ISOLATION AND CHARACTERIZATION POLYHYDROBUTYRATE (PHB) PRODUCING BACTERIA FROM WASTE COOKING OIL USING POMEGRANATE MOLASSES AS CARBON SOURCE

2015 ◽  
Vol 77 (31) ◽  
Author(s):  
Laila Muftah Zargoun ◽  
Nor Azimah Mohd Zain ◽  
Shafinaz Shahir
Keyword(s):  
Carbon Source ◽  
Nile Blue ◽  
Dry Weight ◽  
Waste Cooking Oil ◽  
Biochemical Properties ◽  
Resonance Spectroscopy ◽  
Rrna Gene ◽  
Cooking Oil ◽  
Sudan Black B ◽  
Isolation And Characterization

In this study, a polyhydroxybutyrate (PHB) producing bacterium was isolated from waste cooking oil and characterized for its morphological and biochemical properties. Staining methods utilizing Sudan Black B and Nile Blue A were used on isolated bacterium to demonstrate good capability for synthesizing PHB. It was shown that the isolated bacterium species was related to Bacillus thuringiensis LMA by using 16S rRNA gene sequences analysis. During the stationary phase, the Bacillus strain was subjected to 10 % (w/v) of pomegranate molasses as a carbon source and 5 g/L of peptone as a nitrogen source. 2 ml of batch fermentation was collected. Samples were collected twice during the incubation period for detection of PHB using Sudan Black B. The PHB production accounted for up to 57.45% of the cell dry weight. The PHB produced was characterized using Fourier Transform Infrared Spectroscopy (FTIR) and Nuclear Magnetic Resonance Spectroscopy (NMR). The drastic absorption band at approximately 1717 cm-1 indicated the stretching vibration of C=O group in PHB polyester, while the functional groups of PHB were identified methyl (-CH3) at 1.28 ppm, methylene (-CH2) 2.0 and 2.5 ppm, and methylene doublet group (CH3) at 5.3 ppm.

scholarly journals Polyhydroxyalkanoate Recovery From Newly Screened Bacillus Sp. LPPI-81 Using Various Methods of Extraction From Loktak Lake Sediment Sample

2021 ◽  
Author(s):  
Seid Mohammed Ebu ◽  
Lopamudra Ray
Keyword(s):  
Carbon Source ◽  
Nile Blue ◽  
Wave Length ◽  
Carbon Sources ◽  
Dry Weight ◽  
Acid Group ◽  
Mean Value ◽  
Specific Staining ◽  
Sudan Black B ◽  
Paenibacillus Sp

Abstract Nowadays the conventional plastic wastes are very challenging to environments and its production cost also creates an economic crisis due to petrochemical-based plastic. In order to solve this problem, the current studies were aimed at screening and characterizing these PHA producing isolates and evaluating the suitability of some carbon source for newly screened PHA producing isolates. Some carbon sources such as D-fructose, glucose, molasses, D-ribose and sucrose were evaluated for PHA production. Data were analyzed using SPSS version 20. The 16SrRNA gene sequence of these isolates was performed. This newly isolated taxa was related to Bacillus species. It was designed as Bacillus sp. LPPI-18 and affiliated Bacillus cereus ATCC 14577T (AE01687) (99.10%). Paenibacillus sp. 172 (AF273740.1) was used as an out-group. Bacillus sp. LPPI-18 is a gram-positive, rod-shaped, endospore former, and citrate test positive. This isolate showed positive for amylase, catalase, pectinase, and protease test. They produced intracellular PHA granules when this isolate was stained with Sudan Black B (SBB) and Nile Blue A (NBA) preliminary and specific staining dyes, respectively. Both Temperature and pH used to affect PHA productivity. Bacteria are able to reserve PHA in the form of granules during stress conditions. This isolate produces only when supplied with carbon sources. More PHA contents (PCs) were obtained from glucose, molasses, and D-fructose. In this regard, the maximum mean value of PC was obtained from glucose (40.55±0.7%) and the minimum was obtained from D-Ribose (12.4±1.4%). Great variations (p≤0.05) of PCs were observed among glucose & sucrose, molasses & sucrose and D-fructose & sucrose carbon sources for PHA productivity (PP) of Cell Dry Weight (CDW) g/L. After extraction, PHA film was produced for this typical isolate using glucose as a sole carbon source. Fourier transform infrared spectrum was performed for this isolate and showed the feature of polyester at 1719.64 to 1721.16 wavelength for these extracted samples. The peak of fingerprinting (band of carboxylic acid group) at this wave-length is a characteristic feature of PHB and corresponds to the ester functional group (C=O).

