Yukiko Shinozaki

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.

Ethanol production from ensiled rice straw and whole-crop silage by the simultaneous enzymatic saccharification and fermentation process

Silage production from rice straw and whole-plant forage paddy rice is increasing in Japan because of decrease in rice consumption. One potential use for this silage is bioethanol production. In this study, we analyzed the effectiveness of three different commercially available cellulases at saccharification of sun-dried rice straw, ensiled rice straw, and rice whole-crop silage (WCS). Furthermore, the ethanol productivity of the simultaneous saccharification and ethanol fermentation process (SSF) from the same plant substrates was analyzed. Among the three kinds of cellulases tested (Novozymes NS50013, Genencor GC220, and Acremonium cellulase), Acremonium cellulase showed the highest ethanol production for the three plant substrates, and the WCS produced the highest ethanol level. Analysis of the enzymatic degradation activity of the cellulases revealed that Acremonium cellulase contained remarkably high glucoamylase and pectinase side activities relative to the other cellulase preparations. The addition of glucoamylase and pectinase to the other two cellulases significantly increased ethanol productivity to levels observed for the Acremonium cellulase preparation, which showed little enhanced performance with the addition of the same enzymes. Finally, we tested whether milling and sterilization had an effect on ethanol production and found that sterilized silage produced higher ethanol levels but that the milling process had no significant effect. These results show that (i) silage made from whole-plant rice can be used for bioethanol production and (ii) the proper selection and combination of commercially available enzymes can make SSF more cost efficient by removing the need for a pre-treatment step.

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.

Enzymatic degradation of polyester films by a cutinase-like enzyme from Pseudozyma antarctica: surface plasmon resonance and atomic force microscopy study

Enzymatic degradation of polyester films by a cutinase-like enzyme from Pseudozyma antarctica JCM10317 (PaE) was analyzed by surface plasmon resonance (SPR). The adsorption of PaE and the degradation rate for polyester films were quantitatively monitored by a positive and negative SPR signal shifts, respectively. The decrease in SPR signal and the erosion depth of amorphous poly(l-lactide) (a-PLLA) film measured by atomic force microscopy (AFM) had a linear relationship, and the weight loss was estimated from the AFM data combined with a density of a-PLLA film. Furthermore, SPR sensorgrams for various polyester films showed that degradation rate of poly(ε-caprolactone) and poly(butylene succinate-co-adipate) which contain C6 units was higher than that of other polyesters such as poly(butylene succinate) and a-PLLA. These results suggest that C6 is the preferred chain length as substrates for PaE.

The microbial community in a 2,4-dinitrophenol-digesting reactor as revealed by 16S rDNA gene analysis.

The microbial community of a 2,4-dinitrophenol-digesting reactor was investigated using different molecular biological techniques based on 16S rDNA gene sequences. A PCR-denaturing gradient gel electrophoresis (DGGE) analysis of the bacterial community in the reactor showed that one strong and five minor bands were observed in the DGGE profile. The results of excising and sequencing DGGE bands suggested that members of Rhodococcus, Nocardioides, and Nitrospira species were present in the reactor. Partial sequencing of cloned 16S rDNAs revealed diversity among the six main divisions--the alpha, delta subclasses of Proteobacteria, Nitrospira, Cytophagal Flexibacter/Bacteroides, Verrucomicrobia, and Actinobacteria--in the reactor. Two cloned sequence types were not closely affiliated with any described bacterial divisions. The isolation and phylogenetic analysis of 2,4-DNP-degrading bacteria from the reactor revealed that isolated strains were classified into two types of bacteria having different 16S rDNA sequences. One of these strain types was identified as a relative of Rhodococcus koreensis, and the other was identified as a relative of Nocardioides simplex FJ21-A.

Difference in Degrading p-Nitrophenol between Indigenous Bacteria in a Reactor

p-Nitrophenol (PNP) -degrading bacteria were isolated from a reactor using a mineral salt medium containing a low and high PNP concentration. We isolated two bacterial species, Pseudomonas sp. YTK17 and Rhodococcus opacus YTK32, that utilize PNP as their sole source of carbon and energy. These strains exhibited differences in PNP degradation activity in relation to PNP concentration. Strain YTK17 showed a high level of degradation following pre-exposure to a low PNP concentration, whereas strain YTK32 required a relatively high PNP concentration for degradation to occur. These results indicated that phylogenetically and physiologically different types of PNP-degrading bacteria coexisted in a reactor.

Inhibition of matrix metalloproteinase-2 activity by siderophores of Pseudomonas species.

To obtain a novel matrix metalloproteinase (MMP) inhibitor produced by bacteria, we have focused on the chelating activity of siderophores. Several siderophore-producing bacteria were isolated from soil using chrome azurol S agar plates and then the effect of siderophores on MMP-2 activity was assayed by gelatin zymography. The results showed that partially purified siderophores from ten isolated strains inhibited MMP-2 activity. Among these strains, two were non-fluorescent and eight were fluorescent Pseudomonas species. From these eight strains, pyoverdine-type siderophores were detected. The Zn2+-chelating activity of these siderophores correlated with the inhibition of MMP-2 activity. Therefore, it is considered that siderophores such as pyoverdines inhibit MMP-2 activity by chelating Zn2+ on the active site of MMP-2.

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.

Rapid and simple colorimetric assay for detecting the enzymatic degradation of biodegradable plastic films

We developed a rapid and simple method for evaluating the degradation of solid biodegradable plastics (BPs). Dye-containing BP films were used as substrates and the release of dye caused by the degradation of BPs was confirmed by a color change in the enzyme solution after a reaction time of 24 h.

Silage produces biofuel for local consumption

BackgroundIn the normal process of bioethanol production, biomass is transported to integrated large factories for degradation to sugar, fermentation, and recovery of ethanol by distillation. Biomass nutrient loss occurs during preservation and degradation. Our aim was to develop a decentralized ethanol production system appropriate for farm or co-operative level production that uses a solid-state fermentation method for producing bio-ethanol from whole crops, provides cattle feed, and produces no wastes. The idea is to incorporate traditional silage methods with simultaneous saccharification and fermentation. Harvested, fresh biomass is ensiled with biomass-degrading enzymes and yeast. Multiple parallel reactions for biomass degradation and ethanol and lactic acid production are induced in solid culture in hermetically sealed containers at a ranch. After fermentation, ethanol is collected on site from the vapor from heated fermented products.ResultsThe parallel reactions of simultaneous saccharification and fermentation were induced efficiently in the model fermentation system. In a laboratory-scale feasibility study of the process, 250 g of freshly harvested forage rice with 62% moisture was treated with 0.86 filter paper units/g dry matter (DM) of cellulase and 0.32 U/g DM of glucoamylase. After 20 days of incubation at 28°C, 6.4 wt.% of ethanol in fresh matter (equivalent to 169 g/kg DM) was produced. When the 46 wt.% moisture was gathered as vapor from the fermented product, 74% of the produced ethanol was collected. Organic cellular contents (such as the amylase and pronase degradable fractions) were decreased by 63% and organic cell wall (fiber) content by 7% compared to silage prepared from the same material.ConclusionsWe confirmed that efficient ethanol production is induced in nonsterilized whole rice plants in a laboratory-scale solid-state fermentation system. For practical use of the method, further study is needed to scale-up the fermentation volume, develop an efficient ethanol recovery method, and evaluate the fermentation residue as an actual cattle feed.