Elucidation of ydfW pseuodogene as a functional protein in Escherichia coli during hydrogen metabolism

Hydrogen holds a promise for a clean energy source. In capacity of high energy content, 122 kJ g-1 and classified as a renewable source, hydrogen is expected as the optimum solution for a cleaner energy. Besides, it can be generated by various methods and biological production gains a great interest as it can be produced easily from variety of carbon sources through dark fermentation. However, biohydrogen comes with some of technical barriers, basically about the production, storage and utilization. Other than that, the major drawback in microbe-mediated bioconversion is low hydrogen yield problem. Due to these problems, microorganism need to be studied in detail including the pathway and then do the metabolic engineering in order to improve the hydrogen production. At present, only Escherichia coli bears a high potential to produce hydrogen because it is well studied and easy to be genetic manipulated. Nonetheless, the specific genes involved in hydrogen metabolism are still not truly discovered. In this study, by screening E. coli BW25113 using chemochromic membranes, ydfW is found as one of the potential pseuodogenes that related to the hydrogen metabolism. Then, through conducting several fermentation and computational analysis, it was revealed that ydfW pseuodogene strongly affecting hydrogen production in E. coli

Enhanced Bioenergy Production through Integration of Molecular Biotechnology and Bioprocess Technology Approaches

九州工業大学博士学位論文 学位記番号:生工博甲第209号　学位授与年月日:平成25年9月27日Chapter1 General overview and big picture|Chapter2 Uncharacterized Genes in Escherichia coli Related to Biohydrogen Production|Chapter3 Uncharacterized Escherichia coli Proteins YdjA and YhjY Related to Biohydrogen Production|Chapter4 Metabolic Engineering to Enhance Hydrogen Production Using YhjY in Escherichia Coli|Chapter5 The curiosity a cryptic function of pseudogene in Escherichia coli genome towards hydrogen production|Chapter6 Electricity Generation from Waste Activated Sludge Using Microbial Fuel Cells Application|Chapter7 Influence of Pretreated Activated Sludge for Electricity Generation in Microbial Fuel Cell Application|Chapter8 Exclusive conclusion and suggestion九州工業大学平成25年

Enrichment of anaerobic ammonium oxidation (anammox) bacteria for short start-up of the anammox process: a review

The application of the anaerobic ammonium oxidation (anammox) reaction in a biological nitrogen removal system to treat wastewater has become of great interest since its discovery. The anammox reaction is performed by anammox bacteria that belong to the Planctomycete phylum. The reaction occurs in the presence of ammonium using nitrite as the substrate under anaerobic conditions. However, the bacteria have an extremely slow growth rate and stringent metabolic conditions that cause difficulty in culturing and applying the system for wastewater treatment. Anammox enrichment has a long start-up period for the anammox process that hinders researchers using laboratory and full-scale systems for the first time. Many attempts have been made to culture anammox to establish a successful anammox culture with a shorter start-up period for the anammox reaction and high nitrogen removal activity. This paper reviews previous studies on anammox enrichment with emphasis on (i) inoculum selection, (ii) enrichment techniques and (iii) factors influencing anammox enrichment. This review will assist researchers in planning and designing an appropriate anammox enrichment. The findings should widen the application of anammox in biological nitrogen removal systems for nitrogenous wastewater

Start-up of biohydrogen production from palm oil mill effluent under non-sterile condition in 50 L continuous stirred tank reactor

Feasibility study of biohydrogen production from Palm Oil Mill Effluent (POME) using POME sludge as a mixed culture of natural inoculum was conducted. The experiment was done using a 150 mL serum bottle and 50 L Continuous Stirred Tank Reactor (CSTR) in batch and continuous modes, respectively. The biogas produced from both fermentations was free from methane due to heat treatment of the sludge prior to inoculation. The results obtained showed that the biohydrogen content in 150 mL serum bottle was higher (70%) than that of 50 L CSTR (25%). The biohydrogen rates for serum bottle and 50 L bioreactor were 74 and 33 NmL/h/L, respectively. Butyrate, propionate and acetate were the main soluble metabolites produced during the fermentation and reduced the pH of broth

