Microaerobic Process Research Articles

High-efficiency production of the antimicrobial peptide pediocin PA-1 in metabolically engineered Corynebacterium glutamicum using a microaerobic process at acidic pH and elevated levels of bivalent calcium ions

BackgroundPediocin PA-1 is a bacteriocin of recognized value with applications in food bio-preservation and the medical sector for the prevention of infection. To date, industrial manufacturing of pediocin PA-1 is limited by high cost and low-performance. The recent establishment of the biotechnological workhorse Corynebacterium glutamicum as recombinant host for pediocin PA-1 synthesis displays a promising starting point towards more efficient production.ResultsHere, we optimized the fermentative production process. Following successful simplification of the production medium, we carefully investigated the impact of dissolved oxygen, pH value, and the presence of bivalent calcium ions on pediocin production. It turned out that the formation of the peptide was strongly supported by an acidic pH of 5.7 and microaerobic conditions at a dissolved oxygen level of 2.5%. Furthermore, elevated levels of CaCl2 boosted production. The IPTG-inducible producer C. glutamicum CR099 pXMJ19 Ptac pedACDCg provided 66 mg L−1 of pediocin PA-1 in a two-phase batch process using the optimized set-up. In addition, the novel constitutive strain Ptuf pedACDCg allowed successful production without the need for IPTG.ConclusionsThe achieved pediocin titer surpasses previous efforts in various microbes up to almost seven-fold, providing a valuable step to further explore and develop this important bacteriocin. In addition to its high biosynthetic performance C. glutamicum proved to be highly robust under the demanding producing conditions, suggesting its further use as host for bacteriocin production.

Treatment of coking wastewater by a novel full-scale microaerobic-anoxic-oxic (M/A/O) system: Performance and microbial community analysis.

The aim of this study was to investigate the relationship between system removal performance and microbial community structure in a novel full-scale microaerobic-anoxic-oxic (M/A/O) system for coking wastewater (CWW) treatment. The results showed that 93% of chemical oxygen demand (COD) and 99% of NH4+-N removal efficiency were achieved via M/A/O process, meanwhile, main organic pollutants in CWW including phenolic compounds, heterocyclic compounds and polycyclic aromatic hydrocarbons were basically removed. Four dominant phyla of Proteobacteria, Firmicutes, Bacteroidetes and Nitrospirae were demonstrated distributed in the system and played significant roles in M/A/O biological treatment process. Major function of the microaerobic process was to partly remove the biodegradable substances such as phenols and hydrolyze the refractory contaminants such as N-heterocyclic compounds to improve the biological oxygen demand/chemical oxygen demand (BOD5/COD) ratio and release ammonia. This work illustrated the structure and function of microbial community in M/A/O system and provided a new choice for high-strength CWW treatment.

Co-Production of Isoprene and Lactate by Engineered Escherichia coli in Microaerobic Conditions.

Lactate and isoprene are two common monomers for the industrial production of polyesters and synthetic rubbers. The present study tested the co-production of D-lactate and isoprene by engineered Escherichia coli in microaerobic conditions. The deletion of alcohol dehydrogenase (adhE) and acetate kinase (ackA) genes, along with the supplementation with betaine, improved the co-production of lactate and isoprene from the substrates of glucose and mevalonate. In fed-batch studies, microaerobic fermentation significantly improved the isoprene concentration in fermentation outlet gas (average 0.021 g/L), compared with fermentation under aerobic conditions (average 0.0009 g/L). The final production of D-lactate and isoprene can reach 44.0 g/L and 3.2 g/L, respectively, through fed-batch microaerobic fermentation. Our study demonstrated a dual-phase production strategy in the co-production of isoprene (gas phase) and lactate (liquid phase). The increased concentration of gas-phase isoprene could benefit the downstream process and decrease the production cost to collect and purify the bio-isoprene from the fermentation outlet gas. The proposed microaerobic process can potentially be applied in the production of other volatile bioproducts to benefit the downstream purification process.

