Degradation of Nitriles by Mixed Biofilms of Nitrile-Hydrolyzing Bacteria in Submerged Packed-Bed Reactor.

Degradation of nitriles by mixed biofilms of nitrile-hydrolyzing bacteria Alcaligenes faecalis 2 and Rhodococcus ruber gt 1 grown on basalt and carbon carriers, in a submerged packed-bed reactor was studied. It was shown the formation of a massive mixed biofilm of Al. faecalis 2 and R. ruber gt 1 and the effective removal of nitriles and products of their degradation from the reaction medium. After the accumulation of carboxylic acid and some of the unprocessed substrate, the system adapts to 600-1000 h of biofilter operation, which is expressed in a decrease in the content of substrate and reaction products in the medium. The rate of acetonitrile and acrylonitrile utilization was 0.072-0.086 and 0.039-0.215 g/h, respectively, and acrylonitrile utilization with maximum rate was realized by a mixed biofilm on carbon fibers. Biofilms grown on mixed fibers in a "sandwich"-type reactor had the best characteristics for the transformation of aceto- and acrylonitrile (removal capacity of 99.6-99.9%, nitrile utilization rate of 0.080-0.095 g/h). Biofilms grown on basalt fiber with a diameter of 4-12 µm are also well suited for the degradation of acetonitrile (removal capacity of 100%, nitrile utilization rate of 0.086 g/h). The results of metagenomic analysis showed the resistance of Al. faecalis 2 and R. ruber gt 1 mixed biofilms against leaching from a biofilter and to competitive growth in an open system, indicating the advantages of biofilms over homogeneous biomass for wastewater treatment from nitrile compounds. Biofilms of two species of nitrile hydrolyzing bacteria on basalt and carbon fibers effectively purify water from nitriles in a submerged packed-bed reactor. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-022-01030-z.

Influence of Acrylamide on Energy Status and Survival of Bacteria of Different Systematic Groups.

We studied the effect of acrylamide on the content of intracellular ATP in the cells of bacteria of the genera Rhodococcus and Alcaligenes, the luminescence of the genetically engineered strain Escherichia coli K12 TG1 (pXen7), and the survival of bacteria of various systematic groups. According to the level of decrease in the concentration of intracellular ATP, it was found that the strain with lower amidase activity (R. erythropolis 6-21) and Gram-negative proteobacteria A. faecalis 2 were the most sensitive to acrylamide after a 20-min exposure, while the strain R. ruber gt 1 was stable, having a high nitrile hydratase activity in combination with a low amidase activity. EC50 of acrylamide for 2 h was 7.1 g/L for E. coli K12 TG1 (pXen7). Acrylamide at a concentration of 10-20 mM added to the culture medium led to a slight decrease in the number of CFUs of Rhodococcus, A. faecalis 2, and E. coli compared to the control. At an acrylamide concentration of 250 mM, from 0.016 to 0.116% of viable bacterial cells remained, and a solution of 500 mM and higher inhibited the growth of the majority of the studied strains. The results confirm that acrylamide is much less toxic to prokaryotes than to eukaryotes.

Stereoselective biotransformation of phenylglycine nitrile by heterogeneous biocatalyst based on immobilized bacterial cells and enzyme preparation.

We studied the effect of a heterogeneous environment on the stereoselectivity of transformation of racemic phenylglycine nitrile. Immobilized biocatalysts were prepared by adhesion of Pseudomonas fluorescens C2 cells on carbon-containing supports and covalent crosslinking of nitrile hydratase and amidase of Rhodococcus rhodochrous 4-1 to activated chitosan as well as by the method of cross-linked aggregates. At a reaction duration of 20 h, the ratio of phenylglycine stereoisomers changes depending on the presence of support in medium. The highest optical purity of the product (enantiomeric excess of L-phenylglycine solution, 98%) is achieved when enzyme aggregates of nitrile hydratase and amidase cross-linked with 0.1% glutaraldehyde are used as a biocatalyst.

[Catalytic and Stereoselective Properties of the Immobilized Amidase of Rhodococcus rhodochrous 4-1].

The amidase of Rhodococcus rhodochrous 4-1 was immobilized by covalent attachment to activated chitosan by physical sorption on carbon adsorbents and by the formation of crosslinked aggregates in the absence of carrier material. Comparative analysis of particular catalytic properties of the free and chitosan-immobilized amidase was performed. It was shown that the enzyme retained 50-60% of its initial activity after covalent immobilization on chitosan and was characterized by increased temperature stability as compared to soluble amidase. Moreover, the immobilized enzyme retained more than 20% of its activity after five 24-h cycles of acrylamide deamination. The effects of different types of immobilization on amidase stereose-lective properties were studied by the model reaction of racemic lactamide hydrolysis to D-lactic acid and L-lactic acid. It was shown that crosslinked amidase aggregates possessed high D-stereoselectivity (up to 77-94%). The immobilized enzyme showed the highest enantioselectivity at 60 degrees C.

[Activated Sludge Bacteria Transforming Cyanopyridines and Amides of Pyridinecarboxylic Acids].

Species diversity of bacteria from the activated sludge of Perm biological waste treatment facilities capable of transformation of cyanopyridines and amides of pyridinecarboxylic acids was investigated. Enrichment cultures in mineral media with 3-cyanopyridine as the sole carbon and nitrogen source were used to obtain 32 clones of gram-negative heterotrophic bacteria exhibiting moderate growth on solid and liquid media with 3- and 4-cyanopyridine. Sequencing of the 16S rRNA gene fragments revealed that the clones with homology of at least 99% belonged to the genera Acinetobacte, Alcaligenes, Delftia, Ochrobactrum, Pseudomonas, Stenotrophomonas, and Xanthobacter. PCR analysis showed that 13 out of 32 isolates contained the sequences (-1070 bp) homologous to the nitrilase genes reported previously in Alcaligenes faecalis JM3 (GenBank, D13419.1). Nine clones were capable of nitrile and amide transformation in minimal salt medium. Acinetobacter sp. 11 h and Alcaligenes sp. osv transformed 3-cyanopyridine to nicotinamide, while most of the clones possessed amidase activity (0.5 to 46.3 mmol/(g h) for acetamide and 0.1 to 5.6 mmol/(g h) for nicotinamide). Nicotinamide utilization by strain A. faecalis 2 was shown to result in excretion of a secondary metabolite, which was identified as dodecyl acrylate at 91% probability.