Metabolic engineered Escherichia coli to enhance polyhydroxyalkanoates production

Polyhydroxyalkanoates (PHAs) are linear polyesters produced through fermentation of sugar or lipid. Biosynthesis of PHA consists of three enzymes which are acetyl-CoA acetyltransferase (phaA), acetoacetyl-CoA reductase (phaB) and PHA synthase (phaC). Comamonas sp. is one of the strains commonly used for PHA production. Under growth conditions PHA is synthesized by excess of carbon sources and other essential nutrients. In order to develop higher PHA production from bacterial respond strategy, PHA biosynthesis operon of Comamonas sp. EB172 was introduced into Escherichia coli BW25113 through pGEMT vector. E. coli was chosen due to the complete genome information and the absence of depolymerisation gene, phaZ. The presence of PHA operon in E. coli has yielded PHA about 46% (w/w) with glucose as the carbon source. Therefore, the aim of this study is to improve the PHA production by screening of specific genes related to metabolic pathway of PHA in E. coli. Single gene deletion strains of keio collection harboring PHA biosynthesis operon of Comamonas sp. EB172 were used. Six genes pgi, frdC, fdnG, gltA, pta, and poxB were found to be associated with PHA metabolism activity. Second genes knockouts were introduced in through P1 transduction in order to improve PHA production and E. coli BW25113 frdC gltA::kan was shown an improvement of PHA production 53% compared to the wild type

Accelerated Start-up of a Semi-commercial Digester Tank Treating Palm Oil Mill Effluent with Sludge Seeding for Methane Production

The modern closed digesters are becoming more popular for treating palm oil mill effluent (POME) and are currently being installed nationwide in Malaysia to replace the conventional open lagoons and tanks treatment system. This paper describes an accelerated start-up of the 500 m semi-commercial anaerobic digester 3 treating POME and methane gas recovery for clean development mechanism (CDM) project. Results showed that by direct seeding through the transfer of the sludge from either top or bottom of the open digester tank, the start-up period was significantly shortened. The bottom seed sludge transfer led to interesting results including a 24 day start-up period, stable pH condition (pH 6.8-7.2), high COD removal efficiency (>90%), satisfactory VFA to Alk ratio (<0.3), satisfactory biogas production of nearly 1.8 kg/m /d) and methane 3 composition of 50 to 60%. The presence of high amount of methanogens in the seed sludge significantly reduced the need for a long acclimatization period and the digester could be fed with POME within less than a day after the seed sludge transfer process was completed. Close examination using scanning electron microscopy (SEM) and fluorescence in situ hybridization (FISH) revealed abundant amount of bacteria and methanogens, in particular Methanosaeta sp., in the seed sludge samples, which are very important for successful acidogenesis and methanogenesis processes

Triple knockout of frdC gltA and pta genes enhanced PHA production in Escherichia coli

Polyhydroxyalkanoate (PHA) is a linear polyester produced through the fermentation of sugar or lipid. Biosynthesis of PHA comprises three enzymes known as acetyl-CoA acetyltransferase (phaA), acetoacetyl-CoA reductase (phaB) and PHA synthase (phaC). Comamonas sp. is one of the strains commonly used for PHA production. In order to develop higher PHA production from bacterial respond strategy, PHA biosynthesis operon of Comamonas sp. EB172 was introduced into Escherichia coli BW25113 through a pGEM-T vector. E. coli was chosen due to the complete genome information available and the absence of depolymerisation gene, phaZ. In this study, the deletion of several single genes, which are frdC, gltA, and pta, was found to be associated with PHA metabolism activity in E. coli BW25113. P1 transduction was performed to construct multiple genes knockout. The engineered strain, E. coli BW25113 frdCgltApta::kan/pGEM’-phaCABCo, yielded the highest PHA production at 64 wt.% with 1.4 fold higher than that of control strain of E. coli BW25113/pGEM’-phaCABCo. This strain is potential for industrial application for higher PHA production from E. coli

