Mikrobiologiczno-chemiczne usuwanie arsenu z zanieczyszczonych wód

INTRODUCTION The purification of arsenic contaminated waters constitutes a serious environmental challenge, since most of the available chemical and physical methods are dedicated to the selective removal of As(V), and are inefficient with regard to As(III). One innovative solution to problems linked with the low effectiveness of As(III) removal may be an application of arsenite oxidation bacteria (AOB) in technological processes. The activity of AOB contributes to the reduction of arsenic toxicity as well as may facilitate removing of As from contaminated water via the physical and chemical remediation methods. A good example of an arsenite-oxidizer is Ensifer (formerly Sinorhizobium) sp. M14. This strain carries the self-transmissible plasmid pSinA, which encodes proteins involved in resistance to arsenic, and for oxidation of As(III) to As(V). pSinA is a broad host range replicon that can be transferred to various representatives of Alpha-, Beta- and Gammaproteobacteria, in which it confers the ability to As(III) oxidation by the host cells, and increases their resistance to arsenic. Adsorption is the one of the most effective, low-cost and environmentally friendly, physico-chemical method for arsenic removal from contaminated waters. The efficiency of the adsorption method is determinate by the quality of the adsorbent, which should be characterized by high adsorption properties with regard to arsenic, short residence time as well as high chemical, physical and biological stability. Many laboratory studies have shown that natural and commonly occurring bog iron ores (BIOs) meet these requirements. They are known for their physical sorption (non-specific) and chemisorption (specific) abilities of many metals and metalloids, including arsenic, forming stable compounds with them. AIM The aim of the study performed in the frame of the PhD dissertation was (i) determination of the bioremediation potential of Ensifer (Sinorhizobium) sp. M14 strain representing AOB and (ii) investigation of the possibilities of the arsenate adsorption (as a result of the biooxidation processes) on the bog iron ores. The performed studies constituted the base to the development the versatile, low-cost and environmentally friendly biological method dedicated to arsenic removal from contaminated waters. RESULTS Analysis of the Ensifer (Sinorhizobium) sp. M14 genome Research article no.1: Genomic and biotechnological characterization of the heavy-metal resistant, arsenic-oxidizing bacterium Ensifer sp. M14. 2018. Genes 9, 379. The first stage of this study involved the obtaining of the draft genome sequence and genome-guided characterization of Ensifer (Sinorhizobium) sp. M14 in the context of its application in environmental studies [diCenzo et al., 2018]. The specific goals involved (i) phylogenetic analysis of the M14 strain, (ii) comparative genomic analysis of the M14 strain with other AOB, (iii) identification of prophage loci and putative antibiotic resistance genes in the M14 genome as well as (iv) in silico metabolic reconstruction and constraint-based modeling. It was shown, that Ensifer (Sinorhizobium) sp. M14 genome consists one chromosome (about 4.4 Mb in size), two additional large replicons (chromids or large megaplasmids - at least 1.6 Mb and 0.6 Mb in size), and the two previously reported smaller megaplasmids pSinA and pSinB (109 kb and 300 kb, respectively) [Drewniak et al., 2013; Romaniuk et al., 2017]. The M14 genome contains 6874 protein coding sequences, including hundreds not found in related strains. Based on the analysis of the sequence of the fragment 16S rRNA described in the previous papers [Drewniak et al. 2008, Drewniak et al. 2013], this strain has been classified to the Sinorhizobium genus. Detailed taxonomic analysis carried out in the frame of the doctoral thesis allowed to reclassify this strain as Ensifer, thus it was proposed to change the strain's name to Ensifer sp. M14. It was shown, that nearly all unique genes that are associated with metal resistance and arsenic oxidation are localized within the pSinA and pSinB megaplasmids. Growth simulations using Flux Balance Analysis suggested, that the M14 strain has a broad metabolic capacity, with a predicted ability to catabolize 72 carbon sources, including 25 sugars, 24 aminoacids, 7 alcohols, and 16 organic acids. Genome and antibiotic sensitivity analyses further suggested that the use of Ensifer sp. M14 in biotechnology does not pose serious biosafety risks. Kinetics of arsenite oxidation by Ensifer (Sinorhizobium) sp. M14 strain Research article no.2: Kinetics of arsenite oxidation by Sinorhizobium sp. M14 under changing environmental conditions. 