Anaerobic Digestion Of Lignocellulosic Biomass Via Cotreatment

Author : Anahita Bharadwaj
Publisher :
ISBN 13 :
Total Pages : pages
Book Rating : 4.:/5 (119 download)

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Download or read book Microbial Adaptation and Cotreatment-Enhanced Biomass Solubilization in Lignocellulosic Anaerobic Digestion written by Anahita Bharadwaj and published by . This book was released on 2020 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Lignocellulose recalcitrance, that is, its resistance to biological degradation, is arguably one of the biggest technical challenges in the biofuel and biochemical production industry. The aim of this research was to study and improve biomass utilization, particularly in undefined mixed culture anaerobic and acidogenic digestion systems. In this dissertation, two approaches were tested and applied for the enhancement of biological solubilization of unpretreated lignocellulose, (i) the application of 'cotreatment', that is, milling of biomass during fermentation (ii) the adaptation of mixed microbiomes involved in the degradation and utilization of lignocellulose in anaerobic conditions. First, a lignocellulose-centric mesophilic methanogenic anaerobic digester was set up with inoculum sources including rumen fluid, compost, and wastewater biosolids. After an initial ramp-up period of several weeks, it was operated as a stable reactor for roughly two years (Appendix E). During that entire period the reactor was fed unpretreated senescent switchgrass as the primary carbon source, supplemented with trace nutrient rich media. The 4-L reactor was operated under semi-continuous conditions by feeding once-per-day with fresh switchgrass at a solids loading rate of 2g (dry basis) (L day)-1 with a 30-day retention time until biogas production stabilized. During stable operations the effluent material from this 4-L reactor, termed 'once-fermented material', was collected daily, incubated at 37C, and subsequently used to test the cotreatment strategy. This "once-fermented" partially digested biomass was tested with two different milling strategies -- a ball mill (Chapter 3 and Appendix A) and a colloid mill (Chapter 4 and Appendix B). Various milling durations were compared with unmilled "status-quo" material as the control. After cotreatment milling, the material was placed in BioMethane Potential (BMP) test bottles and fermented for a second time in batch mode for 18-19 days. The entire experimental set-up was termed "ferment-mill-ferment". In these studies, various measurements were taken immediately after milling, and after the second fermentation period. These included sugars present in the biomass, volatile solids, particle size distribution, gravimetric mass, volatile fatty acids, gas volume and composition, and energy consumed by the cotreatment milling. The results indicate a statistically significant improvement in biogas production supported by a significant improvement in biomass sugar consumption, volatile solids consumption, and total mass change as well as decrease in average particle size of the milling treatments when compared to the unmilled control. These results are indicative of improved biomass solubilization with cotreatment. In general, there was a trend of increasing biomass solubilization with increasing milling duration. The impact of cotreatment on biomass solubilization was more significant for the ball mill than the colloid mill. However, the colloid mill was much more energy efficient and therefore may be a better choice for scale-up. In the next stage of this work, the impact of cotreatment shear stress on the microbiome and its ability to recover from this environmental stress was assessed using DNA sequencing (Chapter 5 and Appendix C). The concept of "robustness" of microbiome was introduced here as 'the ability of a microbiome to change, adapt and sustain itself during and after environmental stress or disturbance, while retaining functionality that is similar to the microbiome present before the disturbance'. A similar ferment-mill-ferment experiment was set up with both ball milling (high intensity) and colloid milling (low and moderate intensity) strategies along with an unmilled control. Along with the previously described measurements, samples were collected for 16s rRNA gene sequencing before milling, immediately after milling and after the second fermentation in the BMP test bottles. Relic DNA and non-viable DNA (from membrane-compromised cells), likely caused due to milling stresses, were inactivated by using propidium monoazide. Chloroplast DNA from the digested plant material was inactivated using pPNA clamp (method development in Chapter 6). The V4 variable region was sequenced using Illumina® MiSeq amplicon sequencing and post-processing was done using QIIME2 and RStudio. Results from this study indicate significant improvement in biomass utilization with cotreatment, thereby supporting the results reported in Chapters 3 and 4. 16s rRNA gene sequencing revealed resistant and resilient microbial populations as the anaerobic microbiome responded to milling stress. There was an enhancement of lignocellulose utilizing bacteria, particularly of Fibrobacterales (family)_BBMC-4 (genus) and Cellulomonadaceae (family)_Actinotalea (genus). This may be indicative of access to freshly exposed surfaces of previously recalcitrant biomass due to cotreatment. Finally, acidogenic digestion of lignocellulosic biomass for the production of small and medium chain carboxylic acid was studied (Chapter 7 and Appendix D). Specifically, the different temperatures and low pH were examined for their impacts on the acidogenic bacteria involved in the utilization of biomass. The inoculum sources for these bacteria were rumen fluid, compost and silage. These sources, along with unpretreated mid-season switchgrass, were placed in batch reactor bottles and incubated at various temperatures. The adapted microbiome from these bottles was then used to set up triplicate batch reactors at different temperatures, and fermentation was conducted for 20 days. Samples for volatile fatty acid measurement and 16s rRNA gene sequencing of V1-V2 region were collected. The mesophilic samples (20 -- 40°C) show the presence of C2-C7 carboxylic acids, but almost no lactic acid or ethanol, while the thermophilic samples (50 -- 60°C) predominantly contain mostly lactic acid. Furthermore, 16s rRNA gene sequencing revealed that the mesophilic samples contained bacteria with the capacity to convert simple sugars and lactic acid into small and medium chain carboxylic acids. Lactic acid producing bacteria were detected in these samples, so the absence of this acid may indicate that lactic acid utilizers may have converted it into other carboxylic acids. The thermophilic samples contained bacteria known to utilize simple sugars and starch, and convert them into lactic acid. Very few, if any, predominantly cellulolytic bacteria were detected at both temperature ranges, most likely due to the extremely low pH and difficult to digest unpretreated lignocellulose. Therefore, it is speculated that the bacteria at both temperature ranges utilized the more easily accessible simple sugars, organic material and starch originating from the inoculum sources instead of the lignocellulosic substrate to produce organic acids initially, and very quickly the resulting low pH conditions did not encourage further solubilization of biomass. Ultimately, this dissertation advances two possible strategies that may be employed to accelerate lignocellulosic biomass utilization for the production of value-added biofuels and biochemicals. It provides some ground work for the application of cotreatment and microbiome adaptation in mixed culture fermentation systems that may better inform the efficient design and functioning of dedicated-lignocellulose fermentation systems that may contribute towards a more sustainable future.