Potential of Biological Sulphur Recovery Under Haloalkaline Conditions

Massa- ja paperiteollisuuden (P&P) sekä petrokemian teollisuuden prosesseissa esiintyy rikkipitoisia virtoja ja rikkiyhdisteitä sisältäviä jätevesiä. Ylimäärärikin talteenotto mekaanisesti erotettavana alkuainerikkinä emäksisistä ja suolaisesta liuoksesta haloalkalifiilisiä rikinhapettajabakteereita käyttäen ei edellytä korkeaa painetta ja lämpötilaa kuten fysikaalis-kemialliset menetelmät. Orgaanisten raaka-aineiden jalostamisen jätevedet sisältävät kuitenkin orgaanisia yhdisteitä, jotka saattavat olla haitallisia kemolitoautotrofisille bakteereille. Tämän työssä tutkittiin biologista alkuainerikin tuottoa tiosulfaattia sisältävistä liuoksista. Kemolitoautotrofisten ja haloalkalifiilisten rikinhapettajabakteerien (Thioalkalivibrio versutus ja T. denitrificans) tiosulfaatin (S2O32-) biotransformaatioiden ja kasvun kinetiikkaa tutkittiin ja mallinnettiin. Jatkuvatoimista bioprosessia rikin tuottoon tiosulfaatista tutkittiin leijupetibioreaktoria (FBBR) ja T. versutus -puhdasviljelmää käyttäen. Lopuksi tutkittiin orgaanisten yhdisteiden ja P&P-jätevesien vaikutuksia S2O32- biotransformaatioihin. Aerobinen S0 tuottava tiosulfaatin biotransformaationopeus T. versutus bakteerilla oli suurempi (qm=0,083 h-1) kuin T. denitrificans -bakteerilla (qm=0,024 h-1) suurilla substraattipitoisuuksilla. T. denitrificans ei tuottanut alkuainerikkiä dentirifikaatiolla. FBBR:ssä saavutettiin lähes 100 % S2O32- biotransformaatio kuormituksella 19 g S2O32--S/L/d ja suurin S0 -saanto (27±2 %) kuormituksella 22 g S2O32--S/L/d. Ei-aseptinen FBBR-järjestelmä soveltui puhdasviljelmän ylläpitoon ja tiosulfaatin biotransformaatioon. Tuotetun S0:n erotukseen sentrifikaatio ja FeCl2 -koagulaatio olivat tehokkaammat menetelmät. T. versutus kasvoi ja tuotti S0:a tiosulfaatista valkaisun primäärisuodoksen ja sellutehtaan komposiittijäteveden sekä niiden orgaanisten aineosien läsnäollessä. Hiivauute (2,5-5 g/l) tehosti S2O32- biotransformaatioita ja kasvua. Täässä työssä osoitetaan S2O32- biotransformaatio alkuainerikiksi synteettisissä emäksisissä (pH 10) ja suolaisissa (14-26 g/L Na+) panosviljeilyissä ja jatkuvatoimisissa reaktoreissa käyttäen T. versutus -puhdasviljelmää aerobisissa olosuhteissa. T. versutus sieti massa- ja paperiteollisuuden jätevesien orgaanisia aineosia, mikä viittaa mahdollisuuteen kehittää bioprosessia erityisesti massa- ja paperiteollisuuden jätevesien rikin talteenottoon.Process industries, such as pulp and paper (P&P) and petrochemical, generate concentrated sulphurous process streams and wastewaters. The process streams, which are saline and alkaline (haloalkaline), require careful management as they potentially increase operational costs, related to chemical balancing and corrosion. Sulphur recovery by haloalkaliphilic sulphur oxidizing bacteria (SOB) instead of physicochemical methods would be a cost-efficient approach as it works at ambient pressure and temperature and produces separable elemental sulphur (S0). However, the wastewaters of organic raw-material processing industries can contain organic compounds that may harm chemolithoautotrophic SOB. The aim of this work was to study the biological S0 recovery potential by chemolithoautotrophic SOB from haloalkaline sulphurous solutions for possible use in industrial process streams and wastewaters. Therefore, the kinetics of thiosulphate (S2O32-) biotransformation and growth of model haloalkaliphilic SOB (Thioalkalivibrio versutus and T. denitrificans) were delineated. Also, the S0 recovery by T. denitrificans under anoxic conditions was investigated. Third, the potential of continuous bioprocess with increasing S2O32- loading rates was studied in a T. versutus amended fluidized bed bioreactor (FBBR). Finally, the effects of organic compounds and P&P wastewaters on S2O32- biotransformation were delineated. The kinetic studies showed high-rate S2O32- biotransformation by T. versutus (qm=0.083 h-1) and somewhat lower by T. denitrificans (qm=0.024 h-1) at high initial substrate concentrations under aerobic conditions. S0 was formed by both bacteria in the aerobic batch assays whilst it was not formed by denitrification by T. denitrificans. In the FBBR, 100% S2O32- removal efficiency and 27±2% S0 yield were achieved at loading rates of 19 g S/L/d and 22 g S/L/d, respectively. The non-aseptic FBBR system was suitable to maintain the pure culture but was not suitable for S0 settling. T. versutus showed high tolerance towards P&P mill wastewaters (primary filtrate of bleaching, composite wastewater) and the constituents studied in this work. Yeast extract (2.5-5 g/L) enhanced biotransformation and growth. This work demonstrates efficient S2O32- biotransformation from synthetic solutions (pH 10, 14-26 g/L Na+) and P&P wastewaters under aerobic conditions. The outcomes of this thesis can be used for future bioprocess development