Pseudogene product YqiG is important for pflB expression and biohydrogen production in Escherichia coli BW25113

Pseudogenes in the Escherichia coli genome are assumed to be non-functional. In this study, Keio collection BW25113∆yqiG and YqiG-producing strain (BW25113/pCA24N-YqiG) were used to evaluate the importance of pseudogene yqiG in hydrogen metabolism. Our results show pseudogene protein YqiG was identified as an essential protein in the production of biohydrogen from glucose. The mutant yqiG decreased biohydrogen production from 37 µmol mg−1 protein to 6 µmol mg−1 protein compared to the wild-type strain, and glucose consumption was reduced by 80%. Through transcriptional analysis, we found that the yqiG mutation represses pflB transcription tenfold; pflB encodes pyruvate-formate lyase, one of the key enzymes in the anaerobic metabolism of E. coli. Moreover, production of YqiG stimulated glycolysis and increased biohydrogen productivity 1.5-fold compared to that of the wild-type strain. Thus, YqiG is important for the central glycolysis reaction and is able to influence hydrogen metabolism activity in E. coli

The Effect of Hydraulic Retention Time and Volatile Fatty Acids on Biohydrogen Production from Palm Oil Mill Effluent under Non-Sterile Condition.

The effect of hydraulic retention time and volatile fatty acids produced during fermentation were investigated on biohydrogen production from palm oil mill effluent in a 50 L bioreactor. The fermentation was done in three different hydraulic retention times; HRT 5, HRT 3 and HRT 2 days. Hydraulic retention time and volatile fatty acids concentration showed a vital role in response to the biohydrogen concentration, biohydrogen rate and biohydrogen yield. The maximum biohydrogen concentration was obtained at HRT 2 days with 30% hydrogen content in biogas. The biohydrogen yield and rate were 1054 NmL/L-POME and 44 NmL/h/L-POME, respectively. The lowest biohydrogen yield and rate were observed at HRT 5 days with 557 NmL/L-POME and 5 NmL/h/LPOME, respectively. Meanwhile, the accumulation of propionic acid concentration up to 7 g/L was suggested as a factor that reduced the biohydrogen production.Key words

Ecotoxicological assessment of palm oil mill effluent final discharge by zebrafish (Danio rerio) embryonic assay

Most palm oil mills adopted conventional ponding system, including anaerobic, aerobic, facultative and algae ponds, for the treatment of palm oil mill effluent (POME). Only a few mills installed a bio-polishing plant to treat POME further before its final discharge. The present study aims to determine the quality and toxicity levels of POME final discharge from three different mills by using conventional chemical analyses and fish (Danio rerio) embryo toxicity (FET) test. The effluent derived from mill A which installed with a bio-polishing plant had lower values of BOD, COD and TSS at 45 mg/L, 104 mg/L, and 27 mg/L, respectively. Only mill A nearly met the industrial effluent discharge standard for BOD. In FET test, effluent from mill A recorded low lethality and most of the embryos were malformed after hatching (half-maximal effective concentration (EC50) = 20%). The highest toxicity was observed from the effluent of mill B and all embryos were coagulated after 24 h in samples greater than 75% of effluent (38% of half-maximal lethal concentration (LC50) at 96 h). The embryos in the effluent from mill C recorded high mortality after hatching, and the survivors were malformed after 96 h exposure (LC50 = 26%). Elemental analysis of POME final discharge samples showed Cu, Zn, and Fe concentrations were in the range of 0.10–0.32 mg/L, 0.01–0.99 mg/L, and 0.94–4.54 mg/L, respectively and all values were below the effluent permissible discharge limits. However, the present study found these metals inhibited D. rerio embryonic development at 0.12 mg/L of Cu, and 4.9 mg/L of Fe for 96 h-EC50. The present study found that bio-polishing plant installed in mill A effectively removing pollutants especially BOD and the FET test was a useful method to monitor quality and toxicity of the POME final discharge samples