Production of poly (l-lactide)-degrading enzyme by Actinomadura keratinilytica strain T16-1 under solid state fermentation using agricultural wastes as substrate

Abstract

Poly (l-lactide) (PLLA) is an aliphatic polyester that can be obtained from renewable resources and degraded by various microorganisms. In previous reports, Actinomadura keratinilytica strain T16-1 demonstrated high ability to degrade PLLA under various conditions. PLLA-degrading enzyme production under solid state fermentation has been sparsely studied. PLLA-degrading enzyme production by A. keratinilytica strain T16-1 was investigated using agricultural wastes as substrate under solid state fermentation (SSF). Three agricultural wastes as soybean meal, cassava chips and duckweed were tested as substrates for PLLA-degrading enzyme production by statistical methods using mixture design. Results revealed that using duckweed as the substrate gave the highest enzyme production (138.66 ± 13.57 U/g dry substrate). Maximum enzyme activity of 391.24 ± 15.57 U/g dry substrate was obtained under 10 g duckweed, 10% inoculum size, 7 days of cultivation time, pH 7.0, 2.8% PLLA powder, and 60% moisture content at 45 °C. It can be concluded that duckweed is an inexpensive substrate, which reduces the costs of PLLA-degrading enzyme production, as an alternative to effective water weed management.

Introduction

Plastic waste pollution has become one of the most important global environmental issues. Synthetic materials, generically termed plastics, are widely used in construction due to their durability and low weight (Aciu et al. 2018). However, low biodegradability of these materials and large quantities of plastic waste negatively impact the environment (Sharma and Bansal 2016). Research has focused on developing biodegradable plastics such as PLLA that can be obtained from lactic acid and hydrolytically degraded. Lactic acid is a naturally occurring organic acid that can be produced by fermentation of sugars obtained from renewable resources such as sugarcane (Lopesa et al. 2012). PLLA is a highly versatile, biodegradable, aliphatic polyester derived from 100% renewable resources (Nair and Laurencin 2007). Environmental degradation of PLLA occurs as a two-step process. During the initial phase, water insoluble high molecular weight polyester chains hydrolyze to water soluble low molecular weight oligomers by attacking the chemical bonds in the amorphous phase. During the second step, the degradation process continues through the conversion of low molecular weight PLLA components into carbon dioxide, water and humus (Kolstad et al. 2012; Prema and Uma Maheswari Devi 2015). Commercial enzymes can degrade the PLLA such as trypsin, proteinase K, proteinase and elastase (Lim et al. 2005). Furthermore, microbial enzymes have also been reported to degrade PLLA such as esterase from Rhizopus delemerlipase (Fukuzaki et al. 1989), proteinase K from Tritirachium album (Williams, 1981), lipase (cutinase) from Cryptococcus sp. strain S-2 (Masaki et al. 2005), serine protease from A. keratinilytica strain T16-1 (Sukkhum et al. 2009). Laceyella sacchari strain LP175 (Hanphakphoom et al. 2014) and Bacillus amyloliliquefaciens (Prema and Uma Maheswari Devi 2015). A. keratinilytica strain T16-1 demonstrated high ability to degrade PLLA under various conditions (Sukkhum et al. 2009, 2012; Panyachanakul et al. 2017).

A. keratinilytica strain T16-1 has been studied since 2009. It showed high potential to produce PLLA- degrading enzyme in shaking flasks, airlift fermenter and up to 10-L stirrer fermenter (Sukkhum et al. 2009, 2012; Panyachanakul et al. 2017). Increased PLLA-degrading enzyme activity and productivity were observed in repeated-batch fermentation under submerging fermentation with 942.67 U/mL and 19.64 U/mL h, respectively. Moreover, scaled-up production of the enzyme in a 10-L stirrer bioreactor showed the highest enzyme activity of 578.67 U/mL (Panyachanakul et al. 2017). In addition, the enzyme produced by A. keratinilytica strain T16-1 was subjected to study optimization of Poly(dl-Lactic acid) degradation and evaluation of biological re-polymerization (Youngpreda et al. 2017). Approximately 6700 mg/L PLA powder was degraded by the crude enzyme produced by strain T16-1 under the conditions: an initial enzyme activity of 200 U/mL, incubated at 60 °C for 24 h released 6,843 mg/L lactic acid with 82% conversion, which was similar to the commercial enzyme proteinase K (81%). Panyachanakul et al (2019) reported that PLA degradation after 72 h in a 5 L bioreactor using the enzyme of the strain T16-1 under controlled pH conditions resulted in lactic acid titers (mg/L) of 16,651 mg/L and a conversion efficiency of 89% at a controlled pH of 8.0. The enzyme produced by strain was reported as protease type enzyme (Sukkhum et al. 2009). In the presence of inducers (PLA), the production of PLLA-degrading enzyme and other protease is induced. PLA-degrading enzyme is able to degrade both PLA and protein substrates, but protease degrades only the protein substrates. However, the study of PLLA-degrading enzyme production of strain under solid state fermentation has not been reported so far.

