Yuka Sameshima-Yamashita

Phyllosphere yeasts rapidly break down biodegradable plastics

The use of biodegradable plastics can reduce the accumulation of environmentally persistent plastic wastes. The rate of degradation of biodegradable plastics depends on environmental conditions and is highly variable. Techniques for achieving more consistent degradation are needed. However, only a few microorganisms involved in the degradation process have been isolated so far from the environment. Here, we show that Pseudozyma spp. yeasts, which are common in the phyllosphere and are easily isolated from plant surfaces, displayed strong degradation activity on films made from poly-butylene succinate or poly-butylene succinate-co-adipate. Strains of P. antarctica isolated from leaves and husks of paddy rice displayed strong degradation activity on these films at 30°C. The type strain, P. antarctica JCM 10317, and Pseudozyma spp. strains from phyllosphere secreted a biodegradable plastic-degrading enzyme with a molecular mass of about 22 kDa. Reliable source of biodegradable plastic-degrading microorganisms are now in our hands.

Degradation of biodegradable plastic mulch films in soil environment by phylloplane fungi isolated from gramineous plants

To improve the biodegradation of biodegradable plastic (BP) mulch films, 1227 fungal strains were isolated from plant surface (phylloplane) and evaluated for BP-degrading ability. Among them, B47-9 a strain isolated from the leaf surface of barley showed the strongest ability to degrade poly-(butylene succinate-co-butylene adipate) (PBSA) and poly-(butylene succinate) (PBS) films. The strain grew on the surface of soil-mounted BP films, produced breaks along the direction of hyphal growth indicated that it secreted a BP-degrading enzyme, and has directly contributing to accelerating the degradation of film. Treatment with the culture filtrate decomposed 91.2 wt%, 23.7 wt%, and 14.6 wt% of PBSA, PBS, and commercially available BP polymer blended mulch film, respectively, on unsterlized soil within 6 days. The PCR-DGGE analysis of the transition of soil microbial community during film degradation revealed that the process was accompanied with drastic changes in the population of soil fungi and Acantamoeba spp., as well as the growth of inoculated strain B47-9. It has a potential for application in the development of an effective method for accelerating degradation of used plastics under actual field conditions.

Xylose induces the phyllosphere yeast Pseudozyma antarctica to produce a cutinase-like enzyme which efficiently degrades biodegradable plastics

There is a need to speed up the degradation of used agricultural mulch films that are made of biodegradable plastics (BPs) in the field. Treating them with BP-degrading enzymes could be a solution to this problem. A cutinase-like enzyme of yeast Pseudozyma antarctica (PaE) has wide specificity of BPs degradation, but needs to be produced efficiently. Here we report that the production of PaE by P. antarctica can be increased by using xylose as carbon source. BP-degradation activity was analyzed using a polybutylene succinate-co-adipate (PBSA) emulsion as the substrate. Strain P. antarctica GB-4(1)W was found to be the best PaE producer among the tested strains. Using a 5-L jar fermentor with xylose fed-batch cultivation, high PaE productivity could be maintained and about 21 U/ml of PaE was obtained in 120 h. This amount was 100 times higher than the amount that we obtained previously (0.21 U/ml by flask cultivation using glycerol as carbon source). Under repeated xylose fed-batch cultivation with 24 h intervals, the maximum PaE production rate (0.34 U/ml/h) was maintained and the highest PaE productivity (28,000 U/2 L/d) was repeatedly obtained for 7 intervals. The activity of filtered jar-culture (crude PaE) was stable over 12 weeks at 4°C. Commercially available BP mulch films (20 μm thickness, cut into 1-cm-squares) were completely degraded by submerging them in crude PaE (2 U/ml) at 30°C in 24 h. These results indicated that concentrated PaE can rapidly degrade the strength of BP mulch films in the field so that they do not interfere with plowing.

Production of a biodegradable plastic-degrading enzyme from cheese whey by the phyllosphere yeast Pseudozyma antarctica GB-4(1)W

Cheese whey is a by-product of cheese production and has high concentrations of lactose (about 5%) and other nutrients. Pseudozyma antarctica produces a unique cutinase-like enzyme, named PaE, that efficiently degrades biodegradable plastics. A previous study showed that a combination of 1% oil and 0.5% lactose increased cutinase-like enzyme production by another species of yeast. In this study, to produce PaE from cheese whey, we investigated the effects of soybean oil on PaE production (expressed as biodegradable plastic-degrading activity) by P. antarctica growing on lactose or cheese whey. In flask cultures, the final PaE activity was only 0.03 U/ml when soybean oil was used as the sole carbon source, but increased to 1.79 U/ml when a limited amount of soybean oil (under 0.5%) was combined with a relatively high concentration of lactose (6%). Using a 5-L jar fermentor with lactose fed-batch cultivation and periodic soybean oil addition, about 14.6 U/ml of PaE was obtained after 5 days of cultivation. When the lactose was replaced with cheese whey, PaE production was 10.8 U/ml after 3 days of cultivation.

Enhancement of Biodegradable Plastic-degrading Enzyme Production from Paraphoma-like Fungus, Strain B47-9

To improve the productivity of Paraphoma-like fungal strain B47-9 for biodegradable plastic (BP)-degrading enzyme (PCLE), the optimal concentration of emulsified poly(butylene succinate-co-adipate) (PBSA) in the medium was determined. Emulsified PBSA was consumed as a sole carbon source and an inducer of PCLE production by strain B47-9. Among the various concentrations of emulsified PBSA [0.09-0.9% (w/v)] used in flask cultivation, 0.27% yielded the maximum enzyme activity within a short cultivation period. To evaluate the residual concentration of emulsified PBSA in culture, emulsified PBSA in aliquots of culture supernatant was digested in vitro, and the concentration of released monomerised succinic acid was determined. Regardless of the initial concentration of emulsified PBSA in medium, PCLE activity was detected after residual succinic acid decreased below 0.04 mg/mL in culture broth. Jarfermentation was performed at a 0.27% PBSA concentration. Among the various airflow rates tested, 1 LPM resulted in a PCLE production rate of 1.0 U/mL/day. The enzyme activity in the resulting culture filtrate (4.2 U/2 mL) was shown to degrade commercial BP films (1 × 1 cm, 20 µm thickness) within 8 hours.

Draft Genome Sequence of the Fungus Paraphoma sp. B47-9, a Producer of a Biodegradable Plastic–Degrading Enzyme

ABSTRACT Paraphoma sp. B47-9 is a producer of a biodegradable plastic–degrading enzyme. Here, we report the draft genome sequence of this strain. The draft genome assembly has a size of 39.3 Mb with a GC content of 52.4% and consists of 185 scaffolds.