scholarly journals Isolation and Characterization of a Mutant of the Marine Bacterium Alcanivorax borkumensis SK2 Defective in Lipid Biosynthesis

2010 ◽  
Vol 76 (9) ◽  
pp. 2884-2894 ◽  
Author(s):  
Efraín Manilla-Pérez ◽  
Alvin Brian Lange ◽  
Stephan Hetzler ◽  
Marc Wältermann ◽  
Rainer Kalscheuer ◽  
...  
Keyword(s):  
Carbon Source ◽  
Sole Carbon Source ◽  
Marine Bacterium ◽  
Neutral Lipids ◽  
Nile Red ◽  
Dry Weight ◽  
Wax Ester ◽  
Gene Encoding ◽  
Isolation And Characterization ◽  
Key Enzyme

ABSTRACT In many microorganisms, the key enzyme responsible for catalyzing the last step in triacylglycerol (TAG) and wax ester (WE) biosynthesis is an unspecific acyltransferase which is also referred to as wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT; AtfA). The importance and function of two AtfA homologues (AtfA1 and AtfA2) in the biosynthesis of TAGs and WEs in the hydrocarbon-degrading marine bacterium Alcanivorax borkumensis SK2 have been described recently. However, after the disruption of both the AtfA1 and AtfA2 genes, reduced but substantial accumulation of TAGs was still observed, indicating the existence of an alternative TAG biosynthesis pathway. In this study, transposon-induced mutagenesis was applied to an atfA1 atfA2 double mutant to screen for A. borkumensis mutants totally defective in biosynthesis of neutral lipids in order to identify additional enzymes involved in the biosynthesis of these lipids. At the same time, we have searched for a totally TAG-negative mutant in order to study the function of TAGs in A. borkumensis. Thirteen fluorescence-negative mutants were identified on Nile red ONR7a agar plates and analyzed for their abilities to synthesize lipids. Among these, mutant 2 M131 was no longer able to synthesize and accumulate TAGs if pyruvate was used as the sole carbon source. The transposon insertion was localized in a gene encoding a putative cytochrome c family protein (ABO_1185). Growth and TAG accumulation experiments showed that the disruption of this gene resulted in the absence of TAGs in 2 M131 but that growth was not affected. In cells of A. borkumensis SK2 grown on pyruvate as the sole carbon source, TAGs represented about 11% of the dry weight of the cells, while in the mutant 2 M131, TAGs were not detected by thin-layer and gas chromatography analyses. Starvation and lipid mobilization experiments revealed that the lipids play an important role in the survival of the cells. The function of neutral lipids in A. borkumensis SK2 is discussed.

scholarly journals Isolation and Characterization of Phosphate-Solubilizing Bacteria from Mushroom Residues and their Effect on Tomato Plant Growth Promotion

2017 ◽  
Vol 66 (1) ◽  
pp. 57-65 ◽  
Author(s):  
Jian Zhang ◽  
Peng Cheng Wang ◽  
Ling Fang ◽  
Qi-An Zhang ◽  
Cong Sheng Yan ◽  
...  
Keyword(s):  
Plant Growth ◽  
Growth Promotion ◽  
Wood Flour ◽  
Dry Weight ◽  
Rrna Gene ◽  
Plant Growth Promoting Bacteria ◽  
Phosphate Solubilizing Bacteria ◽  
Sequence Comparisons ◽  
Isolation And Characterization ◽  
Phosphate Solubilizing

Phosphorus is a major essential macronutrient for plant growth, and most of the phosphorus in soil remains in insoluble form. Highly efficient phosphate-solubilizing bacteria can be used to increase phosphorus in the plant rhizosphere. In this study, 13 isolates were obtained from waste mushroom residues, which were composed of cotton seed hulls, corn cob, biogas residues, and wood flour. NBRIP solid medium was used for isolation according to the dissolved phosphorus halo. Eight isolates produced indole acetic acid (61.5%), and six isolates produced siderophores (46.2%). Three highest phosphate-dissolving bacterial isolates, namely, M01, M04, and M11, were evaluated for their beneficial effects on the early growth of tomato plants (Solanum lycopersicum L. Wanza 15). Strains M01, M04, and M11 significantly increased the shoot dry weight by 30.5%, 32.6%, and 26.2%, and root dry weight by 27.1%, 33.1%, and 25.6%, respectively. Based on 16S rRNA gene sequence comparisons and phylogenetic positions, strains M01 and M04 belonged to the genus Acinetobacter, and strain M11 belonged to the genus Ochrobactrum. The findings suggest that waste mushroom residues are a potential resource of plant growth-promoting bacteria exhibiting satisfactory phosphate-solubilizing for sustainable agriculture.