Effect of microaerophilic treatment on swine wastewater (SWW) treatment: Engineering and microbiological aspects

The microaerobic process on swine wastewater (SWW) treatment was investigated, evaluating its effect on organic matter hydrolysis and removal, biogas production, operational stability, and microbial community structure. UASB reactors operating under higher organic loading rates (OLRs) and lower hydraulic retention times (HRTs) than those found in the SWW treatment literature were also assessed. The microaerophilic reactor R2 presented a higher total and particulate organic matter removals and operational stability than the anaerobic reactor R1, reaching CODP removals of 79.4 ± 4.6%. In the specific methanogenic activity (SMA) tests, the microaerobic sludge (R2) showed hydrolytic and acetogenic/methanogenic activity superior to inoculum and anaerobic sludge (R1). The microbiological evaluation of R2 revealed the high presence of hydrolytic microorganisms, therefore justifying the higher hydrolytic activity found in the SMA tests and higher particulate organic matter removal found in the microaerobic reactor.

Efficiency and mechanism of nitrogen removal from piggery wastewater in an improved microaerobic process

Characterized by high ammonium (NH4+ − N) and low ratio of chemical oxygen demand (COD) to total nitrogen (COD/TN), discharge of piggery wastewater has been identified as a primary pollution source resulting in water eutrophication. An improved microaerobic reactor, internal aerating microaerobic reactor (IAMR), was constructed to treat manure-free piggery wastewater without effluent recycle at dissolved oxygen of 0.3 mg/L and 32 °C. A removal rate of COD, NH4+ − N and TN averaged 77.9%, 94.6% and 82.6% was obtained in the reactor, with the concentration of 258.5, 235.5 and 335.2 mg/L in influent, respectively. 16S rDNA amplicon sequencing, carbon and nitrogen mass balance and stoichiometry indicated that heterotrophic nitrification-anammox was the dominant approach to nitrogen removal. Microbiome phenotypes showed that aerobic bacteria were the dominant microorganisms, and the microbiome oxidative stress tolerance was intensified along with the continuous operation of the IAMR, resulting in the survival of various facultative and anaerobic bacteria for nutrients removal. With the good nutrients removal, less energy consumption, and high tolerance to influent fluctuation, the improved IAMR was confirmed as a promising process for treating wastewater with high NH4+ − N and low COD/TN.

Effect of micro-aerobic process on improvement of anaerobic digestion sewage sludge treatment: flow cytometry and ATP assessment.

Micro-aeration as a pretreatment method improves the efficiency of anaerobic digestion of municipal sewage sludge and consequently promotes the methane production. In this study, adenosine triphosphate (ATP) and flow cytometry (FCM) were employed to monitor the performance of the micro-aerobic process and investigate the survival of bacterial cells within the process. At first, the effect of air flow rate (AFR) (0.1, 0.2, 0.3 and 0.5 vvm) on hydrolysis of mixed sludge in 5 aeration cycles (20, 30, 40, 48 and 60 hours) was examined. Then, the effects of the micro aerobic process on methane (CH4) production in anaerobic digestion were surveyed. The highest VSS reduction was 30.6% and 10.4% for 40 hours in the reactor and control, respectively. Soluble COD also fluctuated between 40.87 and 65.14% in micro-aerobic conditions; the highest SCOD was achieved at the time of 40 h. Microbial activities were increased by 597%, 170% and 79.4% for 20, 30 and 40 h pretreatment with the micro-aerobic process, respectively. Apoptosis assay showed that micro-aerobic pre-treatment at 20, 30 and 40 h increased the percentage of living cells by 57.4, 62.8 and 67.9%, respectively. On the other hand, FCM results showed that the highest percentage of viable bacteria (i.e., 67.9%) was observed at 40 h pretreating which was approximately 40% higher the ones for the control. Variation in cumulative methane production shows that methane production was increased by 221% compared to anaerobic digestion (control group). Therefore, ATP and FCM can be employed as two appropriate, accurate, relatively specific indicators for monitoring the process and bacteria viability.

