LIST OF FIGURES AND GRAPHICS
Fluorescence microscopy image of VKTQH... | 5 | |
Figure 1.2. | Diagram of the location of the components of the primary photosynthetic apparatus in VKTQH ................................................................ ................................... | |
10 | ||
Figure 1.3. | Photosynthesis in purple non-sulfur bacteria ............................... | 11 |
Figure 2.1. | Images of different types of substrates .......................................................... | 31 |
Figure 2.2. | Diagram of experimental steps performed in the thesis ................. | 33 |
Figure 2.3. | Diagram of preliminary treatment of different types of substrates ............................................... | 39 |
Figure 2.4. | Details of petroleum hydrocarbon processing model................................. | 40 |
Figure 2.5. | Stages in the petroleum hydrocarbon processing model ......... | 41 |
Figure 3.1. | Oil contaminated sludge samples before and after enrichment .............................. | 44 |
Figure 3.2. | Some VKTQH colonies were isolated from the enrichment sample..... | 45 |
Figure 3.3. | The ability to generate MSH is based on the ability of crystal violet to capture MSH is produced by VKTQH strains....................................... | |
50 | ||
Figure 3.4. | Biofilm formation ability of VKTQH strains petroleum hydrocarbon degradation and Acinetobacter calcoaceticus P23 | |
50 | ||
Figure 3.5. | Growth ability of 10 strains of VKTQH after 7 days of culture cultured at different diesel concentrations .................................. | |
51 | ||
Figure 3.6. | Cultures of 10 strains of VKTQH in 10% diesel oil after 7 culture date ................................................................................................ | |
51 | ||
Figure 3.7. | Growth ability of 10 strains of VKTQH at different concentrations different toluene after 7 days of culture .................................... | |
52 | ||
Figure 3.8. | Cultures of 10 strains of VKTQH at 250 ppm toluene after 7 days of culture ........................................................................ | |
52 | ||
Figure 3.9. | Growth ability of 10 strains of VKTQH after 7 days of culture cultured at different phenol concentrations ........................................ | |
53 | ||
Figure 3.10. | Cultures of 10 strains of VKTQH at 150 ppm phenol after 7 day........................................................................................... | |
53 | ||
Figure 3.11. | Growth ability of 10 strains of VKTQH at different concentrations different naphthalene after 7 days of culture................................. | |
54 | ||
Figure 3.12. | Culture of 10 strains of VKTQH at a concentration of 200 ppm |
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naphthalene after 7 days of culture ............................................... | 54 | |
Figure 3.13. | Growth ability of VKTQH strains at different concentrations pyrene differences after 7 days of culture ..................................... | |
55 | ||
Figure 3.14. | Culture of 10 strains of VKTQH at a concentration of 200 ppm pyrene after 7 days of culture....................................................... | |
55 | ||
Figure 3.15. | Colony shape and cell shape under microscope Electron of strains DD4, DQ41, FO2 ........................................ | |
57 | ||
Figure 3.16. | Phylogenetic tree of 3 strains DD4, DQ41, FO2 ........ | 58 |
Figure 3.17. | Adsorption spectra of cell suspensions of three strains DD4 (A), DQ41 (B), FO2 (C)................................................... .......................... | |
60 | ||
Figure 3.18. | Photosynthetic pigment production ability of VKTQH under two conditions (A) anaerobic, light and (B) aerobic, dark. ........................................ | |
61 | ||
Figure 3.19. | Effect of temperature on biofilm formation Study of VKTQH strains ..................................................................... | |
64 | ||
Figure 3.20. | Effect of pH on biofilm formation of VKTQH strains...................................................................... | |
64 | ||
Figure 3.21. | Effect of NaCl concentration on film formation ability Biology of VKTQH strains .............................................................. | |
66 | ||
Figure 3.22. | Experiment to evaluate the antagonism of strains VKTQH selects................................................................................ | |
70 | ||
Figure 3.23. | Cell density of strains DD4, DQ41 and FO2 in membrane VKTQH biology after 9 days of culture ..................................... | |
71 | ||
Figure 3.24. | Scanning electron microscopy (SEM) images of the scaffolds before and after after VKTQH adhesion ........................................................ | |
72 | ||
Figure 3.25. | Diesel degradation time and cell density of strain VKTQH in single- or multi-strain biofilms without substrate.......................................................................................................... | |
74 | ||
Figure 3.26. | Diesel oil degradation ability and cell density of VKTQH strains DD4, DQ41 and FO2 in multi-strain MSH on the substrate body.......................................................................................................... | |
75 | ||
Figure 3.27. | The decomposition efficiency of n-alkane components (from C8 to C16) has in diesel oil by multi-species biofilm VKTQH on |
different substrates ................................................................................ | 76 | |
Figure 3.28. | PAH degradation efficiency by different types of biofilms each other.......................................................................................................... | |