Solid substrates as fermentable sources were tested due to their low prices, eco-friendly approach, perennial availability, low polluting effluents and easier downstream processing. Several cheap agricultural wastes currently available are used as substrates for the production of pharmaceutically important metabolites. The limitations of solid state fermentation (SSF) includes the chance of contamination, and low degree of aeration due to high solid concentration which can lead to significant yield increase (Mienda et al. 2011; Kamath et al. 2015). PLLA-degrading enzymes can be produced by L. sacchari strain LP175 under SSF using cassava chips and soybean meal as substrates, and inducers containing high contents of starch and protein for their growth (Lomthong et al. 2020). Recently, Lomthong et al. (2021) has been expanded to a large-scale enzyme productivity by L. sacchari strain LP175 in the aerated tray reactor, yielded 518 ± 8.5 U/g dry substrate under conditions of 0.8 L aeration for 24 h and without aeration 48 h at 50 in the tray reactor. Duckweed (Lemna minor L.) is a small floating aquatic plant. It grows and reproduces well in stagnant water such as swamps, marshes, or common ponds that are rich in nutrients, organic matter and in neutral pH (Ishizawa et al. 2017). Duckweed can sexually and asexually multiply, making rapid growth. It is a problem in natural water resources such as water in dams or reservoirs. Duckweed is usually used for fish feed, biofuels, organic fertilizer, and chemical toxicity tests (Ishizawa et al. 2017). Duckweed has been reported the usage as nitrogen source to produce high alkaline protease from Serratia sp. SYBCH (Li et al. 2011) and glycoside hydrolases from Trichoderma asperellum (Bech et al. 2015) and as the inducer for cellulose production in Trichoderma reesei RUT C30 (Li et al. 2019). However, no reports of duckweed on PLLA-degrading enzyme production. Therefore, the aim of this study was to optimize PLLA-degrading enzyme production by A. keratinilytica strain T16-1 using agricultural wastes as substrate under solid state fermentation.

Materials and methods

Microorganism

The actinomycete A. keratinilytica strain T16-1 was isolated and identified by Sukkhum et al. (2009) and stored in the NITE Biological Resource Center (NBRC), Japan, and Biotech Culture Collection (BCC), Thailand. The culture was kept was kept refrigerated at − 20 °C prior to use (Panyachanakul et al. 2017).

Inoculum preparation

One hundred milliliters of ISP-2 broth (International Streptomyces Project-2 medium, containing 4 g/L yeast extract, 10 g/L malt extract and 4 g/L dextrose with pH 7.0) was sterilized into 250 mL Erlenmeyer flasks at 121 °C for 15 min. After sterilization, the medium was inoculated with a loopful of A. keratinilytica strain T16-1 and incubated at 45 °C for 4 days on a rotary shaker (150 rpm). The cell mass was harvested using a centrifuge at speed of 10,000 rpm for 10 min at 4 °C and washed three times. The inoculum was carried out by resuspending in sterile 0.85% NaCl solution and adjusted an optical density at 600 nm (OD600) of 1.0 (1 × 10⁷ CFU/mL) by diluting with Basal broth (0.7% PLLA powder, 0.238% gelatin, 0.4% (NH₄)₂SO₄, 0.4% K₂HPO₄, 0.2% KH₂PO₄ and 0.02% MgSO₄∙7H₂O with pH 8.0) (Panyachanakul et al. 2019).

