PREETHI KUMARAVELAYUTHAM-M.SC THESIS PRESENTATION 5
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PREETHI KUMARAVELAYUTHAM-M.SC THESIS PRESENTATION 5
- 1. Impact of Simple and Complex Substrates on the Composition and Diversity of Microbial Communities and the End-product Synthesis PREETHI KUMARAVELAYUTHAM Master Thesis Presentation 31st July 2015 1
- 3. Mixed acid fermentation Breakdown of organic matter into H2, CH4, CO2 with equal amount of lactate, acetate, succinate, and formate using microorganisms in the absence of O2 3
- 4. Mixed culture technology Mixed culture technology ◦ Undefined microbial communities Advantage: simple operation, ease of bioprocessing in a non- sterile environment and high efficiency with less operational cost Knowledge gap Which type of microorganisms are present ? How abundant are they ? How the microorganisms interact each other ? What type of nutrition they use for metabolic activities ? Solution ◦ Exploiting the nucleic acids and proteins for the identification of microbial communities using molecular tools 4
- 5. Research objective 5 Characterization of microbial communities and associated end-products of mixed acid fermentation from different substrates in anaerobic batch system
- 6. Methodology 6 Pre-treated Dairy manure digestate (DMD) Crude- glycerol with other compounds (fatty acids, methanol) α-Cellulose Raw-wheat straw D-glucose DMD with nutrient medium without a carbon source Substrates vs. Inoculum
- 7. Experimental set up Batch experiment operational conditions ◦ DMD was pre-treated at 70°C; 100°C –30 minutes 6 replicates; 37°C at pH =7.2 Gas and liquid product analysis ◦ Gas Chromatography - H2, CO2, and CH4 ◦ High Performance Liquid Chromatography to identify Volatile fatty acids and alcohols 7
- 8. Microbial community studies DNA Extraction ◦ E.Z.D.N.A soil DNA extraction kit Library preparation ◦ PCR amplification of V3-V4 region of 16S rRNA gene Illumina sequencing 1,669,266 sequences were obtained from the 42 biological replicate samples 8
- 9. Bioinformatic analysis 9 Taxonomies of microbial community analysis QIIME PANDAseq assembler Statistical analysis: Multivariate data analysis (PLS-DA), Mixed procedure, and PERMANOVA analysis
- 10. Sample information 10 Treatment 1- DMD grown without substrates Treatment 2-DMD grown on glucose for 9 days Treatment 3-DMD grown on glycerol for 13 days Treatment 4-DMD grown on α-cellulose for 24 days Treatment 5-DMD grown on wheat straw for 12 days Treatment 6-E-DMD (DMD cultured on α -cellulose mixed with fresh pre- treated DMD at a ratio of 1:1 v/v) grown on α-cellulose for 22 days Treatment 7-E-DMD grown on wheat straw for 22 days
- 12. Alpha-diversity analysis of microbial communities grown on different substrates 12 0 500 1000 1500 2000 2500 3000 3500 4000 4500 0 5000 10000 15000 20000 25000 Chao1Ave Seqs/sample 1 2 3 4 5 6 7 1) Treatment 1, DMD grown without substrates; 2) Treatment 2, DMD grown on glucose for 9 days; 3) Treatment 3, DMD grown on glycerol for 13 days; 4) Treatment 4, DMD grown on -cellulose for 24 days; 5) Treatment 5, DMD grown on wheat straw for 12 days; 6) Treatment 6, E-DMD grown on -cellulose for 22 days; 7) Treatment 7, E-DMD grown on wheat straw for 22 days.
