Effect of cell permeability and dehydrogenase expression on octane activation by CYP153A6-based whole cell Escherichia coli catalysts

BACKGROUND: The regeneration of cofactors and the supply of alkane substrate are key considerations for the biocatalytic activation of hydrocarbons by cytochrome P450s. This study focused on the biotransformation of n-octane to 1-octanol using resting Escherichia coli cells expressing the CYP153A6 operon, which includes the electron transport proteins ferredoxin and ferredoxin reductase. Glycerol dehydrogenase was co-expressed with the CYP153A6 operon to investigate the effects of boosting cofactor regeneration. In order to overcome the alkane supply bottleneck, various chemical and physical approaches to membrane permeabilisation were tested in strains with or without additional dehydrogenase expression. RESULTS: Dehydrogenase co-expression in whole cells did not improve product formation and reduced the stability of the system at high cell densities. Chemical permeabilisation resulted in initial hydroxylation rates that were up to two times higher than the whole cell system, but severely impacted biocatalyst stability. Mechanical cell breakage led to improved enzyme stability, but additional dehydrogenase expression was necessary to improve product formation. The best-performing system (in terms of final titres) consisted of mechanically ruptured cells expressing additional dehydrogenase. This system had an initial activity of 1.67 ± 0.12 U/gDCW (32% improvement on whole cells) and attained a product concentration of 34.8 ± 1.6 mM after 24 h (22% improvement on whole cells). Furthermore, the system was able to maintain activity when biotransformation was extended to 72 h, resulting in a final product titre of 60.9 ± 1.1 mM. CONCLUSIONS: This study suggests that CYP153A6 in whole cells is limited by coupling efficiencies rather than cofactor supply. However, the most significant limitation in the current system is hydrocarbon transport, with substrate import being the main determinant of hydroxylation rates, and product export playing a key role in system stability

Investigating the effect of acid stress on selected mesophilic micro-organisms implicated in bioleaching

During start-up of heap bioleaching, low grade ores are typically treated with acid for agglomeration and to combat the acid neutralising capacity of the gangue minerals. This may stress the bioleaching inocula, particularly upon inoculation during ore agglomeration. Acid addition for agglomeration varies across operations, ore types and their neutralising capacity, with limited information published on recommended concentrations. The initial pH in the agglomeration mix is typically below pH 1.0 and may be as low as pH 0.5. This paper investigates the effect of acid stress in terms of initial acid concentration and exposure duration in submerged culture on mesophilic bacteria typically implicated in mineral sulphide bioleaching and critical for heap colonisation at start-up. Following acid stress, cultures were returned to standard operating conditions in batch stirred slurry reactors and their performance assessed in terms of mineral leach rates, ferrous oxidation and the rate of microbial growth. Increasing acid stress resulted in an increase in the lag period before onset of microbial growth and iron oxidation. Following adaptation, typical growth and ferrous iron oxidation rates were observed under low stress conditions while reduction in the rate and extent of microbial growth and ferrous iron oxidation persisted at extreme conditions. A reduction in yield (microbial cells produced per kg iron oxidised) was observed with increased acid concentration over comparative times. Microbial speciation analysis indicated a substantial decrease in the diversity of surviving bacterial species

The influence of microbial physiology on biocatalyst activity and efficiency in the terminal hydroxylation of n-octane using Escherichia coli expressing the alkane hydroxylase, CYP153A6

Biocatalyst improvement through molecular and recombinant means should be complemented with efficient process design to facilitate process feasibility and improve process economics. This study focused on understanding the bioprocess limitations to identify factors that impact the expression of the terminal hydroxylase CYP153A6 and also influence the biocatalytic transformation of n–octane to 1-octanol using resting whole cells of recombinant E. coli expressing the CYP153A6 operon which includes the ferredoxin (Fdx) and the ferredoxin reductase (FdR). Results: Specific hydroxylation activity decreased with increasing protein expression showing that the concentration of active biocatalyst is not the sole determinant of optimum process efficiency. Process physiological conditions including the medium composition, temperature, glucose metabolism and product toxicity were investigated. A fed-batch system with intermittent glucose feeding was necessary to ease overflow metabolism and improve process efficiency while the introduction of a product sink (BEHP) was required to alleviate octanol toxicity. Resting cells cultivated on complex LB and glucose-based defined medium with similar CYP level (0.20 μmol gDCW -1) showed different biocatalyst activity and efficiency in the hydroxylation of octane over a period of 120 h. This was influenced by differing glucose uptake rate which is directly coupled to cofactor regeneration and cell energy in whole cell biocatalysis. The maximum activity and biocatalyst efficiency achieved presents a significant improvement in the use of CYP153A6 for alkane activation. This biocatalyst system shows potential to improve productivity if substrate transfer limitation across the cell membrane and enzyme stability can be addressed especially at higher temperature. Conclusion: This study emphasises that the overall process efficiency is primarily dependent on the interaction between the whole cell biocatalyst and bioprocess conditions