2017. International Biodeterioration & Biodegradation 119: 476-485. In the second step, the growth rate of the M14 strain and arsenite oxidation efficiency in various chemical and physical conditions were investigated [Debiec et al., 2017a]. Cultures of microorganisms were performed in both batch and continuous conditions in which influence of the pH, temperature, arsenic concentration, hydraulic retention time (HRT) as well as oxygen and growth supplement concentration on the growth rate and arsenite oxidation efficiency were investigated. Analysis of the results from batch cultures showed, that the M14 strain was able to efficient arsenite oxidation in the wide spectrum of physical and chemical conditions (oxidation rate under environmental-like conditions - 0.533 mg/L/h), while increasing the temperature of the culture increases the activity of bacteria and can stimulate this process (up to 1.171 mg/L/h). The collected data also indicate, that the more intensive bacterial growth the higher efficiency of the arsenite oxidation process. Experiments performed in the continuous conditions showed, that the arsenite oxidation by the M14 strain is closely related with the HRT in the flow reactor. The longer HRT, the higher efficiency of the As(III) biooxidation. Moreover, the increasing of the oxygen, as well as yeast extract concentration (used as a growth supplement), caused additional increasing of the microorganisms activity. It was shown, that for a 5 mg/L of As(III), the minimum HRT for the M14 strain was 10 hours (As(V) concentration in culture was between 32% and 75% of the total arsenic content), while the most efficient oxidation of arsenite during 48 hours HRT was observed (87-89% As(V)). From the point of view of the later environmental studies including the increase of the scale of experiments and due to the lower concentration of arsenic in natural water than in the medium tested in the laboratory, it was found that the optimal HRT in environmental research will be 24 hours. For a 24-hour HRT in a laboratory scale, the As(V) content in the culture was 74-77%. Bog iron ores adsorption properties Research article no.3: Granulated bog iron ores as sorbents in passive (bio)remediation systems for arsenic removal. 2018. Frontiers in Chemistry 6: 54. The third step involved investigation of bog iron ores (BIOs) adsorption properties with regard to arsenic [Debiec et al., 2018]. The specific aims concerned (i) determination of the maximal adsorption capacity of BIOs in batch conditions, (ii) investigation of their actual adsorption efficiency in dynamic experiments, (iii) investigation of the chemical stability of BIOs and (iv) estimation of the possibility to re-use of loaded with arsenic BIOs to adsorption of other heavy metals (i.e. zinc). Batch sorption experiments were performed with the use of synthetic arsenic and zinc solutions, while in the dynamic sorption experiments variety of “naturally” arsenic-contaminated waters (Zloty Stok) were used, including: groundwater, surface water and settling pond water containing, approx. 0.3, 2.3 and 5.3 mg As/L, respectively. Batch sorption experiments showed, that in the presence of low concentrations of arsenic (below 50 mg/L), the sorption capacity was definitely lower than at higher concentration of impurities (above 50 mg/L). A similar relationship was noted in the case of the dynamic sorption experiments. The sorption capacity of BIOs with regard to arsenic was 0.0013 mg/g, 0.0085 mg/g and 0.0364 mg/g for contents 0.3, 2.3 and 5.3 mg As/L, respectively. It was also shown, that the sorption capacity tested under batch conditions was much higher than the one tested in flow systems. For the 5 mg/L of arsenic concentration, the batch sorption efficiency was 0.105 mg/g, while the dynamic sorption efficiency for a similar content of this element (5.3 mg/L) was more than three times lower (0.0364 mg/g). Desorption experiments showed the high chemical stability of BIOs and confirmed that As-loaded BIOs may be re-used as an adsorbent for zinc. Most of the available literature data focus on the study of the sorption properties of sorbents in the presence of high concentrations of pollutants that do not naturally occur in the environment. In this paper, the sorption efficiency using natural waters contaminated with arsenic was investigated. These studies allowed to investigate the actual sorption capacity of BIOs with regard to arsenic under environmental conditions and to determine the actual application potential of this adsorbent. Bioweathering of bog iron ores Research article no.4: The influence of thermal treatment on bioweathering and arsenic sorption capacity of a natural iron (oxyhydr)oxide-based adsorbent. 