81 | ||
Figure 3.29. | Chromatogram of crude oil residual composition analysis after 14 days in the oil decomposition experiment using single-strain MSH VKTQH ................................................................................... | |
86 | ||
Figure 3.30. | Chromatogram of crude oil composition analysis after 14 days in oil degradation experiment using multi-strain MSH VKTQH not mounted on a substrate.......................................................................... | |
87 | ||
Figure 3.31. | Chromatogram of crude oil composition analysis after 14 days in oil degradation experiment using multi-strain MSH VKTQH on substrate (light gravel, coconut fiber, foam) ..................................... | |
88 | ||
Figure 3.32. | Chromatogram of crude oil composition analysis after 14 days in the crude oil adsorption capacity experiment of the substrate (gravel) light, coconut fiber, foam) .............................................................. | |
89 | ||
Figure 3.33. | Degradation efficiency of saturated hydrocarbons (%) of crude oil after 14 days of culture by single-strain and multi-strain biofilms VKTQH without substrate...................................................................... | |
91 | ||
Figure 3.34. | Degradation efficiency of saturated hydrocarbons (%) of crude oil after 14 days of culture by multi-strain biofilm VKTQH on the rack body.......................................................................................................... | |
92 |
INTRODUCTION
Petroleum has been used since ancient times and has become increasingly important in society, especially in economics, politics and technology. In addition to economic benefits, petroleum and petroleum products are also one of the serious sources of environmental pollution, discharged from the process of exploiting, using and processing petroleum. Petroleum contains many toxic compounds that are difficult to decompose in nature, are toxic and can cause serious consequences for the ecological environment. In particular, aromatic compounds such as benzene, toluene, naphthalene, pyrene, phenol... have a high solubility in water, toxic to many species of organisms.
Oil pollution treatment can be carried out by mechanical (physical), chemical and biological methods. Of which, physical and chemical methods are often used to treat petroleum hydrocarbon pollution at high concentrations, which are costly. Biological measures using microorganisms (VSV) to decompose petroleum hydrocarbons are effective measures in treating petroleum hydrocarbon pollution at low concentrations, beyond the capacity of mechanical/chemical treatment. Applying VSV capable of decomposing petroleum hydrocarbons while creating biofilms will increase the efficiency of biological treatment.
Purple photosynthetic bacteria (PBL) are anaerobic photosynthetic bacteria, which have been reported to have flexible metabolism, using a variety of substrates, including hydrocarbons. Some PBL have the ability to form biofilms that can play an important role in the degradation and transformation of hydrocarbon compounds in petroleum. PBL are widely distributed in nature, so they have high potential for application in the treatment of petroleum hydrocarbon pollution in situ and ex situ.
The thesis " Research on the ability to decompose petroleum hydrocarbons of some strains of photosynthetic purple bacteria forming biofilms isolated in Vietnam " was carried out with the following objectives and research contents:
Research objectives of the thesis
- Selected some strains of VKTQH from oil-polluted marine areas in Vietnam, which have the ability to create biofilms and decompose petroleum hydrocarbons with high efficiency.
- Evaluate the degradation efficiency of petroleum hydrocarbon compounds by single-strain and multi-strain biofilms of VKTQH on different types of substrates, thereby providing solutions for oil pollution treatment under model conditions.
Research content
1. Select some strains of VKTQH with the ability to form biofilms and decompose petroleum hydrocarbons well; study the biological characteristics and identify the selected strains.
2. Study some physicochemical conditions such as pH, temperature, salt concentration affecting the biofilm formation ability of selected strains.
3. Evaluation of the degradation efficiency of some petroleum hydrocarbon components by single/multi-strain biofilms attached to substrates (coconut fiber, foam, light gravel) or without substrates.
New contributions of the thesis
1) Three strains of VKTQH have been selected in Vietnam that can both create good biofilms and decompose petroleum hydrocarbons with high efficiency.
2) The thesis is the first work to evaluate the efficiency of crude oil and diesel oil decomposition by single-strain and multi-strain VKTQH biofilms on substrates (lightweight gravel, coconut fiber, foam).
CHAPTER 1 OVERVIEW OF DOCUMENTS
1.1. Some basic biological characteristics of photosynthetic purple bacteria
1.1.1. General introduction to photosynthetic purple bacteria
Bacteria (Bacteria) belong to the group of aquatic bacteria (VK) that can grow in anaerobic conditions by photosynthesis but do not release oxygen because they do not receive electrons from the photolysis of water but from some substances such as: hydrogen, simple organic acids, sulfur, hydrogen sulfide, simple sugars and alcohols. VKTQH are often pink to purple in color, photosynthetic pigments all contain bacteriochlorophyll (Bchl) and carotenoids. This group of bacteria has flexible metabolic patterns depending on the living environment conditions, so they are widely distributed in nature [1, 2, 3, 4].
