Elemental sulphur recovery from a sulphate-rich aqueous stream in a single hybrid linear flow channel reactor is mediated through microbial community dynamics and adaptation to reactor zones.

The coupled application of biological sulphate reduction (BSR) and partial sulphide oxidation to treat sulphate-rich wastewater is an effective strategy to mitigate pollution and recover elemental sulphur for repurposing. The recent development of the hybrid linear flow channel reactor (LFCR) achieves simultaneous BSR and partial sulphide oxidation with biosulphur recovery via a floating sulphur biofilm (FSB). Here, we explore the microbial community zoning and dynamics facilitating the process. A total of three continuous LFCRs were used to evaluate the effect of reactor zones, hydraulic residence time (HRT), carbon source, namely lactate and acetate, as well as reactor geometry and scale on process performance and microbial community dynamics. Community composition of sessile and planktonic microbial consortia were resolved at a 5- and 2-day HRT through 16S rRNA amplicon sequencing. Preferential attachment and prevalence of specific phylotypes within the sessile and planktonic communities revealed clear adaptation of key microorganisms to different microenvironments. Key microbial taxa affiliated with sulphate reduction and sulphide oxidation as well as those implicated in fermentation and syntrophic metabolism, fluctuated in response to changes in HRT and process performance. Through understanding the relationship between microbial community dynamics and process performance, this research will inform better process design and optimization of the hybrid LFCR.

Effect of temperature change on the performance of the hybrid linear flow channel reactor and its implications on sulphate-reducing and sulphide-oxidising microbial community dynamics.

Semi-passive bioremediation is a promising strategy to mitigate persistent low volume mine-impacted wastewater containing high sulphate concentrations. Building on the proof of concept demonstration of the hybrid linear flow channel reactor (LFCR), capable of simultaneous biological sulphate reduction and partial sulphide oxidation with elemental sulphur recovery, the impact of key operating parameters, such as temperature, on process performance is critical to real-world application. Temperature fluctuates seasonally and across the diurnal cycle, impacting biological sulphate reduction (BSR) and partial sulphide oxidation. The process is reliant on the metabolic activity and synergistic interactions between sulphate-reducing (SRB) and sulphide-oxidising (SOB) microbial communities that develop within discrete oxic and anoxic microenvironments within the hybrid LFCR. In this study, the impact of operating temperature on process performance was evaluated by decreasing temperature with time from 30 to 10°C in each of three laboratory-scaled hybrid LFCR units operating in pseudo-steady state at 1 g/L sulphate. Using lactate as a carbon source, two reactor sizes (2 and 8 L) were considered, while the impact of lactate vs. acetate as carbon source was evaluated in the 2 L reactors. On incremental decrease in temperature from 30 to 10°C, a decrease in volumetric sulphate reduction rate was observed: from 0.144 to 0.059 mmol/L.h in the 2 L lactate-fed reactor; from 0.128 to 0.042 mmol/L.h in the 8 L lactate-fed reactor; and from 0.127 to 0.010 mmol/L.h in the 2 L acetate-fed reactor. Similarly, sulphate conversion efficiency decreased (2 L lactate-fed: 66% to 27%; 8 L lactate-fed: 61% to 20%; 2 L acetate-fed: 61% to 5%). A decrease in temperature below the critical value (15°C) led to considerable loss in metabolic activity and overall BSR performance. Sessile and planktonic microbial communities were represented by bacterial phyla including Proteobacteria, Synergistetes, Bacteroidetes, and Firmicutes. A diverse group of putative SRB (Deltaproteobacteria) and SOB, including Alpha, Beta, Gamma, and Epsilonproteobacteria phylotypes, were prevalent and shifted in relative abundance and community composition in response to decreasing temperature. Specifically, the decrease in the relative abundance of Deltaproteobacteria with decreasing temperature below 15°C corresponded with a loss of BSR performance across all three reactors. This study demonstrated the impact of low temperature on the physiological selection and ecological differentiation of SRB and SOB communities within the hybrid LFCR and its implications for real-world process performance.

Reactive oxygen species generated in the presence of fine pyrite particles and its implication in thermophilic mineral bioleaching.