scholarly journals Isolation and characterization of a novel Sphingobium yanoikuyae strain variant that uses biohazardous saturated hydrocarbons and aromatic compounds as sole carbon sources

F1000Research ◽  
2020 ◽  
Vol 9 ◽  
pp. 767
Author(s):  
Mautusi Mitra ◽  
Kevin Manoap-Anh-Khoa Nguyen ◽  
Taylor Wayland Box ◽  
Jesse Scott Gilpin ◽  
Seth Ryan Hamby ◽  
...  
Keyword(s):  
Carbon Source ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Aromatic Compounds ◽  
Agar Medium ◽  
Carbon Sources ◽  
Future Research ◽  
Rrna Gene ◽  
Saturated Hydrocarbons ◽  
Isolation And Characterization

Background: Green micro-alga, Chlamydomonas reinhardtii (a Chlorophyte), can be cultured in the laboratory heterotrophically or photo-heterotrophically in Tris-Phosphate-Acetate (TAP) medium, which contains acetate as the carbon source. Chlamydomonas can convert acetate in the TAP medium to glucose via the glyoxylate cycle, a pathway present in many microbes and higher plants. A novel bacterial strain, CC4533, was isolated from a contaminated TAP agar medium culture plate of a Chlamydomonas wild type strain. In this article, we present our research on the isolation, and biochemical and molecular characterizations of CC4533. Methods: We conducted several microbiological tests and spectrophotometric analyses to biochemically characterize CC4533. The 16S rRNA gene of CC4533 was partially sequenced for taxonomic identification. We monitored the growth of CC4533 on Tris-Phosphate (TP) agar medium (lacks a carbon source) containing different sugars, aromatic compounds and saturated hydrocarbons, to see if CC4533 can use these chemicals as the sole source of carbon. Results: CC4533 is a Gram-negative, non-enteric yellow pigmented, aerobic, mesophilic bacillus. It is alpha-hemolytic and oxidase-positive. CC4533 can ferment glucose, sucrose and lactose, is starch hydrolysis-negative, resistant to penicillin, polymyxin B and chloramphenicol. CC4533 is sensitive to neomycin. Preliminary spectrophotometric analyses indicate that CC4533 produces b-carotenes. NCBI-BLAST analyses of the partial 16S rRNA gene sequence of CC4533 show 99.55% DNA sequence identity to that of Sphingobium yanoikuyae strain PR86 and S. yanoikuyae strain NRB095. CC4533 can use cyclo-chloroalkanes, saturated hydrocarbons present in car motor oil, polyhydroxyalkanoate, and mono- and poly-cyclic aromatic compounds, as sole carbon sources for growth. Conclusions: Taxonomically, CC4533 is very closely related to the alpha-proteobacterium S. yanoikuyae, whose genome has been sequenced. Future research is needed to probe the potential of CC4533 for environmental bioremediation. Whole genome sequencing of CC4533 will confirm if it is a novel strain of S. yanoikuyae or a new Sphingobium species.

Isolation and Characterization of Halophilic and Halotolerant Bacteria from Urmia Lake after the Recent Drought Disaster in 2015

2020 ◽  
Vol 9 (2) ◽  
pp. 111-119
Author(s):  
Elham Kazemi ◽  
Vahideh Tarhriz ◽  
Mohammad S. Hejazi ◽  
Mohammad A. Amoozegar
Keyword(s):  
16S Rrna ◽  
Biochemical Properties ◽  
Urmia Lake ◽  
Rrna Gene ◽  
Halotolerant Bacteria ◽  
Thiobacillus Thioparus ◽  
Gram Staining ◽  
Isolation And Characterization ◽  
Plating Method ◽  
Drought Disaster

Background: Due to the last two decades of drought disaster, which resulted in the loss of the main part of Urmia Lake water and changed the natural conditions of an environment, especially ionic strength. Objective: We aimed to isolate and characterize halophilic and halotolerant bacteria in Urmia Lake, Iran, 2015. Urmia Lake is a permanent and salty inland lake located in the Azerbaijan region in northwestern Iran. Methods: Sampling was carried out in multiple water-filled locations of the lake. Liquid basal media for the enrichment of bacteria was successively applied and colonies were isolated by the plating method. Isolates were then distinguished based on differences in colony, Gram staining, microscopic shape, and biochemical properties. Results: One chemolithotrophic isolate belonging to Thiobacillus thioparus and 41 heterotrophic isolates were obtained. The 16S rRNA gene sequences showed that all 42 isolates belong to the genera Kocuria (21.42%), Marinobacter (11.90%), Micrococcus (11.90%), Thalassobacillus (11.90%), Bacillus (11.90%), Halomonas (7.14%) and Thiobacillus (2.38%). Conclusion: Based on 16S rRNA similarity, 5 of 41 isolates showed the potential to be introduced as new species. The dominant genera with abounded frequency were found to be Kocuria, Bacillus and Thalassobacillus genera.