- 13. Dominant taxa detected in microbial communities selected by growth of DMD seed on simple and complex substrates Treatment 2 (Glucose) Treatment 3 (Glycerol) Treatment 4 (-cellulose) Treatment 6 (Wheat straw) Sporolactobacillus (G) Enterobacteriaceae(F) Clostridiaceae (F) Ruminococcus(G) Clostridium (G) Caulobacteraceae (F) Ruminococcus (G) Syntrophomonas (G) Clostridiaceae (F) Clostridium(G) Bacteroidaceae (F) Bacteroidales (O) Natranaerobiales(O) Ruminococcus (G) Sporanaerobacter (G) Clostridum (G) Ruminococcaceae (F) Clostridiaceae (F) Coprococcus(G) Thermoanaerobacterales (O) Enterobacteriaceae(F) Enterococcaceae(F) Ruminococcus (G) Oxobacter (G) Coprococcus (G) Thermoanaerobacterales (O) Paenibacillus (G) Ruminococcaceae (F) Clostridiaceae (F) 13
- 14. Gas and VFA’s synthesis by the selected microbial communities on D-glucose 14 0 5 10 15 20 25 30 35 Formate Lactate Acetate Propionate Isobutyrate Butyrate Ethanol VFA'sing/L 0 500 1000 1500 2000 2500 3000 3500 0 24 48 72 96 120 144 168 192 216 Cumulativegasproductionin mL Time in hour H2 CO2 Clostridiaceae (F)> Clostridium (G)> Sporolactobacillus (G)> Enterobacteriacae (F)> Peptostreptococcaceae (F)> Ruminococcus (G)> Acholeplasma (G)
- 15. Gas and VFA’s synthesis by the selected microbial communities on crude-glycerol 15 0 100 200 300 400 500 600 700 800 0 24 48 72 96 120 144 168 192 216 240 264 288 312 cumulativegasproductioninmL Time in hour H2 CO2 0 5 10 15 20 25 Acetate Propionate Butyrate VFA'sing/L Clostridiaceae (F)> Clostridium (G)> Bacteroidales (O)> Bacteroidaceae (F)> Ruminococcus (G)> Caulobacteraceae(F)> Syntrophomonas (G)
- 16. Gas and VFA’s synthesis by the selected microbial communities on α-cellulose 16 0 20 40 60 80 100 120 140 0 24 48 72 96 120 144 168 192 216 240 264 288 312 336 360 384 408 432 456 480 504 CumilativegasproductioninmL Time in hours H2 CO2 0 1 2 3 4 5 6 7 8 Acetate Butyrate Valerate VFAsing/L Ruminococcaceae (F)> Ruminococcus (G)> Clostridium (G)> Natranaerobiales (O)> Coprococcus (G)
- 17. Comparison of H2 and VFA’s produced by enriched microbial populations grown on α-cellulose 17 0 10 20 30 40 50 60 70 80 90 100 0 48 96 144 192 240 288 336 384 432 480 528 CumilativeH2productioninmL Time in hours EDMD DMD Clostridiaceae (F)> Caulobacteraeceae (F)> Sporolactobacillus (G)> Lactobacillus (G) Ruminococcus (G)> Pseudomonas (G) 0 1 2 3 4 5 6 7 8 9 10 Acetate Butyrate Valerate VFAsing/L EDMD DMD
- 18. Gas and VFA’s synthesis by the selected microbial communities on wheat straw 18 0 10 20 30 40 50 60 70 0 48 96 144 192 240 288 CumilativegasproductioninmL Time in hour H2 CO2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 Acetate Propionate Butyrate VFAsing/L Coprococcus (G)> Ruminococcaceae (F)>Thermoanerobacterales (O)> Enterococcaceae(F)> Ruminococcus(G)> Oxobacter (G)>Paenibacillus(G)> Sporanarobacter (G)
- 19. Comparison of H2 and VFA’s produced by enriched microbial populations grown on wheat straw 19 0 5 10 15 20 25 30 35 40 0 48 96 144 192 240 288 336 384 432 480 528 CumilativeH2productioninmL Time in hours EDMD DMD 0 0.5 1 1.5 2 2.5 3 Acetate Propionate Butyrate VFAsing/L EDMD DMD Ruminococcaceae (F) > Natranaerobiales (O)> Rhodospirillaceae (F) >Pelotomaculum (G) >Pseudomonas (G) Ruminococcus (G)> Sporanaerobacter (G)> Pelotomaculum (G)> Caulobacteraceae (F)
- 20. Conclusion Dairy manure digestate (DMD) contained a wide diversity of bacteria Very different sets of microorganisms were enriched from the DMD by each substrate Greater diversity of bacteria was selected when the DMD seed was cultured with crude-glycerol and raw wheat straw The fermentation end-products (H2, CO2, organic acids, and alcohols) synthesized and associated microbial community structure were determined by the carbon source 20
