in Runs B-1–B-4, where the pure cultured YT1 was inoculated into the PD medium under various cultivation pH 2, 3, 4, and 6, respectively.
Fig. 11. Time courses of relative concentrations of the fragrance components emitted during the cultivation of strain YT1 in Runs B1–B4, where the pure cultured YT1 was inoculated into the PD medium under various cultivation pH 2, 3, 4, and 6, respectively. The concentrations of each fragrance component emitted in Run B-3 were set up as 1.
strain YT1, are summarized in Table 4. The concentrations of each fragrance component emitted at the final stage of Run B-7 was made up as 100, and Table 4 shows the relative values of the fragrance concentrations emitted in each condition when 20 h have elapsed from the start of cultivation, i.e., the final stage. By comparing Runs B-3 and B-5 through B-7, the growth rate of YT1 was the maximum at 30 8C, the second maximum at 40 8C, and the third at 25 8C, though the final cell densities (OD570) at 25 8C were similar to those observed for both the 30
Fig. 12. A pH dependence of the fragrances production for Runs B-1–B-4, where the pure cultured YT1 was inoculated into the PD medium under various cultivation pH 2, 3, 4, and 6, respectively. The concentrations of each fragrance component emitted in Run B-3 were set up as 1 (EA: ethylene acetate; IB: isobutanol; ET: ethanol; IAA: isoamylalcohol.).
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Table 4 Cell growth and fragrances production by pure cultured strain YT1 according to the cultivation conditions Run no.
B-1 B-2 B-3 B-4 B-5 B-6 B-7 B-8 B-9 a b
mmaxa (h 1)
0.148 0.464 0.592 0.567 0.229 0.440 0.468 0.483 0.430
ODmaxb
0.269 2.531 2.533 2.571 0.968 2.450 2.515 2.157 2.408
Concentration (arbitrarily unit) Ethyl acetate
Ethanol
Isobutanol
Isoamylalcohol
0.0 0.0 37.8 5.7 55.4 5.4 19.3 4.7 0.0 0.0 0.2 0.1 100 12.5 0.1 0.2 0.1 0.0
0.3 0.0 47.3 3.5 46.6 2.8 28.1 1.9 0.1 0.2 0.3 0.3 100 17.7 0.5 0.3 11.7 2.6
0.0 0.0 27.6 5.5 52.7 5.2 27.4 2.9 0.0 0.2 2.0 3.3 100 9.6 6.6 6.2 19.4 2.9
0.0 0.0 35.8 9.1 72.8 7.6 43.9 7.7 0.0 0.0 6.0 1.8 100 14.6 26.7 3.6 49.4 7.3
Maximum specific growth rate. Optical density at 20 h.
and 40 8C experiments. The final OD570 value at 20 8C, however, was considerably lower than those of any other temperatures. Although not shown here in detail, the fragrance components were all detected after 12 h and the concentrations of the fragrance components were increased with the progress of cultivation time in Runs B-3 and B-7, in which the temperature was kept over 30 8C. The relative values of the fragrances concentrations at 40 8C were higher than those at 30 8C, as shown in Table 4. In contrast, the fragrances were not detected at all in Run B-5, and the fragrances were emitted at quite a low level in Run B-6, as shown in the same table. It is interesting that the fragrances concentrations were the highest in the cultivation temperature of 40 8C, apparently higher than those at 30 8C, although the final cell density for both temperatures was similar, and, moreover, the specific maximum growth rate was somewhat higher in the 30 8C experiment than in the 40 8C one. This may be because higher temperature makes the vapor pressure of the fragrance components higher. Finally, the effect of the kinds of substrates on the growth of strain YT1 and the production of the fragrances was investigated in Runs B-3, B-8, and B-9. In Run B-3, which used PD medium, ethyl acetate started to be emitted after 12 h, and then increased with the cultivation time, as seen in Fig. 11. However, Run B-8 used Trypticase-soy medium and Run B-9 used ISP No. 2 medium, and ethyl acetate was not detected in either run throughout the cultivation period (see Table 4). A higher amount of the other fragrance components, ethanol, isobutanol, and isoamylalcohol, were produced in the PD medium, ISP No. 2 medium, and Trypticase-soy medium, in that order. By considering the difference in the compositions of these media, it may be suggested that the most suitable substrate for the fragrances production by strain YT1 would be glucose, since the concentration of glucose contained in these media corresponds to the same order for the concentrations of fragrances produced, i.e., PD > ISP No. 2 > Trypticase-soy medium. These results coincided well with the studies on that these strains of yeast could effectively ferment and assimilate the glucose as a carbon source [27,28].