Preparation of solid stat fermentation substrates

Fresh duckweeds were collected from fresh pond, washed three times, crushed in a blender, and dried at 50 °C for 24–48 h. Soybean meal and cassava chip were purchased from local market and mashed with a blender and dried at 50 °C for 24–48 h. All substates were pulverized into 60 mesh powder. The agricultural wastes were used as substrates for PLLA-degrading enzyme production.

Growth determination

Growth in SSF was enumerated using total plate counts calibrated by the spread plate technique. The viable cell was calculated as total plate count (colony forming unit, CFU/g) (Clais et al. 2014).

Mixture design for PLLA-degrading enzyme production under SSF

The SSF process was operated by mixture design method to select the optimal ratio and types of solid substrates. A design containing 7 runs was used, with each component studied at four; namely 0 (0%), 1 (1%), 1/2 (50%) and 1/3 (33.3%), as shown in Table 1. The seven experiments were set at four levels, with different percentages in parentheses for a total of 1 ration as described by de Castro and Sato (2013). Fermentation substrates were selected for the SSF process. Each experiment was performed in an Erlenmeyer flask (250 mL) with 10 g of substrate and 8 mL of Basal medium. The initial moisture content was adjusted of 70% in dry basis with the volume of distilled water (Soares et al. 2005). The flask was plugged with cotton wool before autoclaving at 121 °C for 30 min. The flasks were then inoculated with 6% (v/w) suspension culture. This work was carried out in three replications. Cultivation was then conducted in an incubator at 45 °C for 7 days. After the incubation period, 40 mL of 0.1 M Tris–HCl buffer pH 9.0 was added to the SSF medium and incubated for 3 h at 10 °C on an orbital shaker at 150 rpm. The extract solution was filtered through Whatman filter paper No. 1. The filtrate was centrifuged at 10,000 rpm for 10 min at 4 °C. The supernatant as the crude enzyme source was stored at − 20 °C until required for use.

Table 1.

Seven runs of the fermented three materials for production of PLLA-degrading enzymes using mixture design

Run	Independent variable
	Soybean meal (X1)	Cassava chips (X2)	Duckweed (X3)
1	1	0	0
2	0	1	0
3	0	0	1
4	1/2	1/2	0
5	1/2	0	1/2
6	0	1/2	1/2
7	1/3	1/3	1/3

Amounts of PLLA-degrading enzyme were determined for each run. Experimental design and statistical analyses of the data were performed using SPSS software version 11.5 (IBM Company, USA) and Statistica 10 for Windows™ (StatSoft, Tulsa, OK, USA).

Factors affecting PLLA-degrading enzyme production in a flask

Effect of inoculum size and cultivation time

The optimal substrate combination obtained from mixture design. Ten g of optimal substrate in all Erlenmeyer flasks (250 mL) were added 8 mL of Basal broth. Then, the culture suspension (1X10⁷ CFU/mL) was inoculated with different inoculum size of 2, 6, 10 and 14% (v/w). The initial medium moisture content was adjusted by distilled water to 70% (v/w). Each flask was incubated at 45 °C and harvested sample for 0, 2, 4, 6, 7 and 8 days.

Effect of initial pH

The effect of pH on PLLA-degrading enzyme production by varying initial pH of the fermentation medium from 5 to 10. The culture fermentation was adjusted according to the optimal conditions of the inoculum size and cultivation time.

Effect of temperature

The effects of temperature on PLLA-degrading enzyme production at different temperatures were studied as follows: 35 37 42 45 50 and 60 °C. The culture fermentation was adjusted according to the optimal conditions of the inoculum size and cultivation time.

Effect of PLLA powder concentration

To the optimal substrate combination obtained from mixture design was used with different PLLA powder concentrations of 0.35, 0.7, 1.05, 1.40, 1.75, 2.10, 2.45, 2.80 and 3.15%. The culture fermentation was adjusted according to the optimal conditions of the inoculum size, cultivation time, pH and temperature.

Effect of initial moisture content

The optimal combination of substrates obtained from mixture design method and optimal concentration of PLLA powder was used to test the effect of initial moisture content in the range of 50, 60, 70, 80 and 85% (v/w). The initial moisture content was provided with distilled water in dry basis. The culture fermentation was adjusted according to the optimal conditions of the inoculum size, cultivation time, pH, temperature and PLLA powder concentration.

