Slawomir Ciesielski

Municipal landfill leachate nitrification in RBC biofilm - process efficiency and molecular analysis of microbial structure.

Landfill leachates were treated using a two-stage rotating biological contactor (RBC). At an ammonium load of 1.92gN-NH(4)/m(2)d, complete nitrification was obtained at the first stage; however, at a higher load (3.6gN-NH(4)/m(2)d) a two-stage system was needed to obtain complete nitrification. A further increase in the ammonium load to 4.79gN-NH(4)/m(2)d and 6.63gN-NH(4)/m(2)d caused a decrease of overall nitrification efficiency to 74.4% and 71.6%, respectively. Randomly amplified DNA analysis of bacteria present in the RBC revealed that the bacterial community differed over time. Regardless of the ammonium load, Nitrosomonas europaea and Nitrosomonas eutropha were the dominating species. The performed analyses provide a clear picture of bacterial population changes in response to changes of ammonium load during landfill leachate treatment.

Microcystin-LR induced apoptosis and mRNA expression of p53 and cdkn1a in liver of whitefish (Coregonus lavaretus L.).

There is growing evidence that adverse effects of microcystin-LR (MC-LR) are closely related to oxidative stress processes, free radicals and DNA damage, and involve major gene transcript changes. This study, utilizing gene expression analysis and plasma chemistries was the first to measure the effects of MC-LR in whitefish (Coregonus lavaretus L.), a feasible organism for pollution monitoring in aquatic systems. Fish were injected with different concentrations of MC-LR (0, 10 and 100 microg/kg of body weight) and then sacrificed at either 0, 8, 24, 48 or 72 h later, and their liver tissue were harvested for detailed investigation. Specifically, we were interested whether MC-LR is capable of: (i) modulating expression of two genes, tumor suppressor gene p53 and cdkn1a, p53 direct transcription target, and (ii) inducing apoptosis in whitefish liver. To study these effects, we developed a real-time qPCR assays useful for measuring both p53 and cdkn1a gene transcript levels in liver. To obtain necessary information for the study, either full-length p53 cDNA of whitefish (Wf-p53) was determined, using molecular cloning and rapid amplification of cDNA ends (RACE), or as for Wf-cdkn1a, specific primers were designed based on highly conserved regions of cdkn1a in fish. The Wf-p53 was found to share the same characteristics with a known p53 mRNA sequence of other vertebrates. Whitefish p53 amino acid sequence showed a high degree of homology with the sequences from fishes, amphibians, and mammals. The injection study showed that MC-LR at a higher dose, i.e. 100 microg/kg body weight, up-regulated expression of p53 and cdkn1a genes in whitefish liver, as reflected by the continuous increase in their mRNA levels through the whole experiment. Furthermore, DNA fragmentation was observed in liver cells of whitefish after 24h of exposure to MC-LR (100 microg/kg) that suggests the possibility of apoptosis. Finally, the study confirmed previous observations of severe injury of the liver and loss of normal organ functions as revealed by elevated levels of blood AspAT, AlaAT, and hepatosomatic index (HSI).

Molecular detection and diversity of medium-chain-length polyhydroxyalkanoates-producing bacteria enriched from activated sludge

Aims:  Knowledge of the species composition of complex bacterial communities is still very limited. The main objectives of this study were to identify medium‐chain‐length polyhydroxyalkanoates (mcl‐PHAs)‐producing bacteria from activated sludge fed with methanol as well as to characterize their PHA operon.

Saponified waste palm oil as an attractive renewable resource for mcl-polyhydroxyalkanoate synthesis

The synthesis of mcl-polyhydroxyalkanoates (mcl-PHAs) by Pseudomonas sp. Gl01 using saponified waste palm oil (SWPO) as the sole carbon source was investigated. It was shown that the analyzed strain accumulated biopolymers during the growth phase. Up to 43% of mcl-PHAs at 17 h were produced, when Pseudomonas sp. Gl01 was grown for 48 h in a biofermentor containing 15 g/l of SWPO. The results clearly indicate that lower carbon source supplementation decreased mcl-PHAs production. Furthermore, the obtained results confirmed that nitrogen limitation is unnecessary for the stimulation of biopolymer synthesis. Additionally, in the present study the mcl-PHAs biosynthesis at the molecular level was also investigated. Using the RT real-time PCR technique, the expression of PHA synthase genes (phaC1 and phaC2) and PHA depolymerase gene (phaZ) was analyzed. The data suggest that the phaZ gene could be transcribed together with the phaC1 or phaC2 gene, which means that PHA synthesis and degradation followed simultaneously. Depending on the oily substrate concentration a wide range of repeat-unit components were observed. The purified polymers consisted of monomers ranging from C₆ to C₁₆. Moreover, a differential scanning calorimetric and gel permeation chromatography analysis confirmed that the extracted mcl-PHAs are elastomers with useful physical and chemical properties.

tet genes as indicators of changes in the water environment: Relationships between culture-dependent and culture-independent approaches

The aim of this study was to identify tetracycline resistance determinants that could be used as molecular indicators of anthropogenic changes in aquatic environments. Two parallel approaches were used to examine the prevalence of tet genes: a culture-based method involving standard PCR and a method relying on quantitative PCR. The studied site was the Łyna River in Olsztyn (Poland). The culture-dependent method revealed that the concentrations of doxycycline-resistant bacteria harboring the tet(B) gene were higher in wastewater and downstream river samples than in upstream water samples. The tet(B) gene was transferred from environmental bacteria to Escherichia coli. The results generated by the culture-independent method validated statistically significant differences in tet(B) concentrations between upstream and downstream river sections, and revealed that tet(B) levels were correlated with the presence of other tetracycline resistance genes, dissolved oxygen concentrations, temperature and doxycycline concentrations in water. Our findings indicate that doxycycline-resistant bacteria, in particular E. coli harboring tet(B) or increased concentrations of tet(B), are potentially robust indicators of changes in water environments.