Shake flask methodology for assessing the influence of the maximum oxygen transfer capacity on 2,3-butanediol production

BackgroundProduction of 2,3-butanediol from renewable resources is a promising measure to decrease the consumption of fossil resources in the chemical industry. One of the most influential parameters on biotechnological 2,3-butanediol production is the oxygen availability during the cultivation. As 2,3-butanediol is produced under microaerobic process conditions, a well-controlled oxygen supply is the key parameter to control biomass formation and 2,3-butanediol production. As biomass is on the one hand not the final product, but on the other hand the essential biocatalyst, the optimal compromise between biomass formation and 2,3-butanediol production has to be defined.ResultsA shake flask methodology is presented to evaluate the effects of oxygen availability on 2,3-butanediol production with Bacillus licheniformis DSM 8785 by variation of the filling volume. A defined two-stage cultivation strategy was developed to investigate the metabolic response to different defined maximum oxygen transfer capacities at equal initial growth conditions. The respiratory quotient was measured online to determine the point of glucose depletion, as 2,3-butanediol is consumed afterwards. Based on this strategy, comparable results to stirred tank reactors were achieved. The highest space–time yield (1.3 g/L/h) and a 2,3-butanediol concentration of 68 g/L combined with low acetoin concentrations and avoided glycerol formation were achieved at a maximum oxygen transfer capacity of 13 mmol/L/h. The highest overall 2,3-butanediol concentration of 78 g/L was observed at a maximum oxygen transfer capacity of 4 mmol/L/h.ConclusionsThe presented shake flask approach reduces the experimental effort and costs providing a fast and reliable methodology to investigate the effects of oxygen availability. This can be applied especially on product and by-product formation under microaerobic conditions. Utilization of the maximum oxygen transfer capacity as measure for the oxygen availability allows for an easy adaption to other bioreactor setups and scales.

Nutrient removal from high ammonium swine wastewater in upflow microaerobic biofilm reactor suffered high hydraulic load

To understand the ability of an upflow microaerobic biofilm reactor (UMBR) to remove nutrient from manure-free swine wastewater rich in NH4+ with a COD/TN ratio less than 1.00, effect of hydraulic loading rate (HLR) on the microaerobic process was evaluated with a constant reflux ratio of 25 at 25 °C. The results showed that changes in HLR had a remarkable effect on the performance of the UMBR in nutrient removal from the wastewater. With the favorable HLR 3.0 m3/(m3·d) (Hydraulic Retention Time (HRT) 8 h), average removal of COD, NH4+ and TN in the microaerobic process reached 59.3%, 87.7% and 84.7%, respectively, though the COD/TN ratio was as low as 0.84. With an over HLR of 4.0 m3/(m3·d) (HRT decreased to 6 h), bad performance of the UMBR was observed with an average removal of COD, NH4+ and TN as low as 45.0%, 59.0% and 57.5%, respectively. Since the HLR was decreased to 2.4 m3/(m3·d) (HRT 10 h), the microaerobic process regained the efficiency in nutrient removal with a removal of COD, NH4+ and TN averaged 59.0%, 95.3% and 87.8%, respectively. The microaerobic condition allowed anammox bacteria, ammonia-oxidizing bacteria and archaea, nitrite-oxidizing bacteria and denitrifiers to all thrive in the UMBR, resulting in the efficient synchronous removal of organic carbon and nitrogen. As the dominant approach to nitrogen removal, anaerobic ammonium oxidation (anammox) pathway contributing to the TN removal in the microaerobic process exceeded 59.5% at HLR 3.0 m3/(m3·d). The results demonstrated that the UMBR can remove nitrogen and carbon from swine wastewater, with a suitable HLR.

Effect of temperature on nitrogen removal and biological mechanism in an up-flow microaerobic sludge reactor treating wastewater rich in ammonium and lack in carbon source

Previous study has demonstrated that microaerobic process is effective in nitrogen removal from the wastewater with high ammonium and low carbon to nitrogen ratio. In the microaerobic system, synergistic action of anammox, ammonia oxidizing bacteria (AOB), nitrite oxidizing bacteria (NOB) and denitrifiers was the key issues to remove nitrogen from the wastewater rich in ammonium. Temperature has a significant effect on specific growth rate and activity of various nitrogen removal functional bacteria. In this study, the effect of temperature (35 °C–15 °C) on nitrogen removal were investigated in an up-flow microaerobic sludge reactor (UMSR) at the HRT of 8 h and reflux ratio of 45. Above 71.2% of total nitrogen (TN) and 80.7% of NH4+ removal efficiencies were observed at the temperature no less than 17 °C. With the temperature further decreasing to 15 °C, denitrifiers still dominant the UMSR, but AOB, NOB and Candidatus Brocadia as the predominant anammox bacteria were inhibited revealed by high throughput sequencing, resulting in the decrease of TN and NH4+ removal to 39.7% and 61.8%, respectively. Fortunately, when the temperature rebounded to 20 °C, a higher TN and NH4+ removal of 81.2% and 97.3% were obtained again in the UMSR.