Enzyme activity assay

PLLA-degrading enzyme activity was determined by decreasing in emulsified PLLA turbidity. For this purpose, 0.1% (w/v) PLLA was dissolved in dichloromethane, emulsified with 0.1 M Tris–HCl buffer (pH 9.0) by an ultrasonic processor model VCX 500 (Sonic and Materials, Inc., Newtown, USA), and kept at room temperature to remove the solvent. A reaction mixture containing 2.25 mL of emulsified PLLA and 0.25 mL of enzyme filtrate was incubated at 60 °C for 30 min. The reaction stopped by cooling with ice for 5 min. The decreased in turbidity was measured at 630 nm. One unit of PLLA-degrading activity was defined as a 1-unit decrease in optical density at 630 nm of emulsified PLLA per milliliter under the assay conditions described above (Sukkhum et al. 2009).

Statistical analysis

The data were analyzed using SPSS Statistics version 22 (SPSS, USA). The One way ANOVA was used to analyze at 95% a confidence level of values in different treatments. Mean differences were analyzed using Duncan’s multiple range test.

Results and discussion

Selection of optimal type and ratio of substrate for PLLA-degrading enzyme production using mixture design

Inexpensive agricultural waste residues such as soybean meal, cassava chips and duckweed were used in the mixture design for PLLA-degrading enzyme production. In Table 2, results showed the highest production of PLLA-degrading enzymes at 137.09 ± 28.27 U/g substrate using only duckweed as the substrate, followed by a combination of duckweed and cassava chips showing high PLLA-degradation enzymes (112.53 ± 18.06 U/g substrate), and duckweed mixed with soybean meal (108.67 ± 42.09 U/g substrate).

Table 2.

PLLA-degrading enzyme activity of three substrates from actinomycete A. keratinilytica strain T16-1 at 7 days cultivation using mixture design incubation at 45 °C with initial moisture content of 70%

Run	Independent variable			PLLA-degrading enzyme activity (U/g dry substrate)
	Soybean meal (X1)	Cassava chips (X2)	Duckweed (X3)
1	1	0	0	34.64 ± 10.55
2	0	1	0	24.98 ± 12.03
3	0	0	1	137.09 ± 28.27
4	1/2	1/2	0	44.09 ± 14.97
5	1/2	0	1/2	108.67 ± 42.09
6	0	1/2	1/2	112.53 ± 18.06
7	1/3	1/3	1/3	51.35 ± 32.60

Values are means ± SD (n = 3)

Duckweed, an aquatic plant of the family Lemnaceae, was proposed as a potential feedstock for biofuel production because of its high proportion of carbohydrate (3–75%), low lignin content and high productivity (Zhao et al. 2015). Furthermore, protein content of dry duckweed biomass (15–45%) is mixed with alfalfa (20%) and soybean (41.7%) (Xu et al. 2012; Bech et al. 2015), while cassava chips contain a small amount of protein (3.2%) (Lomthong et al. 2015). Lomthong et al. (2020) reported that PLLA-degrading enzyme production under SSF may be induced by cassava chips and soybean meal as protein and nitrogen sources, respectively. Many proteinaceous substrates are used to induce PLLA-degrading enzyme production such as soybean meal (Lomthong et al. 2017, 2020) and gelatin (Sukkhum et al. 2009).

Furthermore, enzymes produced from some bacteria degrade plant cell wall structures and release sugar. Bech et al. (2015), found that an enzyme cocktail from Trichoderma asperellum degraded plant cell wall structure and released glucose content at 34% from duckweed. Results indicated that high protein contents and sugar from duckweed induced PLLA-degrading enzyme production by A. keratinilytica strain T16-1. Furthermore, duckweed can be used as a low-cost substrate for A. keratinilytica strain T16-1 to produce PLLA-degrading enzymes under SSF.