In the tank bioleaching process, maximising solid loading and mineral availability, the latter through decreasing particle size, are key to maximising metal extraction. In this study, the effect of particle size distribution on bioleaching performance and microbial growth was studied through applying knowledge based on medical geology research to understand the adverse effects of suspended fine pyrite particles. Small-scale leaching studies, using pyrite concentrate fractions (106-75, 75-25, -25 µm fines), were used to confirm decreasing performance with decreasing particle size (D 50 <40 µm). Under equivalent experimental conditions, the generation of the reactive oxygen species (ROS), hydrogen peroxide and hydroxyl radicals from pyrite was illustrated. ROS generation measured from the different pyrite fractions was found to increase with increasing pyrite surface area loading (1.79-74.01 m(2) L(-1)) and Fe(2+) concentration (0.1-2.8 g L(-1)) in solution. The highest concentration of ROS was measured from the finest fraction of pyrite (0.85 mM) and from the largest concentration of Fe(2+) (0.78 mM). No ROS was detected from solutions containing only Fe(3+) under the same conditions tested. The potential of ROS to inhibit microbial performance under bioleaching conditions was demonstrated. Pyrite-free Sulfolobus metallicus cultures challenged with hydrogen peroxide (0.5-2.5 mM) showed significant decrease in both cell growth and Fe(2+) oxidation rates within the concentration range 1.5-2.5 mM. In combination, the results from this study suggest that conditions of large pyrite surface area loading, coupled with high concentrations of dissolved Fe(2+), can lead to the generation of ROS, resulting in oxidative stress of the microorganisms.

The effect of CO2 availability on the growth, iron oxidation and CO2-fixation rates of pure cultures of Leptospirillum ferriphilum and Acidithiobacillus ferrooxidans.

Understanding how bioleaching systems respond to the availability of CO(2) is essential to developing operating conditions that select for optimum microbial performance. Therefore, the effect of inlet gas and associated dissolved CO(2) concentration on the growth, iron oxidation and CO(2) -fixation rates of pure cultures of Acidithiobacillus ferrooxidans and Leptospirillum ferriphilum was investigated in a batch stirred tank system. The minimum inlet CO(2) concentrations required to promote the growth of At. ferrooxidans and L. ferriphilum were 25 and 70 ppm, respectively, and corresponded to dissolved CO(2) concentrations of 0.71 and 1.57 µM (at 30°C and 37°C, respectively). An actively growing culture of L. ferriphilum was able to maintain growth at inlet CO(2) concentrations less than 30 ppm (0.31-0.45 µM in solution). The highest total new cell production and maximum specific growth rates from the stationary phase inocula were observed with CO(2) inlet concentrations less than that of air. In contrast, the amount of CO(2) fixed per new cell produced increased with increasing inlet CO(2) concentrations above 100 ppm. Where inlet gas CO(2) concentrations were increased above that of air the additional CO(2) was consumed by the organisms but did not lead to increased cell production or significantly increase performance in terms of iron oxidation. It is proposed that At. ferrooxidans has two CO(2) uptake mechanisms, a high affinity system operating at low available CO(2) concentrations, which is subject to substrate inhibition and a low affinity system operating at higher available CO(2) concentrations. L. ferriphilum has a single uptake system characterised by a moderate CO(2) affinity. At. ferrooxidans performed better than L. ferriphilum at lower CO(2) availabilities, and was less affected by CO(2) starvation. Finally, the results demonstrate the limitations of using CO(2) uptake or ferrous iron oxidation data as indirect measures of cell growth and performance across varying physiological conditions.

Selection of direct transesterification as the preferred method for assay of fatty acid content of microalgae.

Assays for total lipid content in microalgae are usually based on the Folch or the Bligh and Dyer methods of solvent extraction followed by quantification either gravimetrically or by chromatography. Direct transesterification (DT) is a method of converting saponifiable lipids in situ directly to fatty acid methyl esters which can be quantified by gas chromatography (GC). This eliminates the extraction step and results in a rapid, one-step procedure applicable to small samples. This study compared the effectiveness of DT in quantifying the total fatty acid content in three species of microalgae to extraction using the Folch, the Bligh and Dyer and the Smedes and Askland methods, followed by transesterification and GC. The use of two catalysts in sequence, as well as the effect of reaction water content on the efficiency of DT were investigated. The Folch method was the most effective of the extraction methods tested, but comparison with DT illustrated that all extraction methods were incomplete. Higher levels of fatty acid in the cells were obtained with DT in comparison with the extraction-transesterification methods. A combination of acidic and basic transesterification catalysts was more effective than each individually when the sample contained water. The two-catalyst reaction was insensitive to water up to 10% of total reaction volume. DT proved a convenient and more accurate method than the extraction techniques for quantifying total fatty acid content in microalgae.