4. Conclusions Strain YT1, a type of yeast which we isolated, was effective in suppressing malodors emitted during the storage periods of garbage by producing fragrance components and masking the nuisance odors. The fragrances produced from the garbage by stain YT1 were identified as different kinds of alcohols and esters such as isoamylalcohol, ethyl ester, and so on. It was ascertained that the fragrances production was dependent on the cultivation conditions. The optimum temperature and pH were 40 8C and 4, respectively. Moreover, the fragrances production were strongly dependent on the kinds of substrates, and it was ascertained that strain YT1 prefers the Potato-dextrose medium. This result seems to indicate that the strain YT1 produces more fragrances from glucose than from other substrates. The concentration of glucose in the garbage may be low at first, but it would become high level resulting from the activity of co-existing microorganisms in the garbage such as lactic acid bacteria [26,29] which will provide the glucose derived from sugars or carbohydrates. It is well known that garbage in households or various treatment facilities usually contains high concentration of sugars and carbohydrates, and that many microorganisms possess hydrolysis enzymes of sugars and carbohydrates to convert the glucose. Therefore, it can be considered that C. krusei var. transitoria, YT1 will be effective in preventing the emission of malodors during the storage periods of garbage in households or various treatment facilities. References [1] Chynoweth DP, Owens J, O’Keefe D, Earle JFK, Bosch G, Legrand R. Sequential batch anaerobic composting of the organic fraction of municipal solid waste. Water Sci Technol 1992;25:327–39. [2] Nakasaki K, Yaguchi H, Sasaki Y, Kubota H. Effects of pH control on composting of garbage. Waste Manage Res 1993;1:117–25. [3] Kayhanian M, Hardy S. The impact of four design parameters on the performance of a high-solids anaerobic digestion of municipal solid waste for fuel gas production. Environ Technol 1994;15:557–67. [4] Hwang EJ, Shin HS, Tay JH. Continuous feed, on-site composting of kitchen garbage. Waste Manage Res 2002;20:119–26. [5] Kwon SH, Lee DH. Evaluation of Korean food waste composting with fed-batch operations II: using properties of exhaust gas condensate. Process Biochem 2004;39:1047–55.
K. Nakasaki et al. / Process Biochemistry 41 (2006) 1932–1939 [6] Kim HW, Han SK, Shin HS. The optimisation of food waste addition as a co-substrate in anaerobic digestion of sewage sludge. Waste Manage Res 2003;21:515–26. [7] Tang Y, Shigematsu T, Ikbal, Morimura S, Kida K. The effects of microaeration on the phylogenetic diversity of microorganisms in a thermophilic anaerobic municipal solid-waste digester. Water Res 2004;38:2537– 50. [8] Han SK, Kim SH, Shin HS. UASB treatment of wastewater with VFA and alcohol generated during hydrogen fermentation of food waste. Process Biochem 2005;40:2897–905. [9] Diggelman C, Ham RK. Household food waste to wastewater or to solid waste? That is the question. Waste Manage Res 2003;21: 501–14. [10] Nammari DR, Hogland W, Moutavtchi V, Marques M, Nimmermark S. Physical and chemical processes in baled waste fuel, with emphasis on gaseous emissions. Waste Manage Res 2003;21:309–17. [11] Willetts A, Ugalde U. The production of single-cell protein from whey. Biotechnol Lett 1987;9:795–800. [12] Langrand G, Triantaphylides C, Baratti J. Lipase catalyzed formation of flavor esters. Biotechnol Lett 1988;10:549–54. [13] Gubicza L, Kabiri-Badr A, Keoves E, Belafi-Bako K. Large-scale enzymatic production of natural flavour esters in organic solvent with continuous water removal. J Biotechnol 2000;84:193–6. [14] Vandamme EJ, Soetaert W. Bioflavours and fragrances via fermentation and biocatalysis. J Chem Technol Biotechnol 2002;77:1323–32. [15] Liu SQ, Holland R, Crow VL. Esters and their biosynthesis in fermented dairy products: a review. Int Dairy J 2004;14:923–45. [16] Franssen MCR, Alessandrini L, Terraneo G. Biocatalytic production of flavors and fragrances. Pure Appl Chem 2005;77:273–9. [17] Armstrong DW. Selective production of ethyl acetate by Candida utilis. ACS Symp Ser 1986;317:254–65. [18] Willetts A. Ester formation from ethanol by Candida pseudotropicalis. Anton van Leeuw 1989;56:175–80.
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[19] Janssens L, De Pooter HL, Schamp NM, Vandamme EJ. Production of flavors by microorganisms. Process Biochem 1992;27:195–215. [20] Buzzini P, Martini A, Pagnoni UM, Davoli P. Production of flavoured volatile organic compounds (VOCs) by Candida oleophila GK10. Optimisation using factorial design and response surface analysis. Enzyme Microb Technol 2003;33:668–75. [21] Buzzini P, Martini A, Cappelli F, Pagnoni UM, Davoli P. A study on volatile organic compounds (VOCs) produced by tropical ascomycetous yeasts. Anton van Leeuw 2003;84:301–11. [22] Murray WD, Duff SJB, Lanthier PH, Armstrong DW, Welsh FW, Williams RE. Development of biotechnological processes for the production of natural flavors and fragrances. Dev Food Sci 1988;17:1–18. [23] Yoshimoto T, Takahashi K, Nishimura H, Ajima A, Tamaura Y, Inada Y. Modified lipase having high stability and various enzymic activities in benzene, and its re-use by recovering from benzene solution. Biotechnol Lett 1984;6:337–40. [24] Deleuze H, Langrand G, Millet H, Baratti J, Buono G, Triantaphylides C. Lipase-catalyzed reactions in organic media: competition and applications. Biochim Biophys Acta 1987;911:117–20. [25] Golueke CG. Biological reclamation of solid wastes Emmaus, PA, USA: Rodale Press Inc.; 1977. [26] Wang Q, Narita J, Xie W, Ohsumi Y, Kusano K, Shirai Y, et al. Effects of anaerobic/aerobic incubation and storage temperature on preservation and deodorization of kitchen garbage. Bioresource Technol 2002;84:213–20. [27] Liu Y, Liu D, Su Q, Liu J, Xie D. Effects of phosphorus and nitrogen limitation on continuous production of glycerol in a multistage cascade bioreactor by Candida krusei. Bicochem Eng J 2003;15:101–6. [28] Liu H, Liu D, Zhoung J. Interesting physiological response of the osmophilic yeast Candida krusei to heat shock. Enzyme Microb Technol 2005;36:409–16. [29] Ohkouchi, Y., and Inoue, Y., Direct production of L(+)-lactic acid for starch and food acid wastes using Lactobacillus manihotivorans LMG18011, Bioresource Technol, in press.
