Biodiesel-contaminated wastewater was used to screen for PHAs-producing bacteria by using crude glycerol as the sole carbon source. A gram-negative THA_AIK7 isolate was chosen as a potential PHAs producer. The 16S rRNA phylogeny indicated that THA_AIK7 isolate is a member of Novosphingobium genus which is supported by a bootstrap percentage of 100% with Novosphingobium capsulatum. The 1,487 bp of 16S rRNA gene sequence of THA_AIK7 isolate has been deposited in the GenBank database under the accession number HM031593. Polymer content of 45% cell dry weight was achieved in 72 h with maximum product yield coefficient of 0.29 g PHAs g⁻¹ glycerol. Transmission electron micrograph results exhibited the PHAs granules accumulated inside the bacterial cell. PHAs polymer production in mineral salt media supplemented with 2% (w/v) of crude glycerol at initial pH 7 was extracted by the sodium hypochlorite method. Polymer film spectrographs from Nuclear magnetic resonance displayed a pattern of signal virtually identical to spectra of commercial PHB. Thermal analysis by Differential scanning calorimeter showed a melting temperature at 179°C. Molecular weight analysis by Gel permeation chromatography showed two main peaks of 133,000 and 700 g mol⁻¹ with weight-average molecular weight value of 23,800 and number-average molecular weight value of 755. Endotoxinfree of PHAs polymer was preliminarily assessed by a negative result of the gel-clot formation, Pyrotell® Single test vial, at sensitivity of 0.25 EU ml⁻¹. To our knowledge, this is the first reported test of endotoxin-free PHAs naturally produced from gram-negative bacteria which could be used for biomedical application.

Different sources of wastewater and soil were used to screen for PHA-producing bacteria using biodiesel-derived waste glycerol as a sole carbon source by the Nile red staining method together with polymer determination. Twelve out of twenty-six isolates from biodiesel-contaminated wastewater consortium were screened for their PHA accumulation ability by cultivation in mineral salt medium supplemented with waste glycerol. The AIK7 isolate was chosen as a potential PHA producer. The PHA production on waste glycerol was examined using pure glycerol as a control substrate. The PHA content of AIK7 isolate cultivated in 10 g/L glycerol could reach 35% cell dry weight in 72 hours from waste glycerol and 33% cell dry weight in 120 hours from pure glycerol cultivation. It can be seen that at this content of waste glycerol, AIK7 isolate is effectively capable of biotransforming glycerol into polymer from low-grade glycerol.

Little information is available regarding the effectiveness of water disinfection by CO(2) at low pressure. The aim of this study was to evaluate the use of high levels of dissolved CO(2) at 0.3-0.6 MPa for the inactivation of microorganisms. Bacteriophage T4 was chosen as the model virus and Escherichia coli was selected as the representative bacterium. The results of the study showed a highly effective log inactivation of E. coli and bacteriophage T4 at low and medium initial concentrations by high levels of dissolved CO(2) at 0.3 MPa with a treatment time of 20 min. When the pressure was increased to 0.6 MPa, inactivation of both microorganisms at high initial concentrations was improved to different extents. Neither pressurized air nor O(2) effectively inactivated both E. coli and bacteriophage T4. The pH was not a key factor affecting the inactivation process by this method. The results of scanning electron microscopy of E. coli and transmission electron microscopy of bacteriophage T4 suggested that "CO(2) uptake at high pressure and bursting of cells by depressurization" were the main reasons for lethal effect on microorganisms. This technology has potential for application in the disinfection of water, wastewater, and liquid food in the future.

We developed a system with high levels of dissolved CO2 for water disinfection. Bacteriophages MS2, Qbeta and phiX174 were selected as the inactivation targets. A relatively mild inactivation effect was observed on MS2 and Qbeta at different initial concentrations of dissolved CO2 at 0.3 MPa in 20-30 min. When the pressure was increased to 0.6 MPa, the inactivation of MS2 and Qbeta was differentially improved. However, this system was less effective for the inactivation of phiX174. The capsid surface property is a probable reason for the low inactivation of phiX174. The pH was not a key factor in the inactivation of bacteriophages; moreover, the results obtained using alternative gases (pressurized air and O2) indicated that only CO2 inactivated these bacteriophages. A comparison between the results of real time polymerase chain reaction (RT-PCR) and plaque assay showed that some RNA moved out from the capsid after treatment. Capsid damage by CO2 expansion was the likely mechanism of inactivation with our method.

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