A statistical mixture design of the three agricultural substrates used for PLLA-degrading enzyme production is illustrated by the triangle (simplex) shown in Fig. 1. Relationships between the agricultural substrate features were fitted into a polynomial equation. This was used to predict substrate characteristics for PLLA-degrading enzyme production of mixture designs consisting of three substrates, as shown in an Eq. 1:

$$\begin{array}{cc}\hfill Y_1& =37.6857X_1+28.0254X_2+140.1385X_3\hfill \\ \hfill & +3.8409X_1{X}_2+30.2651X_1{X}_3+65.034X_2{X}_3,\hfill \\ \hfill \end{array}$$ 1

where Y₁ and Y₂ are the predicted responses of PLLA-degrading enzyme production of mixture designs consisting of three substrates, and X₁, X₂ and X₃ represent soybean meal, cassava chips and duckweed, respectively. The predicted coefficient of determination (R²) of this model are 0.925, which the variance of PLLA-degrading enzyme production using the variables in the model was at 92.5%.

Fig. 1.

Triangle plot of PLLA-degrading enzyme production of three substrates by A. keratinilytica strain T16-1 under SFF at 7 days

For quadratic contour area triangle plots of mixture designs of the three agricultural substrates for PLLA-degrading enzyme production, only 10 g of duckweed gave the highest prediction value at 140.14 U/g substrate. The actual experimental value of PLLA-degrading enzyme production was 138.66 ± 13.57 U/g substrate, which was closed the predicted value.

Effect of inoculum size and cultivation time on PLLA-degrading enzyme production

The optimum inoculum concentration on PLLA-degrading production was 10% (v/w) which produced the highest yield of 184.41 ± 1.06 U/g dry substrate at 7 days (Fig. 2A). This optimum inoculum concentration is similar to those described for chitinase production from Streptomyces sp. (Xu et al. 2021). The inoculum concentration increases from 2 to 14%, resulting in an increase in growth and PLLA-degrading production. While, the inoculum concentration higher than 14% decreased in enzyme productivity. This result indicated that the best balance between substrate availability and initial cell concentration led to good oxygen transfer in the solid medium (Soares et al. 2005; Xu et al. 2021). The optimal incubation time for PLLA-degrading production was 7 days (Fig. 2B). These results are different from the report by Lomthong et al. (2021), in that the cultivation time of PLLA-degrading enzyme by L. sacchari LP175 in SSF was 2 days. The cultivation time of A. keratinilytica strain T16-1 in SSF is approximately the same as genus Streptomyces. The most cultivation time for the enzyme production in genus Streptomyces in SSF is 5 days, for example alkaline protease from Streptomyces sp. CN902 (Lazim et al. 2009), cellulase from Streptomyces viridiochromogenes (El-Naggar et al. 2011) and chitinase from Streptomyces olivaceus (MSU3) (Sanjivkumar et al. 2020). The maximum PLLA-degrading yield by A. keratinilytica strain T16-1 in SSF was longer cultivation time than in submerged fermentation (4 days) (Panyachanakul et al. 2017). In contrast, extreme initial inoculum size resulted in growth reduction and enzyme production. This could be the fact that high initial inoculum size may lead to the depletion and imbalance of nutrients within a short period and reduce dissolved oxygen (Ooi et al. 2021; Xu et al. 2021).

Fig. 2.

Effect of inoculum size (A) and cultivation time (B) on PLLA-degrading enzyme activity from actinomycete A. keratinilytica strain T16-1 at 45 °C with initial moisture content of 70%. Values are means ± SD (n = 3)

Effect of pH on PLLA-degrading enzyme production

Optimal initial pH studies for PLLA-degrading enzyme production showed that maximum enzyme activity at initial pH 7.0 (235.00 ± 4.71 U/g dry substrate) followed by initial pH 8.0 (207.50 ± 5.89 U/g dry substrate) on day 7 (Fig. 3). This finding is close to the submerged fermentation for PLLA-degrading production by A. keratinilytica strain T16-1 (Sukkhum et al. 2009). The pH values a pH range from 7.0 to 8.0 were found suitable for optimal produced PLLA-degrading production by A. keratinilytica strain T16-1. While at initial pH 5.0 and 10.0 showed lower enzyme production. The stability of the pH during culture is controlled by phosphate buffers in basal medium, consist of a mixture of K₂HPO₄ and KH₂PO₄. pH plays a key role in the growth of bacteria due to the affects on components transferred across the cell membrane (Krishna 2005).

Fig. 3.

