Search results for: Cupriavidus necator

Authors: Geeta Gahlawat, Sanjeev Kumar Soni

Abstract:

An imprudent use of environmentally hazardous petrochemical-based plastics and limited availability of fossil fuels have provoked research interests towards production of biodegradable plastics - polyhydroxyalkanoate (PHAs). However, the industrial application of PHAs based products is primarily restricted by their high cost of recovery and extraction protocols. Moreover, solvents used for the extraction and purification are toxic and volatile which causes adverse environmental hazards. Development of efficient downstream recovery strategies along with utilization of non-toxic solvents will accelerate their commercialization. In this study, various extraction strategies were designed for sustainable and cost-effective recovery of PHAs from Cupriavidus necator using non-toxic environment friendly solvents viz. 1,2-propylene carbonate, ethyl acetate, isoamyl alcohol, butyl acetate. The effect of incubation time i.e. 10, 30 and 50 min and temperature i.e. 60, 80, 100, 120°C was tested to identify the most suitable solvent. PHAs extraction using a recyclable solvent, 1,2 propylene carbonate, showed the highest recovery yield (90%) and purity (93%) at 120°C and 30 min incubation. Ethyl acetate showed the better capacity to recover PHAs from cells than butyl acetate. Extraction with ethyl acetate exhibited high recovery yield and purity of 96% and 92%, respectively at 100°C. Effect of non-toxic surfactant such as linear alkylbenzene sulfonic acid (LAS) was also studied at 40, 60 and 80°C, and detergent pH range of 3.0, 5.0, 7.0 and 9.0 for the extraction of PHAs from the cells. LAS gave highest yield of 86% and purity of 88% at temperature 80°C and 5.0 pH.

Keywords: polyhydroxyalkanoates, Cupriavidus necator, extraction, recovery yield

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Authors: P. Joris, E. Lombard, X. Cameleyre, G. Navarro, A. Paillet, N. Gorret, S. E. Guillouet

Abstract:

Today, on the international space station, multiple supplies are needed per year to supply food and spare parts and to take out waste. But as it is planned to go longer and further into space these supplies will no longer be possible. The astronaut life support system must be able of continuously transform waste into valuable compounds. Two types of production were identified as critical and could be be supplemented by microorganisms. On the one hand, since microgravity causes rapid muscle loss, single cell proteins (SCPs) could be used as protein rich feed or food. On the other hand, having enough building materials to build an advanced habitat will not be possible only by transporting space goods from earth to mars for example. The bacterium Cupriavidus. necator is well known for its ability to produce a large amount of proteins or of polyhydroxyalkanoate biopolymers (PHAs) depending on its implementation. By coupling the life support system to a 3D-printer, astronauts could be supplied with an unlimited amount of building materials. Additionally, based on the design of the life support system, waste streams have been identified: urea from the crew urine and volatile fatty acids (VFAs) from a first stage of organic waste (excrement and food waste) treatment through anaerobic digestion. Thus, the objective of this, within the Spaceship.Fr project, was to demonstrate the feasibility of producing SCPs and PHAs from VFAs and urea in bioreactor. Because life support systems operate continuously as loops, continuous culture experiments were chosen and the effect of the bioreactor dilution rate on biomass composition was investigated. Total transformation of the carbon source into biomass with high SCP or PHA content was achieved in all cases. We will present the transformation performances of VFAs and urea by the bacteria in bioreactor in terms of titers, yields and productivities but also in terms of the quality of SCP and PHA produced, nucleic acid content. We will further discuss the envisioned integration of our process within life support systems.

Keywords: life support system, space travel, waste treatment, single cell proteins, polyhydroxyalkanoates, bioreactor

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Authors: Jian Yu

Abstract:

Carbo dioxide (CO2) from fossil fuel combustion is a prime green-house gas emission. It can be mitigated by microalgae through conventional photosynthesis. The algal oil is a feedstock of biodiesel, a carbon neutral liquid fuel for transportation. The conventional CO2 fixation, however, is quite slow and affected by the intermittent solar irradiation. It is also a technical challenge to reform the bio-oil into a drop-in liquid fuel that can be directly used in the modern combustion engines with expected performance. Here, an artificial photosynthesis system is presented to produce a biopolyester and liquid fuels from CO2, water, and solar power. In this green process, solar energy is captured using photovoltaic modules and converted into hydrogen as a stable energy source via water electrolysis. The solar hydrogen is then used to fix CO2 by Cupriavidus necator, a hydrogen-oxidizing bacterium. Under the autotrophic conditions, CO2 was reduced to glyceraldehyde-3-phosphate (G3P) that is further utilized for cell growth and biosynthesis of polyhydroxybutyrate (PHB). The maximum cell growth rate reached 10.1 g L-1 day-1, about 25 times faster than that of a typical bio-oil-producing microalga (Neochloris Oleoabundans) under stable indoor conditions. With nitrogen nutrient limitation, a large portion of the reduced carbon is stored in PHB (C4H6O2)n, accounting for 50-60% of dry cell mass. PHB is a biodegradable thermoplastic that can find a variety of environmentally friendly applications. It is also a platform material from which small chemicals can be derived. At a high temperature (240 - 290 oC), the biopolyester is degraded into crotonic acid (C4H6O2). On a solid phosphoric acid catalyst, PHB is deoxygenated via decarboxylation into a hydrocarbon oil (C6-C18) at 240 oC or so. Aromatics and alkenes are the major compounds, depending on the reaction conditions. A gasoline-grade liquid fuel (77 wt% oil) and a biodiesel-grade fuel (23 wt% oil) were obtained from the hydrocarbon oil via distillation. The formation routes of hydrocarbon oil from crotonic acid, the major PHB degradation intermediate, are revealed and discussed. This work shows a novel green process from which biodegradable plastics and high-grade liquid fuels can be directly produced from carbon dioxide, water and solar power. The productivity of the green polyester (5.3 g L-1 d-1) is much higher than that of microalgal oil (0.13 g L-1 d-1). Other technical merits of the new green process may include continuous operation under intermittent solar irradiation and convenient scale up in outdoor.

Keywords: bioplastics, carbon dioxide fixation, drop-in liquid fuels, green process

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Authors: Meenakshi Srivastava, A. K. Mishra

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This study characterizes the metabolic versatility of denitrifying bacterial communities residing in the paddy soil using the GC-MS based Phospholipid Fatty Acid (PLFA) analyses simultaneously with nosZ gene based PCR-DGGE (Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis) and real time Q-PCR analysis. We have analyzed the abundance of nitrous oxide reductase (nosZ) genes, which was subsequently related to soil PLFA profile and DGGE based denitrifier community structure. Soil denitrifying bacterial community comprised majority or dominance of Ochrobactrum sp. following Cupriavidus and uncultured bacteria strains in paddy soil of selected sites. Initially, we have analyzed the abundance of the nitrous oxide reductase gene (nosZ), which was found to be related with PLFA based lipid profile. Chandauli of Eastern UP, India represented greater amount of lipid content (C18-C20) and denitrifier’s diversity. This study suggests the positive co-relation between soil PLFA profiles, DGGE, and Q-PCR data. Thus, a close networking among metabolic abilities and taxonomic composition of soil microbial communities existed, and subsequently, such work at greater extent could be helpful in managing nutrient dynamics as well as microbial dynamics of paddy soil ecosystem.

Keywords: denaturing gradient gel electrophoresis, DGGE, nitrifying and denitrifying bacteria, PLFA, Q-PCR

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Authors: Avinash Vellore Sunder, Shikha Shah, Pramod P. Wangikar

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The enzymatic conversion of nitriles to carboxylic acids by nitrilases has gained significance in the green synthesis of several pharmaceutical precursors and fine chemicals. While nitrilases have been characterized from different sources, the industrial application requires the identification of nitrilases that possess higher substrate tolerance, wider specificity and better thermostability, along with the development of an efficient bioprocess for producing large amounts of nitrilase. To produce large amounts of nitrilase, we developed a fed-batch fermentation process on defined media for the high cell density cultivation of E. coli cells expressing the well-studied nitrilase from Alcaligenes fecalis. A DO-stat feeding approach was employed combined with an optimized post-induction strategy to achieve nitrilase titer of 2.5*105 U/l and 78 g/l dry cell weight. We also identified 16 novel nitrilase sequences from genome mining and analysis of substrate binding residues. The nitrilases were expressed in E. coli and their biocatalytic potential was evaluated on a panel of 22 industrially relevant nitrile substrates using high-throughput screening and HPLC analysis. Nine nitrilases were identified to exhibit high activity on structurally diverse nitriles including aliphatic and aromatic dinitriles, heterocyclic, -hydroxy and -keto nitriles. With fed-batch biotransformation, whole-cell Zobelia galactanivorans nitrilase achieved yields of 2.4 M nicotinic acid and 1.8 M isonicotinic acid from 3-cyanopyridine and 4-cyanopyridine respectively within 5 h, while Cupravidus necator nitrilase enantioselectively converted 740 mM mandelonitrile to (R)–mandelic acid. The nitrilase from Achromobacter insolitus could hydrolyze 542 mM iminodiacetonitrile in 1 h. The availability of highly active nitrilases along with bioprocesses for enzyme production expands the toolbox for industrial biocatalysis.