According to Bergey's classification, VKTQH is divided into 2 groups:
- Sulfur-containing biofilm (PSB): has the ability to accumulate sulfur droplets inside the cell.
- Sulfur-free VKTQH (PNSB): unable to accumulate sulfur droplets inside the cell [2].
1.1.2. Ecology of photosynthetic purple bacteria
Large masses (“blooms”) of PSB are often found in aquatic ecosystems containing sulfides. Most species in this group can grow on sulfide-containing media and can oxidize sulfide to varying degrees to non-toxic sulfur forms such as: S0 , S4O62- or SO42- [ 5 ] .
In the bottom of coastal aquaculture ponds, there is often a significant sulfate content, the sulfate-reducing bacteria group is actively active to form sulfide at the bottom layer, sulfide diffuses from the bottom layer to the top along the water column by the concentration gradient difference. Sulfide stimulates the growth of PSB in the area with light penetration and optimal sulfide content. Different species can be obtained at different depths. If the biomass of VKTQH grows strongly, there will be a "bloom" of the pond, making the pond, lake purple or reddish brown. When the "bloom" in the pond, lake occurs, people can distinguish the characteristic cell morphology of the VKTQH genus under the microscope. In ponds, lakes with "bloom", a mixture of species can be found or only 1 species of VKTQH may appear [6].
The “blooming” phenomenon of PNSB often occurs in environments with low (or no) sulfide concentrations. They are often found in stagnant ponds, wastewater sources, in wastewater treatment systems... Wastewater treatment ponds are considered to have suitable conditions for PNSB to grow. PNSB have been found in wastewater treatment systems such as: Rodobacter (Rba.) capsulatus, (Rba.) sphaeroides, Rhodopseudomonas (Rps.) faecalis, Rhodopseudomonas (Rps.) palustris, Rhodospirillum (Rsp.) photometricum, Blastochloris viridis, Rubrivivax gelatinosus, Rhodocyclustenuis, Rubrivivax gelatinosus... [5, 7, 8, 9].
Okubo et al. (2006) discovered that the PNSB group in a livestock wastewater channel formed a red carpet, in which species such as Rba. sphaeroides , Rba. capsulatus and species in the genus Rhodopseudomonas, especially Rps. palustris [9] appeared.
In addition, representatives of VKTQH can be found in some water bodies with harsh conditions such as: hot springs, sulfur springs, alkaline water bodies, acidic water bodies, in sea areas with high salinity and even in ice-covered lakes. The photosynthesis of VKTQH can take place at the highest temperature up to 57 o C and the lowest at 0 o C; the pH range can be as low as 3 and as high as 11; at salinity up to the saturation value of NaCl (~32%) ... [5].
1.1.3. Diversity of photosynthetic purple bacteria
1.1.3.1. Morphological diversity
VKTQH are Gram-negative, unicellular cells and have spherical, comma, spiral, rod shapes, and can also be found in chains under special environmental conditions. The size of the cells is usually from 0.3 - 0.6 m. Most species reproduce by binary fission, some species have polar vegetative cells that often reproduce by budding (characteristic of the genera Rhodopseudomonas and Rhodomicrobium ). When grown under photosynthetic conditions, the cell suspension is often purple, red, yellow-brown, brown or green [2]. The diversity of cell morphology of VKTQH is an important feature used to classify them.
1.1.3.2. Genetic diversity
VKTQH is divided into 3 families, including (i) Chromatiaceae family : includes all purple sulfur bacteria capable of forming sulfur granules inside the cell,
(ii) family Ectothiorhodospiraceae : includes all purple sulfur bacteria capable of forming extracellular sulfur granules, (iii) family Rhodospirilaceae : includes all photosynthetic bacteria that do not accumulate sulfur granules [2] .
a
b
Figure 1.1. Image taken under a fluorescence microscope by VKTQH
(a) Cells of Thermochromatium tepidum isolated from a New Mexico hot spring. Intracellular sulfur granules refract light (arrows); (b) Cells of Rhodobaca bogoriensis isolated from Lake Bogoria (Kenya) [5]
Phylogenetic analysis based on 16S rRNA gene sequence comparison, VKTQH was classified into 3 subclasses (i) Alphaproteobacteria : including non-sulfur VKTQH (ii) Betaproteobacteria : also including non-sulfur VKTQH and (iii) Gammaproteobacteria : including sulfur VKTQH (Tables 1.1 and 1.2) [5].
To date, 20 genera of PNSB have been published (Table 1.1). Rhodobacter and Rhodopseudomonas species have been pioneers in laboratory studies of anaerobic photosynthesis. In addition, several other species with one or more special metabolic features are also known. For example, species that live in extreme hot, cold, saline, alkaline, and acidic environments have been isolated (Figure 1.1b). As shown in Table 1.1, all PNSB are proteobacteria, and the phylogenetic tree shows that many species are closely related to non-phototrophic species [10].