Suppression of malodor from garbage during storage periods by yeast producing fragrances Kiyohiko Nakasaki *, Sung-Hwan Kwon, Hiroyuki Tanaka, Kumi Oyamada Department of Materials Science and Chemical Engineering, Shizuoka University, 3-5-1 Johoku, Hamamatsu 432-8561, Japan Received 30 October 2005; received in revised form 25 March 2006; accepted 4 April 2006
Abstract A strain of yeast (YT1) that is effective in suppressing malodor emission during the storage of garbage by producing fragrances and masking the malodors was isolated from garbage and identified as Candida krusei var. transitoria. The strain YT1 grew and formed pseudomycelium on the surface of the garbage and produced various fragrances, including alcohols and esters such as isoamylalcohol and ethyl ester. The quantities of the fragrances produced by strain YT1 were dependent on the various cultivation conditions, the temperature, pH, and kinds of substrates. The optimum conditions for fragrance production by strain YT1 were found to be a cultivation temperature of 40 8C, pH 4, and a glucose-rich substrate condition. # 2006 Elsevier Ltd. All rights reserved. Keywords: Suppression of malodor; Garbage storage; Microorganisms producing fragrances; Candida krusei var. transitoria
1. Introduction The treatment of municipal solid wastes (MSW) that are generated by humans poses a very serious problem in many countries. Even though MSW have mainly been treated by landfill or incineration, organic fractions like food wastes have been screened and then treated by biological methods such as composting [1–5] or biogas producing processes [6–8]. An energy recovery process to produce RDF (refuse derived fuel) from these organic fractions has also been considered [9,10]. However, whatever treatment methods were applied to treat these organic fractions, they were easily decomposed by microorganisms, and nuisance malodors were then being produced during the storage period. Generally, many microorganisms putrefy organic materials and emit malodors, but it is known that there are also some microorganisms that produce various fragrances. Willetts and Ugalde [11] reported that whey was metabolized into alcohols and esters by some genera of yeasts in the middle of the production process of single cell proteins (SCP). The production of fragrances is very important in the food and perfume industries [12–16]. In fact, some fragrances are produced by specified microorganisms, and almost all of these
* Corresponding author. Tel.: +81 53 478 1172; fax: +81 53 478 1172. E-mail address: [email protected] (K. Nakasaki). 1359-5113/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2006.04.003
microorganisms belong to different kinds of yeasts [17–21]. Murray et al. [22] reported that Candida utilis could produce selectively significant levels of ethyl acetate or acetaldehyde. Other methods using some enzymes instead of microorganisms have also been applied, and the production of fatty acids esters by these methods has recently attracted interest [12,23,24]. As mentioned above, many researchers have reported on the production of fragrances by some microorganisms or enzymes. However, there are few studies on the production of fragrances by some microorganisms to suppress malodors during the storage period of garbage, although it is empirically known that the fragrances could be emitted by chance when the garbage was laid aside. The objectives of this study were to identify the microorganisms related to the fragrances production from garbage, to analyze the composition of the fragrances, and to investigate the various environmental conditions influencing on the production of these fragrances. 2. Materials and methods 2.1. Raw material As a raw material, the remains of meals (called garbage throughout the rest of the paper) generated from the lunch-supply service industry were used. The garbage was collected in sufficient quantities to use in a series of experiments. It was first ground with a cooking grinder and mixed thoroughly, and then each
K. Nakasaki et al. / Process Biochemistry 41 (2006) 1932–1939 quantity for one experiment were packed separately into plastic bags and frozen once at 20 8C. The frozen raw materials were then thawed out and used when each experiment was begun. The carbon and nitrogen content of the raw material, as determined by element analysis, was 44.9%, and 3.8% on a dry weight basis, respectively. The carbon to nitrogen ratio was 11.8, somewhat smaller than that of the municipal solid waste excluding paper (C/N = 16–20) [25]. The pH and moisture content of the raw material were 5.6 and 77.0%, respectively.