- 21. Future directions 21 Comparison of microbial communities and end- products provided better knowledge to understand the anaerobic fermentation Continuous fermentation reaction and sample collection and end products at various time interval provide insight to the change in the microbial population and end-products over the time
- 22. Thank you 22
- 23. Metagenomic analysis of bacteria grown on D-glucose 23 Taxon Treatment 1 (Blank) Treatment 2 (Glucose) Fold- Enrichment Sporolactobacillus (G) 0.11 17.51 159 Enterobacteriaceae (F) 0.08 10.57 124 Clostridiaceae (F) 4.02 35.36 9 Ruminococcus (G) 0.39 1.75 4 Clostridium (G) 4.70 17.85 4
- 24. Metagenomic analysis of bacteria grown on crude-glycerol 24 Taxon Treatment 1 (Blank) Treatment 3 (Crude-Glycerol) Fold- Enrichment Caulobacteraceae (F) 0.11 7.09 62 Ruminococcus (G) 0.39 5.83 15 Syntrophomonas (G) 0.76 4.46 9 Clostridiaceae (F) 4.02 14.35 4 Clostridium (G) 4.70 7.40 2 Bacteroidaceae (F) 4.16 6.08 1 Bacteroidales (O) 5.84 7.81 1
- 25. Comparative analysis of microbial communities selected by growth on glucose versus crude-glycerol 25 Taxon Treatment 2 (Glucose) Treatment 3 (Crude-Glycerol) Fold- Enrichment Sporolactobacillus (G) 0.001 17.51 17,672 Clostridiaceae (F) 4.56 35.36 7 Clostridium (G) 2.48 17.85 7 Peptostreptococcaceae (F) 0.94 2.39 3 Enterobacteriaceae (F) 4.44 10.57 2
- 26. Metagenomic analysis of bacteria grown on α-cellulose 26 Taxon Treatment 1 (Blank) Treatment 4 (α-Cellulose) Fold- Enrichment Natranaerobiales (O) 0.0098 4.73 481 Ruminococcus (G) 0.98 35.05 35 Sporanaerobacter (G) 0.064 1.66 26 Clostridium (G) 1.35 10.94 8 Ruminococcaceae (F) 9.93 28.61 3 Clostridiaceae (F) 2.27 4.87 2 Coprococcus (G) 5.45 7.64 1 Thermoanaerobacterales (O) 2.18 2.71 1
- 27. Comparisons of enriched microbial population grown on α-cellulose 27 Taxon Treatment 4 (DMD) Treatment 5 (E-DMD) Fold- Enrichment Sporolactobacillus (G) 0.00048 2.09 4,365 Clostridiaceae (F) 4.87 8.12 2 Pseudomonas (G) 0.71 1.52 2 Ruminococcus (G) 35.05 66.27 2
- 28. Metagenomic analysis of bacteria grown on wheat straw 28 Taxon Treatment 1 (Blank) Treatment 6 (Wheat Straw) Fold-Enrichment Enterobacteriaceae (F) 0.085 4.44 52 Enterococcaceae (F) 0.12 5.96 48 Ruminococcus (G) 0.40 9.42 24 Oxobacter (G) 0.049 0.63 12 Coprococcus (G) 3.70 21.68 6 Thermoanaerobacterales (O) 0.14 0.92 6 Paenibacillus (G) 0.11 0.56 5 Ruminococcaceae (F) 5.22 16.97 3 Clostridiaceae (F) 4.02 4.57 1
- 29. Comparisons of enriched microbial populations grown on wheat straw 29 Taxon Treatment 7 (Wheat Straw) Treatment 8 (E-DMD) Fold- Enrichment Ruminococcus (G) 0.015 9.39 606 Natranaerobiales (O) 0.13 3.60 28 Sporanaerobacter (G) 0.46 5.40 12 Pelotomaculum (G) 0.32 3.09 9 Caulobacteraceae (F) 0.31 2.34 8 Pseudomonas (G) 0.092 0.67 7 Ruminococcaceae (F) 0.77 5.01 6
Editor's Notes
- Good morning, My thesis topic is impact of simple and complex substrates on the composition and diversity of microbial communities and the end product synthesis
- Biomass is considered as renewable energy resource because, Carbon in biomass is regarded as part of the neutral carbon cycle, trees use the co2 from atm in the presence of sunlight and produce biomass, That biomass is transformed in different forms through food chain. For example in this figure, wood waste collected was used to produce biofuel by anaerobic fermentation. That biofuel is used for transpotation,then carbon is released back to the environment