Biooxidation of iron in elevated pressures and production of iron oxidizing biomass for a pilot-scale bioreactor

Securing the future´s metal demand through traditional metal recovery methods is often economically not viable because of the low metal content of the readily available ores. Although biological metal recovery from low-grade ores can be potential alternative, the recently used approaches such as heap and tank bioleaching still require the extraction and crushing of the ores. Therefore, an environmentally friendly approach that would work with low-grade ores at the natural occurrence of metals known as deep in situ bioleaching is under investigation. Studying the pressure tolerance of a mixed acidophilic iron oxidizing microbial community (Leptospirillum ferriphilum and Sulfobacillus sp.) that could be used in deep in situ application was the main objective of this thesis. Furthermore, production of activated carbon-bound iron oxidizing biomass for pilot-sale demonstration of in situ bioleaching was also conducted. Experiments with a pressure reactor (1 L) showed pressure tolerance of the acidophilic culture at 40 bar (with initial 0.3 bar oxygen partial pressure (pO2), while the pressure was induced with N2 gas) above atmospheric pressure. The 10 bar/min pressure increase/decrease rate was not inhibitory to the iron oxidation activity of the microorganisms. When the elevated pressure was induced with technical air, the highest tolerated pressure where biotic iron oxidation still occurred was +3 bar (pO2=0.63 bar). From the elevated pressures tested, the highest biotic iron oxidation rate (0.78 g/L/d) was obtained at +3 bar, which was approximately half of the rate obtained at atmospheric pressure (1.7 g/L/d) in shake flask cultures. The abiotic iron oxidation rate linearly increased with the increase of oxygen partial pressure. During the biomass production for the pilot reactor, it was shown that the iron oxidation rate decreased as the reactor volume got larger. In order to reach iron oxidation efficiency of 90% took approximately 0.3, 3 and 4 days in the fluidized bed reactor (900 mL), shake flasks (100 mL) and semi-pilot reactor (~600 L), respectively. This work demonstrated that in situ iron oxidation by acidophilic microbial community of this study in culture suspension is possible up to +3 bar (pO2= 0.63 bar). Abiotic iron oxidation in deep subsurface is an option if oxygen can be provided there. To achieve the highest possible iron oxidation rate and maintain the microbial community structure, fully controlled environment (pH, temperature, pressure, mixing, aeration) and continuous operation are required

High tolerance of chemolithoautotrophic sulphur oxidizing bacteria towards pulp and paper mill wastewaters and their organic constituents supporting sulphur recovery in alkaline conditions