2.2. Storage experiment of the garbage and isolation of microorganisms from the garbage Fig. 1 shows a schematic diagram of the storage bucket for the garbage. The volume of this cylindrical bucket was 15 L, and a perforated plate was set on the bottom of it. The inner cover of the bucket was equipped to drain the drip and delete the air space from the garbage. In this way, the anaerobic condition in the garbage was maintained because it was pressed by the inner cover from the top. The upper cover was made airtight to prohibit infiltration of the surrounding air. The drip was drawn out, if required, through a cock valve installed at the bottom of the bucket. The storage experiment was conducted for 10 d. The state of the garbage and malodor emission was checked once daily when the upper cover had been opened. The garbage was then well mixed and the sample withdrawn from the bucket in order to measure the pH, moisture content, and cell density of the microorganisms. Three grams of sample were suspended with 27 mL of sterilized water and then measured for the pH value using a pH probe. The moisture content was calculated from the weight loss after the sample was dried at 105 8C for 3 d. Both the population densities of the aerobic and anaerobic microorganisms were determined on an agar plate by dilution plating. The medium used was a Potato-dextrose (PD, Nissui) agar medium (potato-dextrose agar, 39 g; distilled water, 1 L), and the pH of the medium was adjusted at 4.0 by the addition of HCl solution. A GaspackTM anaerobic system (Becton-Dickinson Co. Ltd., USA) was used in the case of the anaerobic incubation. This apparatus can maintain an anaerobic condition with the addition of deoxygenate into the jar in which the agar plate was set. The incubation temperature was 30 8C, and the incubation period was 7 d. The population densities were expressed as a colony-forming unit per dry solid weight of the garbage (CFU/g-ds).
2.3. Experimental vessel and the test for fragrances production A kind of bacterium and two kinds of yeasts were isolated from the garbage, and were named for LT1, YT1, and YT2 in this paper, respectively. These microorganisms were purified and then inoculated into the sterilized garbage to demonstrate the contribution of each strain on the fragrances production from the garbage. The isolated microorganism was precultured in PD medium at 30 8C for 24 h. After the preculture, the microorganism was centrifuged at
Fig. 1. A schematic diagram of the garbage storage bucket.
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10,000 rpm for 10 min, washed twice by sterilized physiological saline, and then used as an inoculum. The tests for the fragrances production from the garbage were conducted using a modified sterilizing filtration apparatus (Sterifil1 Aseptic System, Millipore Co. Ltd., USA) to avoid microbial contaminations (see Fig. 2). The capacity of the apparatus was about 300 mL, and it was cylindrical and composed of two parts. A plastic screen was set on the bottom of the upper part to remove the drip from the garbage. Approximately 150 wet-g of garbage sterilized with g-irradiation at 10 kGy/ h for 3 h was packed into the upper part of the vessel. The combinations of all, two, and each single strain of the pure cultured microorganisms, LT1, YT1, and YT2 were inoculated into the sterilized garbage. For all seven combinations of microbial inoculation, the experiments were conducted under both aerobic and anaerobic conditions, as shown in Table 1. After the inoculation of the pure cultured strain(s), the surface of the garbage pile was compacted with a sterilized spatula in order to delete the air space in the pile. Four holes (4 mm in diameter) installed on the lid of the vessel were sealed with rubber caps. In the aerobic experiment, air was introduced into the vessel through one hole and then effused from another hole. The membrane filter of 0.45 mm-pore size (Cellulose nitrate A045047A, Toyo Filter Co. Ltd., Japan) was set on both holes in order to prevent microbial contamination. For the anaerobic experiment, however, the inner air of the vessel was exchanged by N2 gas once daily when the lid of the vessel was taken off for the mixing of the garbage, and then every hole on the vessel was sealed by rubber caps to prevent infiltration of the outer air. The vessel was placed into an incubator controlled at a temperature of 30 8C. The lid of the vessel was taken off every 24 h, and the materials in the vessel were manually mixed well in order to ensure uniformity. At adequate time intervals (once per 2 or 3 days), the samples were withdrawn and the population densities of microorganism in the samples were measured, as were their pH values. The operation was terminated when 9 d had elapsed from the start of the experimental run.
2.4. Qualitative analysis for the fragrances GC/MS analysis for the emitted gas in Run A-11, in which strain YT1 alone was inoculated under aerobic conditions (see Table 1), was conducted to specify the fragrances from the garbage after 1 d incubation and to compare these fragrances with the odorant emitted from the raw garbage itself. Emitted gas was sampled by suctioning the inner air with an air pump into a glass column packed with the absorbents (Tenax GC, Shimadzu Co. Ltd., Japan). The absorbed gas was introduced into the GC/MS (HP-5890, Hewlett-Packard Co. Ltd., USA; M2000A, Hitachi Co. Ltd., Japan) by heating at 250 8C. The capillary column contained BC-WAX (50 m 0.25 mm i.d., GL Science Inc., Japan), and the temperature was incrementally changed from 70 to 220 8C
Fig. 2. A schematic diagram of the modified sterilizing filtration apparatus.
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Table 1 Inoculation conditions of three strains into the garbage sterilized with girradiation and the fragrances production from the garbage Run no.
