Yuka Yajima

Poly-3-hydroxybutyrate (PHB) production from alkylphenols, mono and poly-aromatic hydrocarbons using Bacillus sp. CYR1: A new strategy for wealth from waste

In the present study five different types of alkylphenols, each of the two different types of mono and poly-aromatic hydrocarbons were selected for degradation, and conversion into poly-3-hydroxybutyrate (PHB) using the Bacillus sp. CYR1. Strain CYR1 showed growth with various toxic organic compounds. Degradation pattern of all the organic compounds at 100 mg/l concentration with or without addition of tween-80 were analyzed using high pressure liquid chromatography (HPLC). Strain CYR1 showed good removal of compounds in the presence of tween-80 within 3 days, but it took 6 days without addition of tween-80. Strain CYR1 showed highest PHB production with phenol (51 ± 5%), naphthalene (42 ± 4%), 4-chlorophenol (32 ± 3%) and 4-nonylphenol (29 ± 3%). The functional groups, structure, and thermal properties of the produced PHB were analyzed. These results denoted that the strain Bacillus sp. CYR1 can be used for conversion of different toxic compounds persistent in wastewaters into useable biological polyesters.

Production of poly-3-hydroxybutyrate (P3HB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) P(3HB-co-3HV) from synthetic wastewater using Hydrogenophaga palleronii

In the present study, synthetic wastewater (SW) was used for production of poly-3-hydroxybutyrate (P3HB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) P(3HB-co-3HV) using the bacteria Hydrogenophaga palleronii. SW at various volatile fatty acids concentrations (5-60g/l) was evaluated for the growth and biopolymer production using H. palleronii. Substrate degradation was analyzed using total organic carbon (TOC) analyzer and high pressure liquid chromatography (HPLC). H. palleronii showed highest and lowest removal of TOC at 5g/l (88±4%) and 60g/l (15±6%) respectively. Among all the concentrations evaluated, bacteria showed highest biopolymer production with 20g/l (63±5%), followed by 30g/l (58±3%) and 40g/l (56±2%). Lowest biopolymer production was observed at 5g/l concentration (21±3%). Structure, molecular weight, and thermal properties of the produced biopolymer were analyzed. These results denoted that the strain H. palleronii can be used for degradation of high concentration of volatile fatty acids persistent in wastewaters and their subsequent conversion into useable biopolymers.

Bio-Augmentation of Cupriavidus sp. CY-1 into 2,4-D Contaminated Soil: Microbial Community Analysis by Culture Dependent and Independent Techniques

In the present study, a 2,4-dichlorophenoxyacetic acid (2,4-D) degrading bacterial strain CY-1 was isolated from the forest soil. Based on physiological, biochemical and 16S rRNA gene sequence analysis it was identified as Cupriavidus sp. CY-1. Further 2,4-D degradation experiments at different concentrations (200 to 800 mg l-1) were carried out using CY-1. Effect of NaCl and KNO3 on 2,4-D degradation was also evaluated. Degradation of 2,4-D and the metabolites produced during degradation process were analyzed using high pressure liquid chromatography (HPLC) and GC-MS respectively. The amount of chloride ions produced during the 2,4-D degradation were analyzed by Ion chromatography (IC) and it is stoichiometric with 2,4-D dechlorination. Furthermore two different types of soils collected from two different sources were used for 2,4-D degradation studies. The isolated strain CY-1 was bio-augmented into 2,4-D contaminated soils to analyze its degradation ability. Culture independent methods like denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP), and culture dependent methods like colony forming units (CFU) and most probable number (MPN) were used to analyze the survivability of strain CY-1 in contaminated soil. Results of T-RFLP were coincident with the DGGE analysis. From the DGGE, T-RFLP, MPN and HPLC results it was concluded that strain CY-1 effectively degraded 2,4-D without disturbing the ecosystem of soil indigenous microorganisms.

Polyhydroxyalkanoates (PHA) production from synthetic waste using Pseudomonas pseudoflava: PHA synthase enzyme activity analysis from P. pseudoflava and P. palleronii

Synthetic wastewater (SW) at various carbon concentrations (5-60g/l) were evaluated for polyhydroxyalkanoates (PHA) production using the bacteria Pseudomonas pseudoflava. Bacteria showed highest PHA production with 20g/l (57±5%), and highest carbon removal at 5g/l (74±6%) concentrations respectively. Structure, molecular weight, and thermal properties of the produced PHA were evaluated using various analytical techniques. Bacteria produced homo-polymer [poly-3-hydroxybutyrate (P3HB)] when only acetate was used as carbon source; and it produced co-polymer [poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) P(3HB-co-3HV)] by addition of co-substrate propionate. PHA synthase, the enzyme which produce PHA was extracted from two bacterial strains i.e., P. pseudoflava and P. palleronii and its molecular weight was analysed using SDS-PAGE. Protein concentration, and PHA synthase enzyme activity of P. pseudoflava and P. palleronii was carried out using spectrophotometer. Results denoted that P. pseudoflava can be used for degradation of organic carbon persistent in wastewaters and their subsequent conversion into PHA.