Analysis of the microbial community associated with a bioprocess system for bioremediation of thiocyanate- and cyanide-laden mine water effluents

Gold extraction by cyanidation from refractory gold ores results in the formation of thiocyanate- and cyanide-contaminated wastewater effluents that must be treated before recycle or discard. Activated sludge processes, such as ASTER™, can be used for biodegradation of these effluent streams. The destruction of these compounds is catalyzed by a mixed microbial culture, however, very little is known about the community composition and metabolic potential of the thiocyanate- and cyanide-degrading microorganisms within the community. Here we describe our on-going attempts to better understand the key microorganisms, within the ASTER™ bioprocess, that contribute to the destruction of thiocyanate and cyanide, and how this knowledge relates to further process optimisation

Stable Isotope Imprints during Pyrite Leaching: Implications for Acid Rock Drainage Characterization

The characterization of acid rock drainage (ARD) is traditionally based on mineralogical and geochemical techniques (e.g., Acid Base Accounting tests). The complexity of ARD processes warrants contribution of methods from various disciplines. In the past decade, the increasing role of environmental isotopes in pollution monitoring has enabled the successful application of isotope methods in ARD investigations. While isotopic compositions of different pollutants can refer to their parent mineral, the degree of isotope fractionations are indicative of the mechanisms taking place during the release and transportation of ARD-related contaminants. In natural environments, however, the measured isotope fractionations are predominantly the result of several coexisting or sequential processes. Therefore, the identification and quantification of the distinct contributions of these processes to isotope variations is difficult and requires well-defined laboratory conditions, where the influence of ARD generation on different isotope systems can be assessed with greater certainty. This review provides readers with a single source of information regarding isotopic variations generated by laboratory pyrite leaching

Magnetic resonance imaging characterisation of the influence of flowrate on liquid distribution in drip irrigated heap leaching

Liquid irrigation is one of the key process control parameters following the construction of an ore leaching heap. This study uses 3D magnetic resonance imaging (MRI) to examine non-invasively the effect of liquid flowrate changes on heap hydrology when drip irrigation is used. Experimental results from a vertical column show that the increase in flowrate causes an increase in the number of rivulets in the ore bed. The new rivulets were found to be thicker, and their development caused an increase in liquid–solid contacting area which is considered advantageous for metal ion recovery. Experiments performed on larger samples showed that the effects of flowrate changes were limited to the region directly below the drip emitter because the increase in flowrate caused an increase in macro-pore flow and not capillary retention of liquid. Therefore the increase in flowrate was not found to perturb liquid distribution patterns in a way that would be substantially advantageous to heap leaching recoveries

Evaluation of the ASTERTM process in the presence of suspended solids

The ability to recycle and reuse process water is a major contributing factor toward increased sustainability in the mining industry. However, the presence of toxic compounds has prevented this in most bioleaching operations. The ASTERTM process has been used for the bioremediation of cyanide (CN) and thiocyanate (SCN−) containing effluents at demonstration and commercial scale, increasing the potential for recycling of the treated effluent. The process relies on a complex consortium of microorganisms and laboratory tests have shown that the biomass retention, in suspended flocs or attached biofilm, significantly improved SCN− degradation rates. The current research evaluated the process performance in the presence of suspended solids (up to 5.5% m/v) ahead of implementation at a site where complete tailings removal is not possible. Experiments were performed in four 1 l CSTRs (with three primary reactors in parallel at an 8 h residence time, feeding one secondary reactor at a 2.7 h residence time). Stable operation at the design specifications (5.5% solids, 100 mg/l SCN− feed, effluent SCN− 57 mg/l/h was achieved, despite no obvious floc formation. Microbial ecology studies (16S rRNA clone library) revealed reduced diversity relative to reactors operated without suspended solids

The Free Energy Interviews: Scientists and Journalists Collaborate in a Cross-Disciplinary Research Journey

Many students in high schools and universities view science and scientists as “other”. Students have few mechanisms that they can use to access information about “who” a real scientist is, and “what” they do all day. In 2010 we began a project to address this information gap by (i) producing a series of recorded interviews with working science graduates and (ii) supplying these to undergraduate students in a large mixed-interest biochemistry class. We named the project “Free Energy”. Initially a science academic interviewed other scientists alone, however in the second iteration we included student interviewers as well. To obtain course credit these students, who are all co-authors on this paper, used Free Energy as the basis for their Summer Undergraduate Research Experiences. We present a description of the development and delivery of Free Energy and explain how we used it as the subject of student research projects in a Science faculty. We also explain what we as academics and student interviewers have learned from the process of interviewing science graduates in a working radio studio and delivering these recorded interviews to large groups of undergraduate students