This study reports the tolerance of chemolithoautotrophic biotransformation of sulphurous compounds towards pulp and paper (P&P) mill wastewaters (primary filtrate of bleaching (PFB) and composite wastewater (WW)) and their constituents under haloalkaline conditions. The effects of organic compounds (methanol, acetate, D(+)-xylose, phenol and benzene) that may be present in P&P wastewaters, and yeast extract, a complex organic compound on thiosulphate biotransformation by Thioalkalivibrio versutus were investigated. All experiments were carried out in batch bioassays at pH 10 and 13–23 g Na+/L. Phenol and benzene reduced thiosulphate biotransformation by 88 and 94% at 0.25 and 1 g/L, respectively in 10 days. 20 g/L methanol, 20 g/L yeast extract and 10 g/L xylose reduced the biotransformation by 90, 88 and 56%, respectively. No inhibition of biotransformation occurred with acetate at concentrations up to 20 g/L. The growth was also enhanced by 1 to 10 g/L yeast extract likely serving as additional nutrients. At pH (∼10), the studied organic acids remain mostly unprotonated and, thus control their access through the cell membrane. Therefore, the inaccessibility of these compounds to the cytosol is a likely mechanism for having non-inhibitory effects. The 87% (v/v) WW did not affect thiosulphate biotransformation efficiency while 87% (v/v) PFB reduced it by 36% by day 10. The resistance of T. versutus to common organics present in P&P wastewaters indicates its potential use for sulphur recovery from P&P mill wastewaters at haloalkaline conditions and thus, supports the circular economy approach.publishedVersionPeer reviewe

High-rate and -yield continuous fluidized-bed bioconversion of glucose-to-gluconic acid for enhanced metal leaching

Continuous low-cost bulk biolixiviant production remains as one of the main challenges of heterotrophic bioleaching towards large scale application. This study aimed at developing non-aseptic Gluconobacter oxydans-amended fluidized-bed reactor (FBR) process for continuous production of gluconic acid for efficient leaching of rare earth elements (REEs) and base metals from spent nickel-metal-hydride (NiMH) batteries. In preliminary experiments, the FBR became contaminated and massively overgrown by air-borne fungus, Leptobacillium leptobactrum. In a series of batch bioassays, operational conditions were investigated to discourage the fungal activity i.e., an ecologically engineered niche for gluconic acid production. High gluconate concentration (≥100 g/l) and/or low pH (≤2.5) gave a selective preference for G. oxydans growth over L. leptobactrum and controlled the activity of possible contaminants during FBR continuous operation. The highest gluconic acid production rate of 390 g/l∙d with corresponding glucose-to-gluconic acid conversion yield of 94% was obtained at hydraulic retention time (HRT) of 6.3 h and 380 g/l∙d glucose loading rate. Using the FBR effluents as leaching agents, respectively, total base metals and REEs leaching yields of up to 82% and 55% were achieved within 7 days at 1% (w/v) spent battery pulp density. The obtained glucose-to-gluconic acid conversion rates and yields were one of the highest reported for any glucose biotransformation process. The REE leaching yields were higher than those reported for similar high metal-grade REE secondary sources. The high-rate glucose-to-gluconic acid bioconversion in the non-aseptic system utilizing microbial ecology based FBR operation strategy rather than aseptic chemostats indicates industrial feasibility of gluconic acid production and thus, the applicability of heterotrophic bioleaching.publishedVersionPeer reviewe

Indirect in situ bioleaching is an emerging tool for accessing deeply buried metal reserves, but can the process be managed? – A case study of copper leaching at 1 km depth

Copper is a strategic raw material widely needed for electrification. One possibility to diversify the supply to answer the market demand is to produce copper with in situ technology. In this study, feasibility of in situ bioleaching of copper was tested in a deep subsurface deposit. During in situ bioleaching of copper, copper is leached using a biologically produced ferric iron solution, which is recycled back to the in situ reactor after valuable metals are recovered, after which the solution is re-oxidized by iron-oxidizing microorganisms (IOB). A rock reactor was constructed in the Rudna Mine at ca 1 km depth and the microbiology and hydrogeochemistry of the water circulated through the reactor after blasting for fracturing the rock was monitored over time. The test site was rich in carbonates requiring large quantities of acid to remove the buffering capacity. The bacterial, archaeal and fungal communities in the rock reactor were monitored and characterized by quantitative polymerase chain reaction (qPCR) and amplicon sequencing, and acidophilic, iron oxidizing activity of the microbial communities during operation and pre- and post-operation phases was tested by cultivation. No acidophilic iron oxidizers were detected in the water samples during construction of the pilot reactor. Acidic leaching solution originating from the underground ferric iron generating bioreactor (FIGB) contained acidophilic IOB, which were also viable after the leach liquor was returned from the rock reactor. In the post-operation phase, when the rock reactor was neutralized with CaCO3/Ca(HCO3)2 solution, to inhibit the acidophilic IOB, iron oxidizing microorganisms were still present in the effluent solution one week after termination of the leaching and start of neutralization. Therefore, the post-operation phase needs further attention to completely stop the activity of added microorganisms. Copper was abundantly leached during the acid wash and leaching phases, proving the concept of deep in situ bioleaching.Peer reviewe