Influence of the extent of disruption of Bakers' yeast on protein adsorption in expanded beds.

Expanded bed adsorption chromatography is used to capture the protein product of interest from a crude biological suspension directly, thereby eliminating the need for the removal of the cell debris. While this technique may replace three or four unit operations in a typical downstream process for biological product recovery, the adsorption process is influenced by the interaction between the microbial cells or cell debris and the adsorbent as well as the presence of contaminating solutes. The influence of the extent and nature of disruption of Bakers' yeast on the adsorption of the total soluble protein and alpha-glucosidase was investigated in this study. Two different techniques were used for cell disruption: high pressure homogenisation and hydrodynamic cavitation. Two different adsorbents were chosen: anionic Streamline DEAE and cationic Streamline SP. The settled bed height and the superficial velocity were constant across all experiments. The feedstock was characterised in terms of viscosity, pH, conductivity, particle size distribution of the cell debris and the extent of protein and alpha-glucosidase released. The performance of the adsorption process was found to be influenced by the electrostatic interactions of cell debris with the anionic adsorbent Streamline DEAE and the intraparticle diffusional resistance inside the pores of the adsorbent matrix. The increase in the intensity of disruption resulted in an increase in the dynamic binding capacity (10% feed) of both the total soluble protein and the alpha-glucosidase. However, the increase in the DBC of protein and alpha-glucosidase were not proportional. The amount of protein that could be adsorbed per ml of adsorbent from the samples subjected to a lower intensity of disruption was found to exceed that obtained at a higher disruption intensity on increasing the volume of feed suggesting multilayer adsorption. In this case, selective adsorption of the model protein alpha-glucosidase was reduced, illustrating the compromise of maximising protein recovery through non-specific binding. The study illustrates the need for an interrogation of the intensity of disruption needed and a rigorous understanding of the influence of cell debris and adsorbent-protein interaction, in optimising the selective recovery of intracellular products by EBA.

A study of the influence of yeast cell debris on protein and alpha-glucosidase adsorption at various zones within the expanded bed using in-bed sampling.

Expanded bed adsorption chromatography is used to capture products directly from unclarified feedstocks, thus combining solid-liquid separation, product concentration and preliminary purification into a single step. However, when non-specific ion-exchangers are used as the adsorbent in the expanded bed, there is the possibility that electrostatic interactions of cells or cell debris with the adsorbent may interfere with the adsorption of soluble products. These interactions depend on the particle size of the cell debris and its surface charge, which in turn depend on the extent of disruption used to release the intracellular products. The interactions occurring during expanded bed adsorption between the anionic ion-exchanger STREAMLINE DEAE and particulate yeast homogenates obtained by high pressure homogenisation at different intensities of disruption achieved by operating at different pressures were studied, while maintaining all other parameters constant. In-bed sampling from the expanded bed using ports fitted up the height of expanded bed was used to study the retention of yeast cells and cell debris within the bed and its influence on the adsorption of total soluble protein and alpha-glucosidase within various zones of the expanded bed. The retention of the biomass present in the homogenate obtained at a lower intensity of disruption was found to be high at the lower end of the column (17% from 13.8 MPa sample compared to 1% from 41.4 MPa sample). This interaction of the particulate material with the adsorbent was found to reduce the dynamic binding capacity of the adsorbent for total soluble protein from 3.6 mg/mL adsorbent for 41.4 MPa sample to 3.0 mg/mL adsorbent for 13.8 MPa sample. The adsorption of alpha-glucosidase was found to increase with an increase in the concentration of the enzyme in the feed, which increased with the intensity of disruption. Selective adsorption of 6,732 U alpha-glucosidase per mg of total protein bound, was noticed for the feedstock prepared at a higher disruption intensity at 41.4 MPa compared to adsorption of 1,262 U/mg of total protein bound for that prepared at 13.8 MPa. The selective adsorption of alpha-glucosidase due to its high concentration together with simultaneous high specific activity of the enzyme in the feed indicated the significance of selective release of enzymes during microbial cell disruption for efficient expanded bed adsorption processes.

Characterization of the distribution of glucose oxidase in Penicillium sp. CBS 120262 and Aspergillus niger NRRL-3 cultures and its effect on integrated product recovery.