Inoculum
Condition
Result
A-1 A-2 A-3 A-4 A-5 A-6 A-7 A-8 A-9 A-10 A-11 A-12 A-13 A-14
LT1, YT1, YT2 LT1, YT1, YT2 LT1, YT1 LT1, YT1 LT1, YT2 LT1, YT2 YT1, YT2 YT1, YT2 LT1 LT1 YT1 YT1 YT2 YT2
Aerobic Anaerobic Aerobic Anaerobic Aerobic Anaerobic Aerobic Anaerobic Aerobic Anaerobic Aerobic Anaerobic Aerobic Anaerobic
: produced; : not produced. Fig. 3. A schematic diagram of Jar-fermentor used in this study. at a rate of 4 8C/min. The temperature of the injector was set at 250 8C. Helium (1.5 mL/min) was used as a carrier gas.
2.5. Effects of the cultivation conditions on the fragrances production by strain YT1 The pure cultured strain YT1 was investigated for its fragrances production capability under various cultivation conditions of pH, temperature, and kinds of medium. Details on the experiment conditions are summarized in Table 2. The pH was controlled at 2, 3, 4, and 6 in the PD medium under incubation temperature at 30 8C. In addition, the incubation temperature was changed at 20, 25, 30 and 40 8C with the PD medium under pH 4.0 in order to investigate the effect of cultivation temperature. Finally, the effect of substrates was examined under three different kinds of media, PD, Trypticase-soy, and ISP No. 2 medium, respectively, under pH 4.0 and a cultivation temperature of 30 8C. Strain YT1 was precultured in the PD medium using a shaker (TA-12R, Takasaki Scientific Instruments Co. Ltd., Japan) under 180 rpm and an incubation temperature of 30 8C. The cultivation tests for strain YT1 were conducted using a Jar-fermentor (Fig. 3). One-thousand five-hundred milliliters of the liquid medium was put into the Jar-fermentor and incubated under mixing at 200 rpm and with air supplied at 1 L/min from the bottom of the Jar-fermentor. The temperature of the medium was maintained by a temperature controller. The pH of the medium was continuously measured by a pH sensor set at the Jar-fermentor and then adjusted at the constant value throughout the cultivation by automatically dropping 1N H2SO4 or 5N NaOH solution according to the signal from a pH controller. The inoculation size of strain YT1 at the start of the experiment
was adjusted at around OD570 = 0.05. The incubation period was 20 h, and the OD570 value of the liquid medium was measured every 2 h. The emitted gas was collected every 4 h with a Tedlar bagTM from the exhaust gas outlet of the Jarfermentor and then analyzed for fragrances by a GC-FID (G-3000, Hitachi Co. Ltd., Japan). A capillary column (BC-WAX, 30 m 0.53 mm i.d., GL Science Inc., Japan) was used, and the oven temperature was first maintained at 40 8C for 4 min then increased at 170 8C at a rate of 5 8C/12 min. The temperature of the injector and detector were also set at 250 8C. Nitrogen (8 mL/min) was used as a carrier gas. 1 mL of the collected gas was directly introduced into the GC using a syringe.
3. Results and discussion 3.1. Changes in physicochemical and microbial properties during the storage experiment of the garbage The time courses of the pH and moisture content of the garbage during the storage period are shown in Fig. 4. The pH value of the garbage was decreased until the 4th day from the start of the storage experiment, then leveled off at around 3.9 thereafter. It is deduced that this pH decrease may be due to the
Table 2 Cultivation conditions of pure cultured strain YT1 under various cultivation conditions of pH, temperature, and kinds of medium for the fragrances production Run no.
Medium
pH ( )
Temperature (8C)
B-1 B-2 B-3 B-4 B-5 B-6 B-7 B-8 B-9
PD a PD a PD a PD a PD a PD a PD a Tripticase-soy ISP No. 2
2 3 4 6 4 4 4 4 4
30 30 30 30 20 25 40 30 30
a
Potato-dextrose liquid medium.
Fig. 4. Time courses of pH and moisture content of garbage during the storage period.
K. Nakasaki et al. / Process Biochemistry 41 (2006) 1932–1939
production of organic acids in the garbage [2,5,26]. The moisture content of the garbage was kept in the range of 74% and 77%, and no significant change was observed throughout the experiment. The fragrances were not detected from the garbage when the lid of the vessel was taken off on the 2nd day, but were detected after the 4th day of the experiment. Since the malodor comprised of organic acids was detectable after the mixing of the garbage, even after the 4th day, it was thought that the fragrances were produced only near the surface of the garbage, and that the fragrances masking the malodor associated with the anaerobic degradation of the garbage occurred deep inside the vessel. Fig. 5 shows a comparison of changes in the population densities of the aerobic and anaerobic microorganisms in the garbage with time. In the case of the aerobic microorganisms, two distinctive microbial groups of bacteria and yeasts were detected, whereas in the case of anaerobic microorganisms, the yeasts could not be distinguished from the bacteria on the agar plate, since the colonies of both groups of microorganisms were quite small. Therefore, the total number of colonies on the agar plate was compared for both the aerobic and anaerobic microorganisms. Both the aerobic and anaerobic microorganisms grew rapidly, increasing two orders of magnitude by the 4th day, then finally leveling off around 108 CFU/g-ds. In addition, no significant difference was observed between the aerobic and anaerobic microorganisms. As mentioned above, bacteria and yeasts were observed in the aerobic microorganisms. Among these, it was confirmed from the colony appearance on the agar plate that three strains of microorganisms, a kind of bacterium, LT1, and two kinds of yeasts, YT1, and YT2, predominated (Fig. 6). These microorganisms showed good growth on the PD agar medium adjusted at pH 7 with lime, and the strain LT1 creates a halo related to the acid production around the colony. The other two strains were yeasts with clear differences in the colony appearance and microscopic observation. In addition, these characteristic colony appearances made it possible to distinguish and separately count each microorganism on the agar plate. The time courses of the cell densities of strains LT1, YT1, and YT2, determined by the dilution plating under an aerobic incubation, i.e., the composition of aerobic microorganisms as
Fig. 5. Population densities of aerobic and anaerobic microorganisms in the garbage withdrawn from the garbage storage bucket during the storage period.
