Abstract
A very safe and environmentally friendly solid-state material for bioremediation was prepared using a combination of Aspergillus oryzae and poly(ε-caprolactone) (PCL), a porous biodegradable polymer. The novel material was capable of decomposing 200 ppm formaldehyde solutions to 0 ppm within 7 days. Degradation ability was prolonged by addition of yeast extract-peptone-dextrose medium into the composite; 200 ppm formaldehyde was decomposed to 0 ppm over eight additional decomposition cycles and 100 days. A unique mechanism is proposed where, during PCL degradation, the solid-state composite provides nutrients to A. oryzae.
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Introduction
Bioremediation has been employed in addressing a number of environmental problems, such as contaminated soil and groundwater, toxic industrial effluent, and poor air quality in living and working spaces [1–5]. Bioremediation is typically an efficient and low-cost environmental purification technique [6–8], and involves exploiting microorganism metabolism to remove pollutants. These technologies can be generally classified as in situ or ex situ. In situ bioremediation involves treating the contaminated material on-site, while ex situ involves the removal of the contaminated material for treatment elsewhere [9–11]. In both cases, the technology works by utilizing microorganism metabolism, and the microbes are usually released into rivers, lakes, soil and groundwater. It is imperative that the microbes utilized pose no threat to people at the contamination site or in the community. For this reason, confirmation of microorganism safety is required before carrying out bioremediation [12, 13].
The objective of this study was to produce a very safe and environmentally friendly solid-state material for use in bioremediation. This new type of material combines a safe microorganism and a biodegradable polymer, allowing for a wide range of potential applications, such as a filter or a component of purification systems for the decomposition or removal of harmful chemicals from industrial wastewater or tap water, as well as for treating poor air quality in our living spaces.
There are two types of microorganisms that are commonly known throughout the world to be very safe. One is the fungus Aspergillus oryzae, which has been used in Asian fermentation industries, such as the alcoholic beverage ‘sake’, the soybean paste ‘miso’, as well as for the production of soy sauce and vinegar for more than 1,000 years [14]. A. oryzae is also a widely used organism for the production of amylases, lipases and proteases, and has more recently been employed for the heterologous expression of secondary metabolite genes and non-fungal proteins [15–18]. The second group comprises lactobacilli and probiotic bacteria, which are frequently used as active ingredients in functional foods such as yogurts, dietary adjuncts and health-related products [17]. In previous studies [19, 20], we reported that A. oryzae produced various hydrolytic enzymes, including amylases and proteinases, that decompose biodegradable polymers such as poly(ε-caprolactone) (PCL) and poly(butylene succinate adipate) (PBSA), as well as harmful organic chemicals, e.g. formaldehyde, formic acid, tetrahydrofuran, and acrylamide. Moreover, the fungal filaments were observed to attach strongly to the microstructure of the biodegradable polymer surface. These features allow for the production of a solid-state microorganism/biodegradable polymer composite for use in bioremediation. PCL is a semi-crystalline polymer with a glass transition temperature around −60 °C and a melting temperature around 60 °C. It is a ductile polymer and is, most often used in combination with different fillers [21].
In this study, we prepared an A. oryzae/PCL composite using an environmental friendly process. Formaldehyde was targeted for decomposition using the composite because it is widely used in industrial wood processing [22] and in the production of paper, leather, resins and glues [23], and is a highly reactive compound, with toxic effects on living organisms through its nonspecific reactivity with proteins and nucleic acid. Decomposition capacity and sustainability of the composite for formaldehyde will be discussed in reference to its structure.