Degradation and conversion of toxic compounds into useful bioplastics by Cupriavidus sp. CY-1: relative expression of the PhaC gene under phenol and nitrogen stress

In this study different types of toxic compounds, i.e., alkylphenols, mono and poly-aromatic hydrocarbons were converted into polyhydroxybutyrate (PHB) using the isolated bacteria Cupriavidus sp. CY-1. The influence of Tween-80 on the toxic compound degradation ability of CY-1 was analyzed using high pressure liquid chromatography. Among all the compounds, CY-1 showed the highest removal of naphthalene (100 ± 6%), followed by phenol (96 ± 7%), and the lowest removal of alkylphenols without Tween-80 addition. However, Tween-80 addition enhanced the degradation capacity of CY-1, and showed the highest removal of 4-tertiary-butylphenol (74 ± 5%), followed by phenol (69 ± 5%), 4-chlorophenol (59 ± 3%), 4-tertiary-octylphenol (53 ± 5%), and naphthalene (48 ± 5%). Further experiments were carried out for conversion of toxic compounds into PHB. CY-1 grown with phenol (48 ± 6%) and naphthalene (42 ± 4%) showed the highest PHB production. The functional groups, structure and thermal properties of the produced PHB were analyzed. In addition the expression of the PhaC gene was quantified at the transcriptional level through real time quantitative PCR. The results showed up-regulation of the PhaC gene in the presence of phenol, and up and down-regulations in the presence of nitrogen. The maximum PhaC transcript expression was 5.37 folds at 100 mg l−1 nitrogen concentration.

Improvement of Saccharification and Fermentation by Removal of Endogenous Chemicals from Pretreated Lignocellulosic Biomass (1). Effect of Ion-Exchange Resin Treatment

The effects of removal of enzyme inhibitors on saccharification of lignocellulosic biomass and fermentation of their hydrolysate were examined with the aim of improving bioethanol production. Hydrothermal-mechanochemical treated woody powders lost their cell structures and lignin was widely and randomly distributed in treated woody powders. Enzymatic reaction resulted in the formation of many cracks and pores on the woody powder surface, and these cavities extended to the interior of woody powders. The concentrations of 3 kinds of phenolic enzyme inhibitors (gallic, tannic and trimesic acids) in the supernatant of the reaction mixture gradually increased during the enzymatic hydrolysis. Cracks and pores of hydrolyzed woody powders were probably routes of inhibitor release to the reaction mixture. Enzymatic saccharification was improved 1.4-fold by ion-exchange resin treatment after 24 hours of hydrolysis, and inhibition of cellulase activity was decreased by 60-95% compared to the control. Ethanol yield was improved 2.2-fold and lactic acid by 1.3-fold compared to corresponding yields using normal hydrolysates.

Typhula cf. subvariabilis, new snow mould in Antarctica

ABSTRACT We collected snow blight of moss, Polytrichum juniperinum on King George Island, maritime Antarctica. Host died in a circle of about 10–30 cm after snow melts. Clamp connected hyphae and no sclerotia were observed on tip of host leaves. DNA sequence of ITS region from moss symptoms were perfectly matched with fruit bodies of Typhula sp. on Macquarie Island in the maritime Antarctica and high homology with Typhula cf. subvariabilis from Iran. Therefore, we suggested that T. cf. subvariabilis caused snow blight on moss in Antarctica, and this is first record of Typhula snow blight in Southern Hemisphere. These results also suggested that fungi in same genera gained similar ecological niche in both Polar Regions.

Region of Interest analysis using mass spectrometry imaging of mitochondrial and sarcomeric proteins in acute cardiac infarction tissue

Matrix-assisted laser desorption ionization image mass spectrometry (MALDI-IMS) has been developed for the identification of peptides in various tissues. The MALDI-IMS signal distribution patterns and quantification of the signal intensities of the regions of interest (ROI) with healthy regions were compared for identification of the disease specific biomarkers. We performed a new ROI analysis using the conventional t-test and data number independent Cohen’s d-value analysis. Using these techniques, we analysed heart tissues after acute myocardial infarction (AMI). As a result, IMS signals of mitochondrial adenosine triphosphate synthase alpha subunit (ATP5A), myosin-6/7(MYH6/7), aortic actin, and the myosin light chain 3 (MYL3) were identified in the infarcted region. In particular, the signals of MYH7 are significantly greater in the infarcted region using ROI analysis. ROI analysis using MALDI-IMS may be a promising technique for the identification of biomarkers for pathological studies that involve the comparison of diseased and control areas.

Biodegradation of toxic organic compounds using a newly isolated Bacillus sp. CYR2

The objective of this study was to isolate a new bacterium and investigate its ability for degradation of various toxic organic compounds. Based on 16S rRNA gene sequence and phylogenetic analysis, the isolated strain was identified as Bacillus sp. CYR2. Degradation of various toxic compounds and growth of CYR2 strain were evaluated with 2 and 4% inoculum sizes. All the experiments were conducted for 6 days, flasks were incubated at 30oC under 180 rpm. Among the 2 and 4% inoculum sizes, bacteria showed highest growth and toxic compounds degradation at 4% inoculum size. Especially, compared to 2% inoculum size, growth of the strain CYR2 at 4% inoculum size was increased by 15.1 folds with 4-secondarybutylphenol, 9.1 folds with phenol, and 5.4 folds with 4-tertiary-butylphenol. Strain CYR2 at 4% inoculum size showed highest removal of phenol (84 ± 5%), followed by 4-tertiary-butylphenol (66 ± 3%), 4-secondary-butylphenol (63 ± 5%) and 4-nonylphenol (57 ± 6%). Compared with 2% inoculum size, degradation ability of strain CYR2 with 4% inoculum size was enhanced by 3.45 times with 4-tertiary-octylphenol, and 2.53 times with 4-tertiarybutylphenol. Our results indicated that the newly isolated Bacillus sp. CYR2 can be used for in situ bioremediation of phenol and alkylphenols contaminated water.