Bioremediation of Heavy Metals by using the Microalga Desmodesmus Subspicatus

All around the world natural water bodies are contaminated with heavy metals from previous and recent mining activities. These contaminants not just can reach our drinking water supplies but endanger aquatic ecosystems and also other organisms. The recent technologies used for the removal of heavy metals are expensive and also causing harm to the nature. To avoid the negative impacts caused through the removal of contaminants, environmentally friendly methods, bioremediation should be applied. Using microbial processes for the removal of contaminants is still not widely used and enough studied. The objective of this work was to investigate the bioremediation properties and behavior in different environmental conditions of the microalga Desmodesmus subspicatus. The heavy metal (300µg/l Pb, 30µg/l As, Cd, Hg) solution chosen was similar with the values found in the River Oker, which originate from previous mining site of the Harz Mountain in Lower Saxony, Germany. The heavy metal uptake by Desmodesmus subspicatus biomass was not efficient at pH 5 since the final uptakes were (4.5±0.67) µg/l As, (21.1±2.89) µg/l Pb, (7.33±1.96) µg/l Cd and (6.25±1.28) µg/l Hg (15% As, 7% Pb, 24% Cd, 21% Hg). However, its initial biosorption showed good results with Pb (<79%) and Hg (<63%). In the conditions applied the Desmodesmus subspicatus can be used only for initial biosorption of Pb and Hg. Making changes like adjustment of different pH levels; applying multi culture mix, adding immobilization material, increasing the population and pretreating the cells could increase the efficiency. Also the immediate uptake of Hg and Pb by biosorption is an interesting feature as it could be used with biomass filtering not with growing cultures and lead to immediate results. The bioremediation property of Desmodesmus subspicatus in an environment with one or several of the previously mentioned methods applied should be further investigated

Biooxidation of iron in elevated pressures and production of iron oxidizing biomass for a pilot-scale bioreactor

Securing the future´s metal demand through traditional metal recovery methods is often economically not viable because of the low metal content of the readily available ores. Although biological metal recovery from low-grade ores can be potential alternative, the recently used approaches such as heap and tank bioleaching still require the extraction and crushing of the ores. Therefore, an environmentally friendly approach that would work with low-grade ores at the natural occurrence of metals known as deep in situ bioleaching is under investigation. Studying the pressure tolerance of a mixed acidophilic iron oxidizing microbial community (Leptospirillum ferriphilum and Sulfobacillus sp.) that could be used in deep in situ application was the main objective of this thesis. Furthermore, production of activated carbon-bound iron oxidizing biomass for pilot-sale demonstration of in situ bioleaching was also conducted. Experiments with a pressure reactor (1 L) showed pressure tolerance of the acidophilic culture at 40 bar (with initial 0.3 bar oxygen partial pressure (pO2), while the pressure was induced with N2 gas) above atmospheric pressure. The 10 bar/min pressure increase/decrease rate was not inhibitory to the iron oxidation activity of the microorganisms. When the elevated pressure was induced with technical air, the highest tolerated pressure where biotic iron oxidation still occurred was +3 bar (pO2=0.63 bar). From the elevated pressures tested, the highest biotic iron oxidation rate (0.78 g/L/d) was obtained at +3 bar, which was approximately half of the rate obtained at atmospheric pressure (1.7 g/L/d) in shake flask cultures. The abiotic iron oxidation rate linearly increased with the increase of oxygen partial pressure. During the biomass production for the pilot reactor, it was shown that the iron oxidation rate decreased as the reactor volume got larger. In order to reach iron oxidation efficiency of 90% took approximately 0.3, 3 and 4 days in the fluidized bed reactor (900 mL), shake flasks (100 mL) and semi-pilot reactor (~600 L), respectively. This work demonstrated that in situ iron oxidation by acidophilic microbial community of this study in culture suspension is possible up to +3 bar (pO2= 0.63 bar). Abiotic iron oxidation in deep subsurface is an option if oxygen can be provided there. To achieve the highest possible iron oxidation rate and maintain the microbial community structure, fully controlled environment (pH, temperature, pressure, mixing, aeration) and continuous operation are required