Glucose oxidase (GO) is an important industrial enzyme typically purified from Penicillium and Aspergillus sp. As GO distribution within the cultures influences process design for maximal product recovery, distribution of GO activity in Penicillium sp. CBS 120262 and Aspergillus niger NRRL-3, during mid-exponential and stationary phases, is compared. On progression from mid-exponential to stationary phase, the percentage GO activity in the cytoplasm decreased 1.6- and 1.3-fold in Penicillium sp. and A. niger respectively. In Penicillium sp., a concomitant 1.8- and 1.9-fold decrease in the percentage GO activity in the cell envelope and slime mucilage respectively, translated into a 2.0-fold increase in the extracellular fluid. In A. niger, decreasing cytoplasmic GO activity was accompanied by 1.3-fold increases in the cell envelope and slime mucilage, with a 1.3-fold decrease in the extracellular fluid. Similar trends were observed in specific GO activities. As final GO activity recovered is governed by the purification program, recovery from the extracellular fluid plus cell extract or from the extracellular fluid only were compared through simulating processes of varying complexity. A critical yield for each purification stage was identified above which recovery from the extracellular fluid plus cell extract exceeded that from extracellular fluid alone. These results highlight the influence of microorganism, harvest time and efficiency of downstream process on GO activity delivered. In the systems studied, Penicillium sp. is the organism of choice and should be harvested during stationary phase. The purification process chosen should be informed by both enzyme distribution and individual purification stages yields.

Environmental analysis of plastic production processes: comparing petroleum-based polypropylene and polyethylene with biologically-based poly-beta-hydroxybutyric acid using life cycle analysis.

Polymers based on olefins have wide commercial applicability. However, they are made from non-renewable resources and are characterised by difficulty in disposal where recycle and re-use is not feasible. Poly-beta-hydroxybutyric acid (PHB) provides one example of a polymer made from renewable resources. Before motivating its widespread use, the advantages of a renewable polymer must be weighed against the environmental aspects of its production. Previous studies relating the environmental impacts of petroleum-based and bio-plastics have centred on the impact categories of global warming and fossil fuel depletion. Cradle-to-grave studies report equivalent or reduced global warming impacts, in comparison to equivalent polyolefin processes. This stems from a perceived CO(2) neutral status of the renewable resource. Indeed, no previous work has reported the results of a life cycle assessment (LCA) giving the environmental impacts in all major categories. This study investigates a cradle-to-gate LCA of PHB production taking into account net CO(2) generation and all major impact categories. It compares the findings with similar studies of polypropylene (PP) and polyethylene (PE). It is found that, in all of the life cycle categories, PHB is superior to PP. Energy requirements are slightly lower than previously observed and significantly lower than those for polyolefin production. PE impacts are lower than PHB values in acidification and eutrophication.

A study of the relative dominance of selected anaerobic sulfate-reducing bacteria in a continuous bioreactor by fluorescence in situ hybridization.

The diversity and the community structure of sulfate-reducing bacteria (SRB) in an anaerobic continuous bioreactor used for treatment of a sulfate-containing wastewater were investigated by fluorescence in situ hybridization. Hybridization to the 16S rRNA probe EUB338 for the domain Bacteria was performed, followed by a nonsense probe NON338 as a control for nonspecific staining. Sulfate-reducing consortia were identified by using five nominally genus-specific probes (SRB129 for Desulfobacter, SRB221 for Desulfobacterium, SRB228 for Desulfotomaculum, SRB660 for Desulfobulbus, and SRB657 for Desulfonema) and four group-specific probes (SRB385 as a general SRB probe, SRB687 for Desulfovibrioaceae, SRB814 for Desulfococcus group, and SRB804 for Desulfobacteriaceae). The total prokaryotic population was determined by 4',6-diamidino-2-phenylindole staining. Hybridization analysis using these 16S rRNA-targeted oligonucleotide probes showed that, of those microbial groupings investigated, Desulfonema, Desulfobulbus, spp., and Desulfobacteriaceae group were the main sulfate-reducing bacteria in the bioreactor when operated at steady state at 35 degrees C, pH 7.8, and a 2.5-day residence time with feed stream containing 2.5 kg m-3 sulfate as terminal electron acceptor and 2.3 kg m-3 acetate as carbon source and electron donor.

Study of physical and biological factors involved in the disruption of E. coli by hydrodynamic cavitation.