Enhanced nitrogen removal from piggery wastewater with high NH4+ and low COD/TN ratio in a novel upflow microaerobic biofilm reactor

To enhance nutrient removal more cost-efficiently in microaerobic process treating piggery wastewater characterized by high ammonium (NH4+-N) and low chemical oxygen demand (COD) to total nitrogen (TN) ratio, a novel upflow microaerobic biofilm reactor (UMBR) was constructed and the efficiency in nutrient removal was evaluated with various influent COD/TN ratios and reflux ratios. The results showed that the biofilm on the carriers had increased the biomass in the UMBR and enhanced the enrichment of slow-growth-rate bacteria such as nitrifiers, denitrifiers and anammox bacteria. The packed bed allowed the microaerobic biofilm process perform well at a low reflux ratio of 35 with a NH4+-N and TN removal as high as 93.1% and 89.9%, respectively. Compared with the previously developed upflow microaerobic sludge reactor, the UMBR had not changed the dominant anammox approach to nitrogen removal, but was more cost-efficiently in treating organic wastewater with high NH4+-N and low COD/TN ratio.

ANAEROBIC DIGESTION OF WASTEWATER WITH HIGH SULFATE CONCENTRATION USING MICRO-AERATION AND NATURAL ZEOLITES

Abstract - The behavior of anaerobic digestion in batch and UASB reactors using a microaerobic process and natural zeolites was studied. Laboratory assays were carried out across 4 sets of variables: different COD/SO 42- ratios, different airflow levels, with and without natural zeolites and room and mesophilic controlled temperatures. The microaerobic process demonstrated hydrogen sulfide removal levels exceeding 90% in most cases, while maintaining the flammable condition of the generated biogas. The level of COD removal exceeded 75% in UASB reactors despite their operation under very low hydraulic retention times (2.8–4.8 hours). The effectiveness of natural zeolites in accelerating UASB reactor startup was demonstrated. Results showing the positive influence of zeolites on the granulation process in UASB reactors were also achieved. Keywords : Granulation process; Micro-aeration; Sulfate; Sulfide; Temperature; UASB; Zeolite. INTRODUCTION The advantages of anaerobic technology in or-ganic matter removal from wastewater have been fully demonstrated in multiple studies and full-scale implementations (Pabon

Technical, Economical, and Microbiological Aspects of the Microaerobic Process on H2S Removal for Low Sulfate Concentration Wastewaters.

We studied the feasibility of the microaerobic process, in comparison with the traditional chemical absorption process (NaOH), on H2S removal in order to improve the biogas quality. The experiment consisted of two systems: R1, biogas from an anaerobic reactor was washed in a NaOH solution, and R2, headspace microaeration with atmospheric air in a former anaerobic reactor. The microaeration used for low sulfate concentration wastewater did not affect the anaerobic digestion, but even increased system stability. Methane production in the R2 was 14% lower compared to R1, due to biogas dilution by the atmospheric air used. The presence of oxygen in the biogas reveals that not all the oxygen was consumed for sulfide oxidation in the liquid phase indicating mass transfer limitations. The reactor was able to rapidly recover its capacity on H2S removal after an operational failure. Bacterial and archaeal richness shifted due to changes in operational parameters, which match with the system functioning. Finally, the microaerobic system seems to be more advantageous for both technical and economical reasons, in which the payback of microaerobic process for H2S removal was 4.7months.