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Fig. 6. A photograph of the colony appearances of microorganisms in the garbage withdrawn from the garbage storage bucket that were cultivated aerobically on the potato-dextrose agar medium adjusted at pH 7 with the addition of lime.
already seen in Fig. 5, are shown in Fig. 7. The strain LT1 grew rapidly with the progression of time, and the cell density was up to 108 CFU/g-ds by the 4th day. The cell density of strain YT1 increased at around 107.5 CFU/g-ds by the 6th day, and the values held up. Strain YT2 was attained at 107.5 CFU/g-ds by the 4th day, but had decreased gradually by one order of magnitude by the 8th day. And it was observed that strain YT1 formed pseudomycelium on the surface of the garbage when the fragrances were produced from the garbage. 3.2. Microorganisms responsible for the fragrances production The fragrances production was observed in the experiments inoculated with strain YT1 alone, and the combination with YT1 and LT1 as well as YT2, and also all three strains, irrespective of aerobic or anaerobic conditions (Table 1). In all of the experiments which produced the fragrances, strain YT1 was involved. Therefore, strain YT1 can be considered the main contributor to the fragrances production from garbage. In addition, it was ascertained that the fragrances were produced
Fig. 7. Time courses of cell densities of the strains, LT1, YT1 and YT2 in the garbage determined by dilution plating under an aerobic condition during the storage period.
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whether oxygen was present or not. The morphological and physiological characteristics of strain YT1 are summarized in Table 3. Based on those characteristics, strain YT1 was identified as Candida krusei var. transitoria, by Deutche Sammlung von Mikroorganismen und Zellkuturen (DSMZ) GmbH, Germany. It was known that some kinds of yeast strains including the genus Candida could produce flavors via bioconversion called specific conversions of appropriate precursor-compounds, and denovo process, which means by fermentation staring from simple nutrients such as sugars and amino acids [17,19,21]. It was however, characteristic that the strain YT1 isolated in this study could produce fragrances from garbage rather than the selected substrates such as sugars and amino acids. The time courses of the cell densities for strain YT1 during Run A-11 are shown in Fig. 8. The strain YT1 increased immediately after the inoculation on the garbage. The cell density was about 109.5 CFU/g-ds by the 5th day, and was maintained thereafter. Although the cell density of strain YT1 was not measured on the 1st day when the fragrances began to be produced, it could be assumed that YT1 had grown at around 107 CFU/g-ds from the curve of the YT1 cell density. As mentioned above, the fragrances were detected at the 4th day in the experiment using the unsterilized garbage, when strain YT1 grew at around 106.5 CFU/g-ds level (see Fig. 7). It was thus considered that the ranges of the cell density for strain YT1 to initiate the fragrances production were similar for both sterilized and unsterilized garbage. These results seem to indicate that the threshold cell density of strain YT1 for the fragrances production is at around 107 CFU/g-ds. Table 3 Morphological and physiological characteristics of the strain YT1 Morphology Colony on potato-dextrose agar butyrous, smooth, creme colored, fringed with mycelium, blastospores ellipsoidal; pseudomycelium present; no true mycelium; no sexual reproduction detected; dry pellicle in liquid culture Condition
Sources
Growth
Sources
Fig. 8. Time course of cell density of the strain YT1 during Run A-11, where the pure cultured YT1 was inoculated into the garbage sterilized with girradiation and cultivated at 30 8C for 9 d under an aerobic condition.
3.3. Analytical results of the fragrance components Fig. 9 shows the comparisons of the GC/MS analytical results of the emitted gas from the raw garbage and the 1st day garbage of Run A-11. By comparing the two chromatograms, it was confirmed that the concentrations of some chemical components, the esters ethyl acetate, prophyl acetate, isobutyl acetate, and isoamyl acetate and the alcohols isopentanol, isobutanol, and isoamylalcohol, were apparently different each other and were higher in the 1st day garbage than in the raw garbage. The fragrances could be smelled in the gas collected from the 1st day garbage. Therefore, it was considered that the fragrances were composed of these chemicals. It was demonstrated that these chemicals were produced by yeast strains [20,21]. In addition, Buzzini et al. [20] reported that isoamylalcohol and isoamyl acetate were main compounds of VOC produced and accounted for the banana smell, which was detected during the manipulation of some yeast culture. In this study, the smell of the fragrances was similar to that of isoamlyalcohol. This may be because the peak of isoamylalcohol was the largest among all fragrance components.