Effect of pH on PLLA-degrading enzyme activity from actinomycete A. keratinilytica strain T16-1 at 45 °C for 7 days with initial moisture content of 70%. Values are means ± SD (n = 3)

Effect of temperature on PLLA-degrading enzyme production

The effect of incubation temperature on PLLA-degrading enzyme production is shown in Fig. 4. The highest enzyme activity was achieved at 45 °C (241.81 ± 6.16 U/g dry substrate) followed by at 42 °C (228.00 ± 2.86 U/g dry substrate). This result is similar to the optimum temperature in submerge fermentation by A. keratinilytica strain T16-1 (Panyachanakul et al. 2017). At temperatures less than 42 °C (35 and 37 °C) and greater than 45 °C (50 °C) showed decreased enzyme production. At 60 °C, no enzyme production was detected. The growth of A. keratinilytica strain T16-1 has related to the PLLA-degrading enzyme production. While at a temperature of 60 °C, growth was continuously declining which was not observed on day 6. Therefore, high temperature affects the inhibition of growth and enzyme activity due to the temperature affects protein degradation (Ooi et al. 2021).

Fig. 4.

Effect of temperature on PLLA-degrading enzyme activity from actinomycete A. keratinilytica strain T16-1 at 45 °C for 7 days with initial moisture content of 70%. Values are means ± SD (n = 3)

Effect of PLLA powder content on PLLA-degrading enzyme production

PLLA powder was used to induce the production of PLLA-degrading enzyme. As shown in Fig. 5, optimal PLLA powder content at 2.80% (w/w) gave highest enzyme activity of 285.50 ± 14.34 U/g dry substrate, while further increasing concentration resulted in declining enzyme production. Lomthong et al. (2020) reported that optimal content of PLLA powder for PLLA-degrading enzyme production from L. sacchari strain LP175 under SFF was 2% (w/w), similar to these results. According to the previous report, PLLA powder (0.035% w/v) was used as an inducer to produce PLLA-degrading enzyme from A. keratinilytica strain T16-1 under submerged fermentation (Sukkhum et al. 2009). Interestingly, PLLA-degrading enzyme from A. keratinilytica strain T16-1 produced under SFF (138.66 ± 13.57 U/g dry substrate) was higher than enzyme from the submerged fermentation (44.6 U/mL) reported by Sukkhum et al. (2009).

Fig. 5.

PLLA-degrading enzyme activity of PLLA powder from actinomycete A. keratinilytica strain T16-1 at 45 °C for 7 days with initial moisture content of 70%. Values are means ± SD (n = 3)

Effect of initial moisture content on PLLA-degrading enzyme production

The effect of different initial moisture contents on PLLA-degrading enzyme production under SSF was investigated (Fig. 6). Optimal initial moisture content at 60%, gave the highest PLLA-degrading activity of 391.24 ± 15.57 U/g dry substrate. At moisture content up to 60% enzyme production and load showed increases in nutrient solubility and substrate swelling with increase in surface area (Aljammas et al. 2018). Higher moisture content levels (70–80%) led to reduction in enzyme production, decreases in porosity, loss of particulate structure, development of stickiness, reduction in gas volume, decreases of gas exchange and enhancing formation of aerial mycelium (Pandey et al. 2000). These results concurred with previous findings that moisture content at 60% gave the highest lovastatin yield (3.50 mg/g DWS) produced by Aspergillus terreus (KM017693) (Sharma and Bansal 2016). However, results differed from previous findings regarding optimal initial moisture content (70%) for PLLA-degrading enzyme production by L. sacchari strain LP175 (Lomthong et al. 2020).

Fig. 6.

Effect of different initial moisture contents on production of PLLA-degrading enzyme under SSF by A. keratinilytica T16-1 after incubation at 45 °C for 7 days. Values are means ± SD (n = 3)

Conclusions

Results revealed that A. keratinilytica strainT16-1 produced PLLA-degrading enzyme using SSF. The statistical mixture design method revealed that duckweed showed a higher enzyme production (138.66 ± 13.57 U/g dry substrate). Optimization of influencing factors using the one factor at a time method in flask scale was performed for PLLA powder concentration and initial moisture content. Maximum enzyme activity of 391.24 ± 15.57 U/g dry substrate were obtained under conditions of 10% inoculum size, 7 days of cultivation time, pH 7.0, 2.80% PLLA powder and 60% moisture contents at 45 °C (Table 3). By comparing the PLLA-degrading enzyme production, it was found that L. sacchari LP175 had higher enzyme activity whereas cultivation time was less than A. keratinilytica strain T16-1 (Table 4). In this study, only one duckweed, was used as substrate, while Lomthong et al. (2020) used both cassava chips and soybean meal as substrates in the production of PLLA-degrading enzyme by L. sacchari LP175 under solid state fermentation, possibly due to duckweed is cheaper than cassava chips and soybean meal.