Experimental
Poly(ε-caprolactone) (PCL; Daicel Chemical Co. Ltd., CELGREEN PH-7) (Mn = 68,000, Mw = 125,000) was used for the preparation of the porous biodegradable polymer, with pore sizes ranging from 20 to 106 μm and 20 to 60 vol% porosity. The value of porosity was calculated from the bulk density (ρ b) and particle density (ρ p) as follows:
Sodium chloride (solid) was pounded in a mortar, and the powder was screened through various sizes of mesh wire (20–106 μm). The screened salt and PCL pellet were combined using a mixer (IMC-1A65; IMOTO Co. Ltd.) at 100 °C for 20 min. The mixture was hot pressed at 120 °C for 5 min and rinsed with distilled water until the sample weight was to be constant. A. oryzae was incubated on PDA (Potato Dextrose Agar; Difco™ Becton Dickinson and Company) plates for a week at 30 °C. The spores of A. oryzae were collected as a spore suspension by adding sterile water on the plate. Then the spore concentration was adjusted to 1 × 106 spores/mL. 500 μL of the spore suspension was dropped on the porous PCL. Next, the porous PCL containing spores was dried under ambient conditions. Then, the spores on the PCL were germinated in YPD (yeast extract peptone dextrose; Difco™ YPD Broth, Becton Dickinson and Company) medium in shaking condition of 100 rpm. The process is shown in Fig. 1.
To obtain PCL sample containing nutrition, YPD powder (40 wt%) and PCL pellets were combined at 80 °C using a mixer (IMC-1A65, Imoto Co. Ltd.) In order to enhance the attachment of A. oryzae fungal filaments, a micro-scale pattern was imprinted on the surface of the sheets by hot-pressing with 20 μm wire mesh at 80 °C. The composite containing YPD was also observed using scanning electron microscope (SEM).
The concentration of formaldehyde was measured by the Nash method [24] and HPLC (Separation Model 2695; Waters) using a column (CAPCELL PAK C8 type SG120, 5 μm, 4.6 mm ID × 250 mm: Shiseido Co.). The flow rate and temperature were 1 mL/min and 40 °C. 0.1% (v/v) phosphate buffer was used as running buffer. The degradation tests were carried out in 50 mL of formaldehyde solution adding 500 mg of A. oryzae /PCL composite with shaking (120 rpm) and static conditions.
Results and Discussion
Figure 2 shows SEM images of cross sections of nine PCL sheet samples with various pore sizes and porosity. It seems that the porosities of 40 % indicated that most of pore connected each other. For the sample of porosity of 20 %, most of pores are not completely connected each other. Some of salts are isolated in the sample. Figure 3 shows SEM images of A. oryzae/ PCL composites after spore germination in YPD medium under shaking condition for 1 day. A large amount of A. oryzae obviously adhered to the samples with 20–53 μm pore size and with 40 and 60 % porosities, and filament growth and spore production was observed. Table 1 provides the amounts of A. oryzae immobilized on the nine PCL samples under shaking and static incubation conditions for one day. The porous PCL with 20–53 μm pore size and 60 % porosity was suitable for immobilization of A. oryzae filaments.
Figure 4 shows the capacity of A. oryzae/PCL composites to degrade formaldehyde at 30 °C. The composites (500 mg pieces) were added to 50 mL of 200 ppm formaldehyde solution. The decomposition rate depended on the material’s porosity and on the incubation conditions. The 60 % porosity sample with shaking exhibited the highest degradation rate of formaldehyde and completely decomposed it within 5–7 days. When A. oryzae alone was added to formaldehyde, the concentration did not decrease over the 12 days, clearly indicating that A. oryzae attachment to the PCL composite is important for its effectiveness [25–27]. In general, spores showed high germination and attached strongly to the solid scaffolds [27], as shown in Fig. 3. It was observed that degradation ability continued when the composite was exposed to a second 200 ppm formaldehyde solution, although the degradation rate was lower than upon initial exposure. The results indicate that this novel material is useful for the purification of formaldehyde dissolved in water.
Previously, it has been reported that 400 ppm of formaldehyde has been degraded by bacterium [28]. However, we were considering that bacterium couldn’t very well adhere to the matrix. The organisms grows attached to the matrix is a needful methods when make use of it for the environmental.
It is a known fact [29], that the formaldehyde is degraded by Aspergillus flavus. In this regard, since it is an unsafe organism, compared with existing the method by A. flavus, this method allowed safe bioremediation use of A. oryzae.