Removal of petroleum pollutants and monitoring of bacterial community structure in a membrane bioreactor

The long-term operational stability (159 d) in removal of organics and ammonia from synthetic wastewater was investigated. The experiment was carried out in two identical plug flow membrane bioreactors (MBR) (each with a submerged A4 Kubota membrane) operated under aerobic conditions. The vacuum distillate of a crude oil fraction in the emulsified state, which was used to model the petroleum pollutants, was added into the feed medium. The performance of biological treatment was evaluated by physicochemical analyses such as nitrogen forms, COD, and BOD. Additionally, monitoring of PAHs in the wastewaters was performed using HPLC-diode array detector. Moreover, the community structure of bacteria was analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis. The MBR treatment was very effective with reduction by more than 90% of COD and Total Organic Carbon. Nearly complete removal of petroleum originated non-polar micropollutants was observed. The influence of the highest dosage of petroleum pollutants (1000 μLL(-1)) on the bacterial community was noted.

The influence of nitrogen limitation on mcl-PHA synthesis by two newly isolated strains of Pseudomonas sp.

The nucleotide composition of key enzymes involved in medium-chain-length polyhydroxyalkanoates (mcl-PHA) synthesis was analyzed in two newly isolated strains of Pseudomonas. The isolated strains were tested for their abilities to synthesize polyhydroxyalkanoates using three different substrates as a carbon source: sodium octanoate, oleic acid, and sodium gluconate. Both analyzed strains were able to accumulate mcl-PHA in a range from 2.07 to 21.40%, which depended on the substrate used. Potential nitrogen-dependent regulation of mcl-PHA synthesis was analyzed by cell cultivation in nitrogen-limiting and non-limiting conditions. The analyzed strains demonstrated an incremental increase of mcl-PHAs in response to nitrogen starvation when oleic acid and sodium gluconate were applied as the carbon source. The transcriptional analysis showed that the induction of gene coding for PHA synthases was correlated with an increment in mcl-PHAs content. Both analyzed strains revealed differences in terms of the studied genes expression, showing a dependence on the carbon source used.

Molecular insight into activated sludge producing polyhydroxyalkanoates under aerobic–anaerobic conditions

One of the options enabling more economic production of polyhydroxyalkanoates compared to pure cultures is the application of mixed cultures. The use of a microbial community in a sequencing batch reactor has a few advantages: a simple process control, no necessity for sterile processing, and possibilities of using cheap substrates as a source of carbon. Nevertheless, while cultivation methods to achieve high PHAs biomass concentration and high productivity in wild and recombinant strains are defined, knowledge about the cultivation strategy for PHAs production by mixed culture and species composition of bacterial communities is still very limited. The main object of this study was to characterize on the molecular level the composition and activity of PHAs producing microorganism in activated sludge cultivated under oxygen limitation conditions. PHAs producers were detected using a PCR technique and the created PHA synthase gene library was analyzed by DNA sequencing. The obtained results indicate that PHAs-producers belonged to Pseudomonas sp., and possessed genes coding for mcl-PHA synthase. The kinetics of mcl-PHA synthase expression was relatively estimated using real-time PCR technology at several timepoints. Performed quantitative and qualitative analysis of total bacterial activity showed that there were differences in total activity during the process but differential expression of various groups of microorganisms examined by using DGGE was not observed.

The Diversity of Bacteria Isolated from Antarctic Freshwater Reservoirs Possessing the Ability to Produce Polyhydroxyalkanoates

The diversity of polyhydroxyalkanoates-producing bacteria in freshwater reservoirs in the Ecology Glacier foreland, Antarctica, was examined by a cultivation-dependent method. Isolated strains were analyzed phylogenetically by 16S rRNA gene sequencing, and classified as members of Alpha-, Beta-, or Gammaproteobacteria classes. Polymerase chain reaction was used to detect PHA synthase genes. Potential polyhydroxyalkanoates (PHAs) producers belonging mainly to Pseudomonas sp., and Janthinobacterium sp. were isolated from all five sampling sites, suggesting that PHA synthesis is a common bacterial feature at pioneer sites. All Pseudomonas strains had the genetic potential to synthesize medium-chain-length PHAs, whereas some isolated Janthinobacterium strains might produce short-chain-length PHAs or medium-chain-length PHAs. It is the first report revealing that Janthinobacterium species could have the potential to produce medium-chain-length PHAs.

Pulsed feeding strategy is more favorable to medium-chain-length polyhydroxyalkanoates production from waste rapeseed oil.

This article presents the results of production and characterization of medium‐chain‐length polyhydroxyalkanoates (mcl‐PHAs) using Pseudomonas sp. Gl01. Studies have been carried out to find suitable feeding strategies for mcl‐PHAs production and, for the first time, to investigate in‐depth the properties of biopolyesters obtained under controlled conditions with waste rapeseed oil as a substrate. Up to 44% mcl‐PHAs of cell dry weight was produced at 41 h of biofermentor culture by employing pulsed feeding of waste rapeseed oil. GC analysis showed a polymer composition with monomer length of C6 to C12 with C8 and C10 as the principal monomers. The monomeric structure of the extracted polyesters did not depend on the cultivation time and the feeding strategy. Molecular weight of the mcl‐PHAs was found to be ranging from 57 to 154 kDa. Thermal analyses showed the obtained mcl‐polyhydroxyalkanaotes to be semi‐crystalline biopolymer with promising thermal stability, having a glass transition temperature of −38 to −50°C.