Isolation and Characterization of Pepsin-Solubilized Collagen from Skin of Argentine Shortfin Squid (Illex argentinus)

2011 ◽  
Vol 236-238 ◽  
pp. 19-26 ◽  
Author(s):  
Jun Jie Zhang ◽  
Rui Duan ◽  
Yi Feng Chen ◽  
Xiao Jing Xu
Keyword(s):  
Dry Weight ◽  
Biochemical Properties ◽  
Commercial Fish ◽  
Isolation And Characterization ◽  
Collagen Denaturation ◽  
Sds Page ◽  
Skin Collagen ◽  
Illex Argentinus ◽  
Peptide Maps

Argentine shortfin squid (Illex argentinus) is one of the most important commercial fish in the world. Pepsin-solubilized collagen (PSC) was isolated from squid skin and biochemical properties of PSC were studied. The yields of skin PSC was about 18.5 % (on the dry weight basis). SDS-PAGE patterns showed that the collagen consisted of at least two different polypeptides (α1 and α2 chains). The peptide maps of PSC digested by pepsin were distinct from those of carp skin collagen. Denaturation temperatures, measured by melting point using circular dichroism, was 26.5 °C.The results suggest that squid skin collagen has potential as a possible underutilized resource used in various fields.

scholarly journals Endogenously Synthesized (−)-proto-Quercitol and Glycine Betaine Are Principal Compatible Solutes of Schizochytrium sp. Strain S8 (ATCC 20889) and Three New Isolates of Phylogenetically Related Thraustochytrids

2007 ◽  
Vol 73 (18) ◽  
pp. 5848-5856 ◽  
Author(s):  
Anita N. Jakobsen ◽  
Inga M. Aasen ◽  
Arne R. Strøm
Keyword(s):  
Glycine Betaine ◽  
Optical Rotation ◽  
Compatible Solutes ◽  
Docosapentaenoic Acid ◽  
Dry Weight ◽  
Defined Medium ◽  
18S Rrna Gene ◽  
Resonance Spectroscopy ◽  
Rrna Gene ◽  
Specific Optical Rotation

ABSTRACT We report that endogenously synthesized (−)-proto-quercitol (1d-1,3,4/2,5-cyclohexanepentol) and glycine betaine were the principal compatible solutes of Schizochytrium sp. strain S8 (ATCC 20889) and three new osmotolerant isolates of thraustochytrids (strains T65, T66, and T67). The compatible solutes were identified and quantified by use of nuclear magnetic resonance spectroscopy, and their identity was confirmed by mass spectroscopy and measurement of the specific optical rotation. The cellular content of compatible solutes increased with increasing NaCl concentration of a defined medium. (−)-proto-Quercitol was the dominating solute at all NaCl concentrations tested (0.25 to 1.0 M), e.g., cells of S8 and T66 stressed with 1.0 M NaCl accumulated about 500 μmol (−)-proto-quercitol and 100 μmol glycine betaine per g dry weight. To our knowledge, (−)-proto-quercitol has previously been found only in eucalyptus. The 18S rRNA gene sequences of the four (−)-proto-quercitol-producing strains showed 99% identity, and they displayed the same fatty acid profile. The only polyunsaturated fatty acids accumulated were docosahexaenoic acid (78%) and docosapentaenoic acid (22%). A less osmotolerant isolate (strain T29), which was closely phylogenetically related to Thraustochytrium aureum (ATCC 34304), did not contain (−)-proto-quercitol or glycine betaine. Thus, the level of osmotolerance and the osmolyte systems vary among thraustochytrids.

scholarly journals Engineering the oleaginous yeast Yarrowia lipolytica to produce limonene from waste cooking oil