Hydrodynamic cavitation results in flow restriction in a flow system causing rapid pressure fluctuations and significant fluid forces. These can be harnessed to mediate microbial cell damage. Hydrodynamic cavitation was studied for the partial disruption of E. coli and selective release of specific proteins relative to the total soluble protein. The effects of the cavitation number, the number of passes, and the specific growth rate of E. coli on the release of periplasmic and cytoplasmic proteins were studied. At the optimum cavitation number of 0.17 for this experimental configuration, 48% of the total soluble protein, 88% of acid phosphatase, and 67% of beta-galactosidase were released by hydrodynamic cavitation in comparison with the maximum release attained using multiple passes through the French Press. The higher release of the acid phosphatase over the total soluble protein suggested preferred release of periplasmic compounds. This was supported by SDS-PAGE analysis. The absence of micronization of cell material resulting in the potential for ease of solid-liquid separation downstream of the cell disruption operation was confirmed by TEM microscopy. E. coli cells cultivated at a higher specific growth rate (0.36 h(-1)) were more easily disrupted than slower grown cells (0.11 h(-1)). The specific activity of the enzyme of interest released by hydrodynamic cavitation, defined as the units of enzyme in solution per milligram of total soluble protein, was greater than that obtained on release by the French Press, high-pressure homogenization, osmotic shock, and EDTA treatment. The selectivity offered indicates the potential of enzyme release by hydrodynamic cavitation to ease the purification in the subsequent downstream processing.

Disruption of Brewers' yeast by hydrodynamic cavitation: Process variables and their influence on selective release.

Intracellular products, not secreted from the microbial cell, are released by breaking the cell envelope consisting of cytoplasmic membrane and an outer cell wall. Hydrodynamic cavitation has been reported to cause microbial cell disruption. By manipulating the operating variables involved, a wide range of intensity of cavitation can be achieved resulting in a varying extent of disruption. The effect of the process variables including cavitation number, initial cell concentration of the suspension and the number of passes across the cavitation zone on the release of enzymes from various locations of the Brewers' yeast was studied. The release profile of the enzymes studied include alpha-glucosidase (periplasmic), invertase (cell wall bound), alcohol dehydrogenase (ADH; cytoplasmic) and glucose-6-phosphate dehydrogenase (G6PDH; cytoplasmic). An optimum cavitation number Cv of 0.13 for maximum disruption was observed across the range Cv 0.09-0.99. The optimum cell concentration was found to be 0.5% (w/v, wet wt) when varying over the range 0.1%-5%. The sustained effect of cavitation on the yeast cell wall when re-circulating the suspension across the cavitation zone was found to release the cell wall bound enzyme invertase (86%) to a greater extent than the enzymes from other locations of the cell (e.g. periplasmic alpha-glucosidase at 17%). Localised damage to the cell wall could be observed using transmission electron microscopy (TEM) of cells subjected to less intense cavitation conditions. Absence of the release of cytoplasmic enzymes to a significant extent, absence of micronisation as observed by TEM and presence of a lower number of proteins bands in the culture supernatant on SDS-PAGE analysis following hydrodynamic cavitation compared to disruption by high-pressure homogenisation confirmed the selective release offered by hydrodynamic cavitation.

Location of glucose oxidase during production by Aspergillus niger.

The production of the enzyme glucose oxidase by Aspergillus niger is well documented. However, its distribution within the fungal culture is less well defined. Since the enzyme location impacts significantly on enzyme recovery, this study quantifies the enzyme distribution between the extracellular fluid, cell wall, cytoplasm and slime mucilage fractions in an A. niger NRRL-3. The culture was separated into the individual fractions and the glucose oxidase activity was determined in each. The extracellular fluid contained 38% of the total activity. The remaining 62% was associated with the mycelia and was distributed between the cell wall, cytoplasm and slime mucilage in the proportions of 34, 12 and 16%, respectively. Intracellular cytoplasmic and cell wall sites were confirmed using immunocytochemical labelling of the mycelia. In the non-viable cell, the mycelial-associated enzyme was distributed between these sites, whereas in the viable cell, it was predominantly associated with the cell wall. The distribution of the enzyme activity indicates that recovery from the solids would result in a 38% loss, whereas recovery from the extracellular fluid would result in a 62% loss. The results also suggest, however, that this 62% loss could be reduced to around 34% by disintegrating the solids prior to separation due to the contribution of the enzyme in the cytoplasm and slime mucilage. This was confirmed by independently establishing the percentage activity in the liquid and solid portions of a disintegrated culture as 62 and 38%, respectively.