Keywords: biocatalysis, isonicotinic acid, iminodiacetic acid, mandelic acid, nitrilase

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Authors: Vivek B. Ravindran, Aravind Surapaneni, Rebecca Traub, Sarvesh K. Soni, Andrew S. Ball

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Parasitic diseases have a devastating, long-term impact on human health and welfare. More than two billion people are infected with soil-transmitted helminths (STHs), including the roundworms (Ascaris), hookworms (Necator and Ancylostoma) and whipworm (Trichuris) with majority occurring in the tropical and subtropical regions of the world. Despite its low prevalence in developed countries, the removal of STHs from wastewater remains crucial to allow the safe use of sludge or recycled water in agriculture. Conventional methods such as incubation and optical microscopy are cumbersome; consequently, the results drastically vary from person-to-person observing the ova (eggs) under microscope. Although PCR-based methods are an alternative to conventional techniques, it lacks the ability to distinguish between viable and non-viable helminth ova. As a result, wastewater treatment industries are in major need for radically new and innovative tools to detect and quantify STHs eggs with precision, accuracy and being cost-effective. In our study, we focus on the following novel and innovative techniques: -Recombinase polymerase amplification and Surface enhanced Raman spectroscopy (RPA-SERS) based detection of helminth ova. -Use of metal nanoparticles and their relative nanozyme activity. -Colorimetric detection, differentiation and enumeration of genera of helminth ova using hydrolytic enzymes (chitinase and lipase). -Propidium monoazide (PMA)-qPCR to detect viable helminth ova. -Modified assay to recover and enumerate helminth eggs from fresh raw sewage. -Transcriptome analysis of ascaris ova in fresh raw sewage. The aforementioned techniques have the potential to replace current conventional and molecular methods thereby producing a standard protocol for the determination and enumeration of helminth ova in sewage sludge.

Keywords: colorimetry, helminth, PMA-QPCR, nanoparticles, RPA, viable

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Authors: Mohammad Al-Refai, Majed Wakid

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Introduction: Soil-transmitted diseases (STD) are caused by intestinal worms that are transmitted via various routes into the human body resulting in various clinical manifestations. The intestinal worms causing these infections are known as soil transmitted helminths (STH), including Hook worms, Ascaris lumbricoides (A. lumbricoides), Trichuris trichiura (T. trichiura), and Strongyloides sterocoralis (S. sterocoralis). Objectives: The aim of this study was to investigate the prevalence of STH among newly arrived expatriate labors in Jeddah city, Saudi Arabia, using three different techniques (direct smears, sedimentation concentration, and real-time PCR). Methods: A total of 188 stool specimens were collected and investigated at the parasitology laboratory in the Special Infectious Agents Unit at King Fahd Medical Research Center, King Abdulaziz University in Jeddah, Saudi Arabia. Microscopic examination of wet mount preparations using normal saline and Lugols Iodine was carried out, followed by the formal ether sedimentation method. In addition, real-time PCR was used as a molecular tool to detect several STH and hookworm speciation. Results: Out of 188 stool specimens analyzed, in addition to STH parasite, several other types were detected. 9 samples (4.79%) were positive for Entamoeba coli, 7 samples (3.72%) for T. trichiura, 6 samples (3.19%) for Necator americanus, 4 samples (2.13%) for S. sterocoralis, 4 samples (2.13%) for A. lumbricoides, 4 samples (2.13%) for E. histolytica, 3 samples (1.60%) for Blastocystis hominis, 2 samples (1.06%) for Ancylostoma duodenale, 2 samples (1.06%) for Giardia lamblia, 1 sample (0.53%) for Iodamoeba buetschlii, 1 sample (0.53%) for Hymenolepis nana, 1 sample (0.53%) for Endolimax nana, and 1 sample (0.53%) for Heterophyes heterophyes. Out of the 35 infected cases, 26 revealed single infection, 8 with double infections, and only one triple infection of different STH species and other intestinal parasites. Higher rates of STH infections were detected among housemaids (11 cases) followed by drivers (7 cases) when compared to other occupations. According to educational level, illiterate participants represent the majority of infected workers (12 cases). The majority of workers' positive cases were from the Philippines. In comparison between laboratory techniques, out of the 188 samples screened for STH, real-time PCR was able to detect the DNA in (19/188) samples followed by Ritchie sedimentation technique (18/188), and direct wet smear (7/188). Conclusion: STH infections are a major public health issue to healthcare systems around the world. Communities must be educated on hygiene practices and the severity of such parasites to human health. As far as drivers and housemaids come to close contact with families, including children and elderlies. This may put family members at risk of developing serious side effects related to STH, especially as the majority of workers were illiterate, lacking the basic hygiene knowledge and practices. We recommend the official authority in Jeddah and around the kingdom of Saudi Arabia to revise the standard screening tests for newly arrived workers and enforce regular follow-up inspections to minimize the chances of the spread of STH from expatriate workers to the public.

Keywords: expatriate labors, Jeddah, prevalence, soil transmitted helminths

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