Growth
Utilization of carbon and nitrogen sources Anaerobic Glucose + Aerobic
Additional test Growth at 40 8C
Glucose Galactose Sorbose Rhamnose Dulcit Inositol Mannitol Sorbitol Glycerol Erythritol D-Arabinose L-Arabinose Ribose D-Xylose L-Xylose Adonitol Nitrate
+
+
+
a-Methylglycoside Salicin Cellobiose Maltose Lactose Melibiose Sucrose Trehalose Inulin Melezitose Raffinose Starch Xylitol Gluconate 2-Keto-gluconate 5-Keto-gluconate Acetate production
Fig. 9. Gas chromatograms of the emitted gas from the raw garbage and the 1st day garbage of the storage experiment of the garbage in Run A-11, where the pure cultured YT1 was inoculated into the garbage sterilized with g-irradiation and cultivated at 30 8C under an aerobic condition.
K. Nakasaki et al. / Process Biochemistry 41 (2006) 1932–1939
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3.4. Effect of cultivation conditions for strain YT1 on fragrance production The effects of cultivation conditions such as pH, temperature, and kinds of medium for the fragrance production by strain YT1 were investigated using the Jar-fermentor (see Table 2). The time courses of the OD570 for Runs B-1–B-4 are compared in Fig. 10. In Runs B-3 and B-4, the values of OD570 began to increase at almost the same time from the start of the cultivation experiment. The values then became nearly constant after 10 h. Even though the growth rate of strain YT1 in Run B-2 was a little late, the OD570 for Run B-2 reached similar values for both Runs B-3 and B-4 after about 18 h. However, the OD570 in Run B-1, in which the pH was controlled at 2, increased very slowly, and finally reached the lowest value. These results suggest that the low pH was not suitable for the growth of strain YT1. The maximum specific growth rates of strain YT1 for Runs B-1–B-4 were 0.148, 0.464, 0.592, and 0.567 h 1, respectively. Fig. 11 shows the time courses of the relative concentrations of the fragrance components emitted during Runs B-1–B-4. Four kinds of fragrance components: ethyl acetate, ethanol, isobutanol, and isoamylalcohol were analyzed in these experiments, and the relative concentrations of each fragrance component were calculated with dividing by those emitted in Run B-3. The relative concentrations of every component were the highest in Run B-3 among all experimental runs. In Runs B2–B-4, all of the fragrance components began to be detected after 12 h and then their concentrations increased gradually, whereas these fragrances were not detected in Run B-1 throughout the cultivation period. Fig. 12 summarizes the pH dependence of the fragrances production for Runs B-1–B-4. The relative concentrations of each fragrance component at 20 h of cultivation time are shown here. The value for every fragrance component was the largest in the cultivation condition of pH 4, and the values decreased distinctly in the conditions of pH 6 or 2. Therefore, it was ascertained that the quantities of the fragrances produced by strain YT1 depended on the pH conditions, and that pH 4 was the optimum for the production of the fragrances. The effects of the cultivation temperature and kinds of medium used, together with the results of the pH dependence of
Fig. 10. Time courses of OD570 during the cultivation experiments of strain YT1 in Runs B-1–B-4, where the pure cultured YT1 was inoculated into the PD medium under various cultivation pH 2, 3, 4, and 6, respectively.
Fig. 11. Time courses of relative concentrations of the fragrance components emitted during the cultivation of strain YT1 in Runs B1–B4, where the pure cultured YT1 was inoculated into the PD medium under various cultivation pH 2, 3, 4, and 6, respectively. The concentrations of each fragrance component emitted in Run B-3 were set up as 1.
strain YT1, are summarized in Table 4. The concentrations of each fragrance component emitted at the final stage of Run B-7 was made up as 100, and Table 4 shows the relative values of the fragrance concentrations emitted in each condition when 20 h have elapsed from the start of cultivation, i.e., the final stage. By comparing Runs B-3 and B-5 through B-7, the growth rate of YT1 was the maximum at 30 8C, the second maximum at 40 8C, and the third at 25 8C, though the final cell densities (OD570) at 25 8C were similar to those observed for both the 30
Fig. 12. A pH dependence of the fragrances production for Runs B-1–B-4, where the pure cultured YT1 was inoculated into the PD medium under various cultivation pH 2, 3, 4, and 6, respectively. The concentrations of each fragrance component emitted in Run B-3 were set up as 1 (EA: ethylene acetate; IB: isobutanol; ET: ethanol; IAA: isoamylalcohol.).
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K. Nakasaki et al. / Process Biochemistry 41 (2006) 1932–1939
Table 4 Cell growth and fragrances production by pure cultured strain YT1 according to the cultivation conditions Run no.