Elemental sulphur production from thiosulphate under haloalkaline conditions in a Thioalkalivibrio versutus amended fluidized bed bioreactor

Concentrated sulphurous and saline streams, produced for example by pulp and paper and petrochemical industries, pose challenges for both environmental and processes management. In this study, the potential of biological recovery of S0 from haloalkaline thiosulphate solution in a Thioalkalivibrio versutus amended continuous-flow fluidized bed bioreactor (FBBR) was investigated using different (12−5 h) hydraulic retention times (HRT) as well as physico-chemical means to separate the S0 produced. S2O32− was biotransformed to SO42− and S0 with the highest biotransformation efficiency of 99.9 %. At 7 h HRT, the capacity of the FBBR was reached, seen as incomplete thiosulphate conversion. S0 production rate increased up to 6.3 ± 0.6 g S/l/d at HRT 7 h, whilst the average S0 yield was 27 ± 2 %. The presence of biologically produced S0 was visual and identified by scanning electron microscopy. Separation of S0 from the effluent by centrifugation at 3417 relative centrifugal force (rcf) resulted in 93 % separation, while among the four tested coagulants, FeCl2 at 0.5 g/l resulted in 40 % separation. Also, FeCl2 enhanced thiosulphate biotransformation rates. In summary, continuous biological S0 production followed by separation by centrifugation indicates potential for sulphur recovery from alkaline and saline industrial streams.publishedVersionPeer reviewe

Kinetics and modelling of thiosulphate biotransformations by haloalkaliphilic Thioalkalivibrio versutus

Biotransformation of thiosulphate by Thioalkalivibrio versutus was studied under haloalkaline conditions (pH 10, 0.66–1.2 M Na+) using batch assays and modelling tools for possible sulphur recovery from haloalkaline industrial streams. The thiosulphate was fully biotransformed to sulphate or to sulphate and elemental sulphur at initial S2O32−-S concentrations of 25–550 mM within 10 days. The highest biotransformation rate of 2.66 mM [S2O32−-S] h−1 was obtained at initial S2O32−-S concentration of 550 mM with half saturation constant (Ks) of 54.5 mM [S2O32−-S]. At initial concentrations below 100 mM S2O32−-S, the main product was sulphate whilst at above 100 mM also elemental sulphur was produced with up to 29% efficiency. The model approach developed incorporated S2O32− biotransformation to SO42− and S0. The kinetic modelling results were compatible (R2 > 0.90) with the experimental data. The maximum growth rate (µm) was 0.048 h−1 (0.47 mM C5H7NO2 h−1) and the maximum growth yield 0.18 mM C5H7NO2/mM S2O32−-S (20 g cell/mol S2O32−-S). The high rate thiosulphate biotransformation and elemental sulphur recovery results together with the developed kinetic model can be used for bioprocess design and operation. The potential industrial applications would aim at sustainable resource recovery from industrial haloalkaline and sulphurous process and/or effluent streams.acceptedVersionPeer reviewe

Effects of metal extraction liquors from electric vehicle battery materials production on iron and sulfur oxidation by heap bioleaching microorganisms

This study reports the effects of metal extraction liquors that are used for production of electric vehicle batteries on biological iron and sulfur oxidation. These liquors include ammonium sulfate and organic solvent constituents, and thus are potentially or inhibitory for heap bioleaching microorganisms. The effects of the liquors and their potential constituents were studied in batch bioassays at pH 2 and 27 ± 2 °C. Both metal extraction liquors had a negative effect on biological iron oxidation at >2% (v/v), whereas biological sulfur oxidation was enhanced with ≤8% (v/v) metal extraction liquor 1. Biological iron oxidation was negatively affected by ammonium sulfate at above 20 g/L. From the studied low-solubility organic solvents (neodecanoic acid, Nessol D100, Cyanex 272, and Baysolvex D2EHPA), neodecanoic acid was the only one negatively affecting biological iron oxidation, and this effect occurred at ≥ 6.3 mg/L (2.5% of its aqueous solubility). Since these extraction liquors and some of their potential constituents inhibited biological iron oxidation, they may also inhibit heap bioleaching and have adverse impacts in recipient waters, if released to the environment. With ammonium limited culture, iron oxidation was stimulated with ≤ 1% (v/v) of metal extraction liquor 1 and 2, and therefore, would also likely enhance heap bioleaching.publishedVersionPeer reviewe