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Yaru Pang ◽  
Yakun Zhao ◽  
Shenglong Li ◽  
Yu Zhao ◽  
Jian Li ◽  
...  
Keyword(s):  
Carbon Source ◽  
Yarrowia Lipolytica ◽  
Oleaginous Yeast ◽  
Waste Cooking Oil ◽  
Biologically Active ◽  
Citrus Limon ◽  
Mentha Spicata ◽  
Cooking Oil ◽  
Limonene Synthase ◽  
Pharmaceutical Industries

Abstract Background Limonene is an important biologically active natural product widely used in the food, cosmetic, nutraceutical and pharmaceutical industries. However, the low abundance of limonene in plants renders their isolation from plant sources non-economically viable. Therefore, engineering microbes into microbial factories for producing limonene is fast becoming an attractive alternative approach that can overcome the aforementioned bottleneck to meet the needs of industries and make limonene production more sustainable and environmentally friendly. Results In this proof-of-principle study, the oleaginous yeast Yarrowia lipolytica was successfully engineered to produce both d-limonene and l-limonene by introducing the heterologous d-limonene synthase from Citrus limon and l-limonene synthase from Mentha spicata, respectively. However, only 0.124 mg/L d-limonene and 0.126 mg/L l-limonene were produced. To improve the limonene production by the engineered yeast Y. lipolytica strain, ten genes involved in the mevalonate-dependent isoprenoid pathway were overexpressed individually to investigate their effects on limonene titer. Hydroxymethylglutaryl-CoA reductase (HMGR) was found to be the key rate-limiting enzyme in the mevalonate (MVA) pathway for the improving limonene synthesis in Y. lipolytica. Through the overexpression of HMGR gene, the titers of d-limonene and l-limonene were increased to 0.256 mg/L and 0.316 mg/L, respectively. Subsequently, the fermentation conditions were optimized to maximize limonene production by the engineered Y. lipolytica strains from glucose, and the final titers of d-limonene and l-limonene were improved to 2.369 mg/L and 2.471 mg/L, respectively. Furthermore, fed-batch fermentation of the engineered strains Po1g KdHR and Po1g KlHR was used to enhance limonene production in shake flasks and the titers achieved for d-limonene and l-limonene were 11.705 mg/L (0.443 mg/g) and 11.088 mg/L (0.385 mg/g), respectively. Finally, the potential of using waste cooking oil as a carbon source for limonene biosynthesis from the engineered Y. lipolytica strains was investigated. We showed that d-limonene and l-limonene were successfully produced at the respective titers of 2.514 mg/L and 2.723 mg/L under the optimal cultivation condition, where 70% of waste cooking oil was added as the carbon source, representing a 20-fold increase in limonene titer compared to that before strain and fermentation optimization. Conclusions This study represents the first report on the development of a new and efficient process to convert waste cooking oil into d-limonene and l-limonene by exploiting metabolically engineered Y. lipolytica strains for fermentation. The results obtained in this study lay the foundation for more future applications of Y. lipolytica in converting waste cooking oil into various industrially valuable products.

ISOLATION AND CHARACTERIZATION OF POLYHYDROXYALKANOATES (PHAS) PRODUCING BACTERIA FROM BRACKISH STREAM

2016 ◽  
Vol 78 (7) ◽  
Author(s):  
Nor Azimah Mohd Zain ◽  
Laila Muftah Ali Zargoun ◽  
Nur Fatihah Elias ◽  
Mohd Firdaus Abdul-Wahab ◽  
Mohd Suardi Suhaimi
Keyword(s):  
Nile Blue ◽  
Blue Staining ◽  
Pseudomonas Sp ◽  
Property A ◽  
Identification Procedure ◽  
Sudan Black B ◽  
Isolation And Characterization ◽  
Ft Ir ◽  
Lipid Granules

Polyhydroxyalkanoates (PHAs) are biopolymers which have similar characteristics with petrochemical plastics but a step better due to its biodegradable property. A total of 23 strains were isolated from two different brackish sources. In order to detect the PHAs granules, the PHAs producing bacteria were first screened with Sudan Black B staining. Twenty strains were observed with lipid granules and were subjected to further confirmation with Nile blue staining.  From the Nile blue staining, only 10 strains have the ability in producing PHAs and 2 were identified as strong PHAs producers. This study focuses on the 2 strains named S1 and L1. Further identification procedure was carried out and found that strain S1 and L1 belongs to Pseudomonas sp. L1 strain was found to be promising for PHAs production since it accumulated PHAs for about 88.3%. The PHAs produced by this strain was analyzed using Fourier Transform Infrared Spectroscopy (FT-IR) and Nuclear Magnetic Resonance (NMR) analysis and was identified as poly-3-hydroxybutyrate (P-3HB).

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