B-1 B-2 B-3 B-4 B-5 B-6 B-7 B-8 B-9 a b
mmaxa (h 1)
0.148 0.464 0.592 0.567 0.229 0.440 0.468 0.483 0.430
ODmaxb
0.269 2.531 2.533 2.571 0.968 2.450 2.515 2.157 2.408
Concentration (arbitrarily unit) Ethyl acetate
Ethanol
Isobutanol
Isoamylalcohol
0.0 0.0 37.8 5.7 55.4 5.4 19.3 4.7 0.0 0.0 0.2 0.1 100 12.5 0.1 0.2 0.1 0.0
0.3 0.0 47.3 3.5 46.6 2.8 28.1 1.9 0.1 0.2 0.3 0.3 100 17.7 0.5 0.3 11.7 2.6
0.0 0.0 27.6 5.5 52.7 5.2 27.4 2.9 0.0 0.2 2.0 3.3 100 9.6 6.6 6.2 19.4 2.9
0.0 0.0 35.8 9.1 72.8 7.6 43.9 7.7 0.0 0.0 6.0 1.8 100 14.6 26.7 3.6 49.4 7.3
Maximum specific growth rate. Optical density at 20 h.
and 40 8C experiments. The final OD570 value at 20 8C, however, was considerably lower than those of any other temperatures. Although not shown here in detail, the fragrance components were all detected after 12 h and the concentrations of the fragrance components were increased with the progress of cultivation time in Runs B-3 and B-7, in which the temperature was kept over 30 8C. The relative values of the fragrances concentrations at 40 8C were higher than those at 30 8C, as shown in Table 4. In contrast, the fragrances were not detected at all in Run B-5, and the fragrances were emitted at quite a low level in Run B-6, as shown in the same table. It is interesting that the fragrances concentrations were the highest in the cultivation temperature of 40 8C, apparently higher than those at 30 8C, although the final cell density for both temperatures was similar, and, moreover, the specific maximum growth rate was somewhat higher in the 30 8C experiment than in the 40 8C one. This may be because higher temperature makes the vapor pressure of the fragrance components higher. Finally, the effect of the kinds of substrates on the growth of strain YT1 and the production of the fragrances was investigated in Runs B-3, B-8, and B-9. In Run B-3, which used PD medium, ethyl acetate started to be emitted after 12 h, and then increased with the cultivation time, as seen in Fig. 11. However, Run B-8 used Trypticase-soy medium and Run B-9 used ISP No. 2 medium, and ethyl acetate was not detected in either run throughout the cultivation period (see Table 4). A higher amount of the other fragrance components, ethanol, isobutanol, and isoamylalcohol, were produced in the PD medium, ISP No. 2 medium, and Trypticase-soy medium, in that order. By considering the difference in the compositions of these media, it may be suggested that the most suitable substrate for the fragrances production by strain YT1 would be glucose, since the concentration of glucose contained in these media corresponds to the same order for the concentrations of fragrances produced, i.e., PD > ISP No. 2 > Trypticase-soy medium. These results coincided well with the studies on that these strains of yeast could effectively ferment and assimilate the glucose as a carbon source [27,28].
4. Conclusions Strain YT1, a type of yeast which we isolated, was effective in suppressing malodors emitted during the storage periods of garbage by producing fragrance components and masking the nuisance odors. The fragrances produced from the garbage by stain YT1 were identified as different kinds of alcohols and esters such as isoamylalcohol, ethyl ester, and so on. It was ascertained that the fragrances production was dependent on the cultivation conditions. The optimum temperature and pH were 40 8C and 4, respectively. Moreover, the fragrances production were strongly dependent on the kinds of substrates, and it was ascertained that strain YT1 prefers the Potato-dextrose medium. This result seems to indicate that the strain YT1 produces more fragrances from glucose than from other substrates. The concentration of glucose in the garbage may be low at first, but it would become high level resulting from the activity of co-existing microorganisms in the garbage such as lactic acid bacteria [26,29] which will provide the glucose derived from sugars or carbohydrates. It is well known that garbage in households or various treatment facilities usually contains high concentration of sugars and carbohydrates, and that many microorganisms possess hydrolysis enzymes of sugars and carbohydrates to convert the glucose. Therefore, it can be considered that C. krusei var. transitoria, YT1 will be effective in preventing the emission of malodors during the storage periods of garbage in households or various treatment facilities. References [1] Chynoweth DP, Owens J, O’Keefe D, Earle JFK, Bosch G, Legrand R. Sequential batch anaerobic composting of the organic fraction of municipal solid waste. Water Sci Technol 1992;25:327–39. [2] Nakasaki K, Yaguchi H, Sasaki Y, Kubota H. Effects of pH control on composting of garbage. Waste Manage Res 1993;1:117–25. [3] Kayhanian M, Hardy S. The impact of four design parameters on the performance of a high-solids anaerobic digestion of municipal solid waste for fuel gas production. Environ Technol 1994;15:557–67. [4] Hwang EJ, Shin HS, Tay JH. Continuous feed, on-site composting of kitchen garbage. Waste Manage Res 2002;20:119–26. [5] Kwon SH, Lee DH. Evaluation of Korean food waste composting with fed-batch operations II: using properties of exhaust gas condensate. Process Biochem 2004;39:1047–55.
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