- Mixed acid fermentation is an anaerobic fermentation carried out by microorganisms that convert organic matters into lactata,formate, succinate with equal amount of H2,CO2 . There are three steps involved in the process, Through hydrolysis complex substance into sugars, aminoacids, fatty acids, In Acidogenesis step - monomers are converted into carbonic acid and H2. CO2; In third stage Acteogenisis-carbonic acids are further converted into acetic acid and H2, co2 4.methanogenisis- ch4 and co2
- MCT defined as idea of using mixture of microorganisms for the fermentation process. It is an undefinde culture obtained from environment, The main advantage of using mixed culture is simple operation, no need to worry about sterilization, the outcome of the process is efficient with low operational cost. But, when we use undefined culture we have few knowledge gap, To fill the knowledge gap and improve the process- the study of microbial communities is necessary, The best way to understand the microbial communities through molecular techniques by exploring nucleicacids or proteins.
- To understand the mixed acid fermentation process, we set an objective to characterize the microbial communities and associated end products produced from different substrates using an batch experiments
- I chose Four different substrates which are categorized into simple and complex substrates. My source of inoculum was DMD collected from CSTR which fed with dairy manure for methane production by Elsie
- Moving on to experimental set up, The DMD was pre-treated at 70 and 100 degree for 30 min, and used for batch experiments, The experiment was carried out in anaerobic respirometer at 37 degree and PH- 7.2, pH was adjusted by adding nutrient medium; Six biological replicates were used for the experiment for each substarte along with 2 blank without substrates Advantage of respirometer: Measures the gas production rate online, Reduction of Partial pressure disturbance, Online removal of hydrogen reduce the growth of methanogens
- At the end of the batch experiment, 1g of digestate was removed from each bottle to extract DNA, For illumina sequencing, The V3-V4 region of 16S rRNA gene was amplified using PCR to construct the DNA library, Then the DNA libraries were subjected to sequencer to sequence the DNA
- To determine the taxonomy of microbial population, PANDAseq assembler was used to assemble the paired end sequences of DNA QIIME a open source bioinformatic pipeline used to perform the microbial communities analysis through series of steps. Then different statistical analysis were carried out to compare and study the relationship between the taxa and the factors involved in the experiment
- The result of batch experiments were divided into 7 treatments by use of four different substrates and two different source of inoculum. Treatment 1- is a blank which means the DMD was incubated with nutrient medium alone, In other bottles the DMD was incubated with different substrates and nutrient medium, we hypothesised that, using grown culture will improve the process for complex substrates. In treatment 6 and 7 enriched inoculum was used, that was prepared by mixing equal ratio of old and new DMD
- I Will be discussing my experiment results
- Alpha- mean diversity of microorganisms in a particular habitat. The graph is constructed by seqs/sample Chao 1 ave (Species richness), The blank- have highest number of diversity, Treatment 3 and Treatment 5 are second highest diversity, Other treatments increased in a similar fashion
- Here is the list of dominant taxa which play important role in the end product synthesis. Clostrium genus, known H2 producer was present in all the substrates except wheat straw.