The thermophilic (55 °C) microaerobic pretreatment of corn straw for anaerobic digestion

Microaerobic process has been proven to be an alternative pretreatment for the anaerobic digestion (AD) process in several studies. In this study, the effect of thermophilic microaerobic pretreatment (TMP) on the AD of corn straw was investigated. Results indicated that TMP process obviously improved the methane yield. The maximum methane yield was obtained at the oxygen loads of 5ml/g VSsubstrate, which was 16.24% higher than that of untreated group. The modified first order equation analysis showed the TMP process not only accelerated the hydrolysis rates but also reduced the lag-phase time of AD process. The structural characterization analysis showed cellulosic structures of corn straw were partly disrupted during TMP process. The crystallinity indexes were also decreased. In addition, large or destroyed pores and substantial structural disruption were observed after pretreatment. The results showed that TMP is an efficient pretreatment method for the AD of corn straw.

Model‐based derivation, analysis and control of unstable microaerobic steady‐states—Considering Rhodospirillum rubrum as an example

Microaerobic (oxygen-limited) conditions are critical for inducing many important microbial processes in industrial or environmental applications. At very low oxygen concentrations, however, the process performance often suffers from technical limitations. Available dissolved oxygen measurement techniques are not sensitive enough and thus control techniques, that can reliable handle these conditions, are lacking. Recently, we proposed a microaerobic process control strategy, which overcomes these restrictions and allows to assess different degrees of oxygen limitation in bioreactor batch cultivations. Here, we focus on the design of a control strategy for the automation of oxygen-limited continuous cultures using the microaerobic formation of photosynthetic membranes (PM) in Rhodospirillum rubrum as model phenomenon. We draw upon R. rubrum since the considered phenomenon depends on the optimal availability of mixed-carbon sources, hence on boundary conditions which make the process performance challenging. Empirically assessing these specific microaerobic conditions is scarcely practicable as such a process reacts highly sensitive to changes in the substrate composition and the oxygen availability in the culture broth. Therefore, we propose a model-based process control strategy which allows to stabilize steady-states of cultures grown under these conditions. As designing the appropriate strategy requires a detailed knowledge of the system behavior, we begin by deriving and validating an unstructured process model. This model is used to optimize the experimental conditions, and identify properties of the system which are critical for process performance. The derived model facilitates the good process performance via the proposed optimal control strategy. In summary the presented model-based control strategy allows to access and maintain microaerobic steady-states of interest and to precisely and efficiently transfer the culture from one stable microaerobic steady-state into another. Therefore, the presented approach is a valuable tool to study regulatory mechanisms of microaerobic phenomena in response to oxygen limitation alone. Biotechnol. Bioeng. 2014;111: 734-747. © 2013 Wiley Periodicals, Inc.

The role of the headspace in hydrogen sulfide removal during microaerobic digestion of sludge

The role of the headspace (HS) in the microaerobic removal of hydrogen sulfide from biogas produced during sludge digestion was studied. Research was carried out in a pilot reactor with a total volume of 265 L, under mesophilic conditions. Biogas was successfully desulfurized (99%) by introducing pure oxygen (0.46 NL/L(fed)) into the recirculation stream when the HS volume was both 50.0 and 9.5 L. The removal efficacy dropped sharply to ≈15% when the HS was reduced to 1.5 L. The system responded quickly to the operational changes imposed: micro-oxygenation stops and variations in supply, as well as HS volume reductions and increases. As the final result, the microaerobic process required a minimum surface into the gas space to occur, which along with the elemental sulfur deposition in this area indicated that the oxidation took place there. Additionally, the pattern of sulfur accumulation suggested that the removal occurred preferentially on certain materials, and pointed to a significant biological contribution.

Stepwise reduction of the culture redox potential allows the analysis of microaerobic metabolism and photosynthetic membrane synthesis in Rhodospirillum rubrum