Sterilized A. oryzae never decompose formaldehyde under the same condition of Fig. 4. Therefor A. oryzae still grow even for long time after 100 days. The results clearly show that the composite materials slowly deliver the nutrients to A. oryzae, successfully.
TOF-MS analysis of the enzyme produced by A. oryzae produce an enzyme to be assigned to a SOD oxidation enzyme under the same condition of our prior study by using of TOF-MS analysis, the enzyme might be oxidize formaldehyde [30].
Two strategies were employed in an attempt to further improve the process: increasing the sustainability of degradation and enhancing the degradation rate. The first strategy involved the gradual and continuous supply of nutrients to A. oryzae. For this purpose, we prepared a PCL composite containing A. oryzae spores and YPD medium. The second strategy involved A. oryzae growth enhancement on the composite by making concave and convex patterns on the surface, thereby promoting A. oryzae germination and attachment. We previously reported that the fungus grew and the filaments attached strongly to the 20-μm mesh pattern imprinted of the PCL surface [31]. The micro-pattern would enhance the A. oryzae attachment on the surface, leading to enhance rate of formaldehyde degradation.
Figure 5 shows a schematic of composite production using the above-mentioned two strategies. In the first step, A. oryzae spores germinate and grow on the PCL. In the second step, the surface-attached A. oryzae begins to degrade the PCL. As a result, nutrients are released from the YPD medium within the PCL and supplied to A. oryzae. This process continues until the composite is completely degraded, allowing for continued decomposition of formaldehyde.
Figure 6 shows SEM images (cross-section and surface) of the composite containing 40 wt% nutrient YPD powder and imprinted with a mesh micro-pattern on the surface. The YPD is partially isolated in the PCL sheets (cross-section). A. oryzae filaments grew on the material’s surface, covering the composite (surface). The composite has potential for decomposing formaldehyde or other chemicals using enzymes produced by the surface-attached A. oryzae.
The sustainability of formaldehyde degradation by the composite containing YPD is shown in Fig. 7. During the first addition/degradation cycle, the composite reduced 200 ppm formaldehyde to 0 ppm within 18 days; a degradation rate much lower than for the A. oryzae/porous PCL composite (6 days) shown in Fig. 4. In contrast, during the second addition/ degradation cycle, the degradation rate increased and was nearly the same as for the A. oryzae/PCL composite. Formaldehyde degradation was maintained over eight addition/degradation cycles and 100 days. The results show the composite supplied nutrients to the surface-attached A. oryzae, allowing for A. oryzae to decompose the formaldehyde. We have reported that A. oryzae hydrolyzes PCL and assimilates 6-hydroxyl hexanoic acid. During PCL degradation, the composite supplies sufficient nutrients to A. oryzae, leading to high sustainability of formaldehyde degradation.
Conclusion
We prepared a very safe and environmentally friendly solid-state material for use in bioremediation, by combining A. oryzae, a microorganism known to be safe, and a porous biodegradable PCL polymer. The composite showed a high capacity for decomposing formaldehyde solution. Degradation was strongly improved by the addition of a nutrient medium (YPD) to the composite. The mechanism by which the A. oryzae/nutrient/biodegradable polymer composite undergoes degradation and continues to supply nutrients to A. oryzae is useful for long-term formaldehyde decomposition.
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Acknowledgments
We appreciate the financial support from Grants-in-Aid for Scientific Research No. 23510113 and Ministry of Education, Culture, Sports, Science and Technology-Supported Program for the Strategic Research Foundation at Private Universities in Japan, 2009–2013.
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Tanida, I., Sakaue, A. & Osawa, S. Development of a Safe Solid-State Microorganism/Biodegradable Polymer Composite for Decomposition of Formaldehyde. J Polym Environ 22, 329–335 (2014). https://doi.org/10.1007/s10924-014-0644-0
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DOI: https://doi.org/10.1007/s10924-014-0644-0