- Here is the result of gas and VFA synthesis of glucose, The plot shows the cumulative H2 and co2 production, The batch experiment produced H2- 2,905 mL; CO2- 1725mL; The over all hydrogen yield was 1.11molH2/mol glucose. Enterobacter cloaceae –enterobacteriacea family Lactate-14.28 g/L, Sporolactobacillus sp. Ethanol-28.0 g/L- cow dung compost, +ve partial hydrogen pressure; Acetate-4.35; Butyrate- 6.572
- The result of end-products produced by DMD on crude-glycerol, The H2-142 mL; CO2- 702 mL ; propionate was the highest VFA observed with glycerol substrate (15.53 g/L), acetate (4.8 g/L) and butyrate (0.46 g/L) Family Caulobacteraceae (Phylum Proteobacteria) based on redox reaction towards propionate that Syntrophomonas genus have the ability to convert butyrate into H2, CO2, and CH4. The other dominant taxa were reported as H2 producers on glucse, but no evidence for glycerol
- Moving on the results of complex substrate cellulose produced by DMD; The first graph shows the gas production- Biphasic H2 production- The net H2 yield was 40 mmol H2 / mol cellulose; Acetate- 6.81 0.20 g/L, butyrate- 2.3 0.5 g/L Ruminococcaceae (F); Ruminococcus (G); Clostridium (G); Natranaerobiales (O)- Follwed acetate/butyrate pathway; -Ren et al., (2010) observed the same pattern greater amounts by cow dung compost.
- Here is the comparison of H2 and VFA production between enriched culture and DMD. Biphasic growth was observed in H2 production by both the cultures- DMD-5.25 mL and EDMD-19.0 mL at 72hr;The cumulative H2 production was 78ml-DMD; EDM-95ml Acetate, butyrate and valerate were major VFA produced by both the culture, But in case of e-dmd -lactate, formate, isobutyrate, and isovalerate were also observed in low quantity. Clostridiaceae and the Genera Pseudomonas and Ruminococcus – E-DMD Sporolactobacillus- lactate-0.5g/L
- The gas and acid production of raw wheat straw, The cumulative H2-25mL; CO2- 60mL- Ruminococcaceae (F); Ruminococcus(G)-colse assosiation with clostridum, Thermoanerobacterales (O); Oxobacter (G); Paenibacillus(G)-well known for H2, co2, and VFA synthesis- 16S rDNA analysis- collins-1994 Enterococcaceae(F)- enterobacter aregnosa acetate (1.0 g/L), butyrate (0.46 g/L), and propionate (0.05 g/L) –acetate/butyrate pathway; Fan et al-cow dung compost on wheat straw; zhao et al observed these microbes in biofloculant production from rice straw Sporanarobacter (G)-increase the acetate production
- Comparison of end-products produced by E-DMD and DMD.DMD produced 27mL But EDMD- 37mL first phase; 25ml-consumption of H2 by sporoanaerobacter Acetate high- sporoanaerobacter and other microbes increase in the population
- The take home message from the anaerobic batch experiment is DMD was highly diversity source of inoculum for mixed acid fermentation, Very different microorganisms were enriched in each substrate, Greater diversity of microbial population was observed in glycerol and wheats traw Substrate has higher influence on microbial diversity and end products synthesis
- The comparison of microbial diversity and end-products helps to understand the dark fermentation reaction using mixed culture. Based on the results obtained from batch experiment, We can move to the next step of conducting fermentation reaction in continuous system, Sample collection to investigate the microbial communities and end products together at different time intervals will give clear idea of change in the system, it will pave the way to find microbial species for fermentation process
- PLS-regression is a multivariate statistical method used to correlate the different variables. PLS da loading plot display the relationship b/w the y variable and influence of Y on X variable. SIMCA software used univariance to scale the data and cross validation to determine components. Triangle signifies the high degree of influence of treatment in the graph, According to relative abundance; Phylum-Firmicutes; Proteobacteria
- Phylum-Firmicutes, Bacteroides, Proteobacteria-Clau
- metabolized via glycolysis and share a similar stoichiometry, end product are different. Phylum bacteroides significant to glycerol; high diversity; Glucose-1.11 molH2/mol substrate ; 15-fold greater than the H2 yield (0.07 mol H2/mol substrate)
- Total 41 OTUS
- Hypothesis: 50% of old DMD and 50% of new DMD improve the h2 yield Major dominant- Firmicutes; Proteobacteria
- Total -63 OTUs; Dominant-Phylum-Firmicutes
- Natranaerobiales (O), Ruminococcaceae (F) and Rhodospirillaceae (F), Pelotomaculum (G) and Pseudomonas (G).