Bacterial growth under oxygen-limited (microaerobic) conditions is often accompanied by phenomena of great interest for fundamental research and industrial application. The microaerobic lifestyle of anoxygenic photosynthetic bacteria like Rhodospirillum rubrum harbors such a phenomenon, as it allows the formation of photosynthetic membranes and related interesting products without light. However, due to the technical difficulties in process control of microaerobic cultivations and the limited sensitivity of available oxygen sensors, the analysis of microaerobic growth and physiology is still underrepresented in current research. The main focus of the present study was to establish an experimental set-up for the systematic study of physiological processes, associated with the growth of R. rubrum under microaerobic conditions in the dark. For this purpose, we introduce a robust and reliable microaerobic process control strategy, which applies the culture redox potential (CRP) for assessing different degrees of oxygen limitation in bioreactor cultivations. To describe the microaerobic growth behavior of R. rubrum cultures for each of these defined CRP reduction steps, basic growth parameters were experimentally determined. Flux variability analysis provided an insight into the metabolic activity of the TCA cycle and implied its connection to the respiratory capacity of the cells. In this context, our results suggest that microaerobic growth of R. rubrum can be described as an oxygen-activated cooperative mechanism. The present study thus contributes to the investigation of metabolic and regulatory events responsible for the redox-sensitive formation of photosynthetic membranes in facultative photosynthetic bacteria. Furthermore, the introduced microaerobic cultivation setup should be generally applicable for any microbial system of interest which can be cultivated in common stirred-tank bioreactors.

Production of High Calorific Biogas from Organic Wastewater and Enhancement of Anaerobic Digestion

Anaerobic digestion is a widely applied technology to produce biogas from organic wastewater. The biogas calorific value depends on the methane-content. For biogas flows >100 m3/h, the two-step process is usually used for production of high calorific biogas from organic wastewater: the first step, anaerobic digestion; the second step, biogas purification. However, for biogas flows 3/h, biogas purification is not economical, and one-step process according to the big gap between methane and non-methane-gas in solubility at higher pressure or lower temperature, should be condidered. New anaerobic digestion processes, such as micro-aerobic process, electrolysis enhancing methane production process, process of internal circulation anaerobic digester (ICAD) with sewage source heat pump, may all enhance biogas producton or lower biogas production cost. In addition, suitable environmental conditions, such as organic loading rate (OLR), solid retention time (SRT), hydraulic retention time (HRT) and surface area, are all beneficial to enhance methane fermentation. Furthermore, new operation modes and optimal dose of trace metals might be selected.

Flocculation, Anaerobic and Microaerobic Treatment Process of Normal Temperature Low Concentration Domestic Sewage

Domestic sewage was treated by adopting flocculation, anaerobic and microaerobic combined process under normal temperature. The result demonstrates: total hydraulic retention time is 5.5h (chemical flocculation 1.0h, UASB reactor 2.0h and MUSB reactor 2.5h). UASB reactor can achieve anaerobic sludge granulation under normal temperature and is fairly feasible for low concentration domestic sewage treatment. Compared with single stage UASB reactor, hydraulic retention time of flocculation-UASB combined technique reduces from 4h to 2h. COD removal rate rises from 45% to 50%-60% and suspended COD is mainly removed. DO of microaerobic MUSB technique after anaerobic treatment is 0.2mg/L-0.5mg/L with air and water ratio of 1:1. Effluent quality is stable, in accordance with first standard of Synthetical Draining Standard of Sewage, GB8978—1996.

Microaerobic steroid biosynthesis and the molecular fossil record of Archean life

The power of molecular oxygen to drive many crucial biogeochemical processes, from cellular respiration to rock weathering, makes reconstructing the history of its production and accumulation a first-order question for understanding Earth's evolution. Among the various geochemical proxies for the presence of O(2) in the environment, molecular fossils offer a unique record of O(2) where it was first produced and consumed by biology: in sunlit aquatic habitats. As steroid biosynthesis requires molecular oxygen, fossil steranes have been used to draw inferences about aerobiosis in the early Precambrian. However, better quantitative constraints on the O(2) requirement of this biochemistry would clarify the implications of these molecular fossils for environmental conditions at the time of their production. Here we demonstrate that steroid biosynthesis is a microaerobic process, enabled by dissolved O(2) concentrations in the nanomolar range. We present evidence that microaerobic marine environments (where steroid biosynthesis was possible) could have been widespread and persistent for long periods of time prior to the earliest geologic and isotopic evidence for atmospheric O(2). In the late Archean, molecular oxygen likely cycled as a biogenic trace gas, much as compounds such as dimethylsulfide do today.