2017. Chemosphere 188: 99-109. The next step was related with the investigation of the biological stability of BIOs [Debiec et al., 2017b]. The aim of this study was to determine the effect of organic matter removal by thermal transformation (roasting) on the bioavailability of BIOs and their arsenic sorption capacity. For this purpose, the influence of bacterial growth and activity on untreated and treated BIOs, unloaded and loaded with arsenic, was studied. In these studies chemolithoautotrophic Ensifer (Sinorhizobium) sp. M14 strain as well as heterotrophic strain Pseudomonas sp. OS29R were cultured in the presence of previously prepared samples of BIOs. The performed experiments showed, that microorganisms are able to colonize BIOs, while the organic matter removal from the sorbent limits their growth. Moreover, it was shown, that in the presence of As(III)-loaded BIOs, chemolithoautotrophic bacterial activity is higher since that microbes may use arsenite as an electron donor. Microbes may also contribute to BIOs destabilization through iron and adsorbed arsenic release, however, due to the high stability of BIOs, the concentration of the desorbed elements was negligible. Despite the significant activity of microorganisms in the presence of BIOs, the desorption of both As and Fe in all experimental variants did not exceed 2% of the total content of elements in the deposit. The thermal treatment of the deposit decreased its bioavailability, and additionally, limited desorption of both arsenic and iron. Based on these results, it can be concluded, that both untreated and treated BIOs are excellent sorbents of arsenic, and thermal treatment of the deposit may prove to be beneficial for applications where microbial growth is especially intensive. Verification of the technology assumptions in the pilot-scale installation Research article no.1: Genomic and biotechnological characterization of the heavy-metal resistant, arsenic-oxidizing bacterium Ensifer sp. M14. 2018. Genes 9, 379. The final step involved the verification of the laboratory-scale experiments concern both AOB activity and adsorption efficiency of BIOs in the environmental studies. For this purpose, a novel two-stage installation for remediation of arsenic-contaminated waters was developed [diCenzo et al., 2018]. It consisted of a microbiological module, where the M14 strain oxidized As(III) to As(V) ions, followed by an adsorption module for As(V) removal using granulated bog iron ores. During a 40-day pilot-scale test in an abandoned gold mine in Zloty Stok (Poland), water leaving the microbiological module (flow rate 0.2 m3/day) generally contained approx. 2.5 mg/L of As, and dramatic decreases in total arsenic concentrations were observed after passage through the adsorption module. It was shown, that similarly as in the laboratory-scale experiments, the efficiency of arsenite biooxidation in the bioreactor was positively correlated with the quantity of microorganisms. In turn, adsorption module studies showed that the use of a series column system allowed for the significant reduction of arsenic in water, which after the treatment was below 0.1 mg/L for 12 days of the experiment. This value is an acceptable limit of arsenic in waters intended for technological purposes. CONCLUSIONS The most important results obtained in this PhD thesis were: (i) deciphering of the draft genome sequence of the Ensifer (Sinorhizobium) sp. M14 and demonstrating of the absence of contraindications for the use of this strain in environmental technologies, (ii) revealing of the growth rate and kinetics of arsenite oxidation, and demonstrating of high efficiency of oxidation of As(III) compounds by the M14 strain, (iii) confirmation of the high sorption properties and significant chemical and biological stability of bog iron ores, (iv) development of technological guidelines for the use of the M14 strain and bog iron ores in environmental technologies, (v) verification of the obtained results concerning of the AOBs activity and adsorption of arsenates in a pilot-scale installation tested under actual environmental conditions and (iv) reduction of arsenic concentration in water up to the acceptable limit of arsenic in technological waters. To summarize, both arsenic oxidation by the M14 strain and adsorption of arsenates on bog iron ores surface have a high application potential and can be use in technologies dedicated to removing of arsenic from contaminated waters. The results obtained in the frame of the proposed doctoral thesis may be a starting point for further basic and applied research on the development of a universal, low-cost and environmentally friendly (bio)technology dedicated to the purification of arsenic contaminated waters