Table 3.

Summary of steps of optimal conditions for production of PLLA-degrading enzymes by A. keratinilytica strain T16-1 under SSF

Step	Method	Substrate	Condition	Medium	PLLA-degrading enzyme production (U/g dry substrate)
1	Optimization of substrate composition using mixture design	Duckweed	50% inoculum size, 7 days, Initial pH 8.0, Temperature at 45 °C, 70% initial moisture content	0.7% PLA powder, 0.238% gelatin, 0.4% (NH4)2SO4, 0.4% K2HPO4, 0.2% KH2PO4, 0.02% MgSO4.7H2O	138.66 ± 13.57
2	Optimization of inoculum size and cultivation time using one factor at a time method	Duckweed	60% inoculum size, 7 days, Initial pH 8.0, Temperature at 45 °C, 70% initial moisture content	0.7% PLA powder, 0.238% gelatin, 0.4% (NH4)2SO4, 0.4% K2HPO4, 0.2% KH2PO4, 0.02% MgSO4.7H2O	184.41 ± 1.06
3	Optimization of pH using one factor at a time method	Duckweed	60% inoculum size, 7 days, Initial pH 7.0, Temperature at 45 °C, 70% initial moisture content	0.7% PLA powder, 0.238% gelatin, 0.4% (NH4)2SO4, 0.4% K2HPO4, 0.2% KH2PO4, 0.02% MgSO4.7H2O	235.00 ± 4.71
4	Optimization of temperature using one factor at a time method	Duckweed	60% inoculum size, 7 days, Initial pH 7.0, Temperature at 45 °C, 70% initial moisture content	0.7% PLA powder, 0.238% gelatin, 0.4% (NH4)2SO4, 0.4% K2HPO4, 0.2% KH2PO4, 0.02% MgSO4.7H2O	241.81 ± 6.16
5	Optimization of PLA powder using one factor at a time method	Duckweed	60% inoculum size, 7 days, Initial pH 7.0, Temperature at 45 °C, 70% initial moisture content	2.8% PLA powder, 0.238% gelatin, 0.4% (NH4)2SO4, 0.4% K2HPO4, 0.2% KH2PO4, 0.02% MgSO4.7H2O	285.50 ± 14.34
6	Optimization of initial moisture content using one factor at a time method	Duckweed	60% inoculum size, 7 days, Initial pH 7.0, Temperature at 45 °C, 60% initial moisture content	2.8% PLA powder, 0.238% gelatin, 0.4% (NH4)2SO4, 0.4% K2HPO4, 0.2% KH2PO4, 0.02% MgSO4.7H2O	391.24 ± 15.57

Values are means ± SD (n = 3)

Table 4.

Comparative of PLLA-degrading enzyme production under SFF in a flask scale

Microorganism	Solid substrate	Liquid medium	Physical condition	PLLA-degrading enzyme production (U/g dry substrate)	References
Laceyella sacchari LP175	Cassava chips and soybean meal	2.8% PLA, 2% peptone	Temperature at 50 °C, Initial pH of 6.5, 70% initial moisture content, 3 days	467	Lomthong et al. (2020)
Actinomadura keratinilytica strain T16-1	Duckweed	2.8% PLA, 0.238% gelatin, 0.4% (NH4)2SO4, 0.4% K2HPO4, 0.2% KH2PO4, 0.02% MgSO4.7H2O	Temperature at 45 °C, Initial pH 7.0, 60% initial moisture content, 7 days	391.23 ± 26.62	This study

Funding

The National Research Council of Thailand: NRCT.

Declarations

Conflict of interest

The authors declare no conflicts of interest.

Ethical approval

This article does not contain any studies with human or animal subjects.