This work was supported by the National Natural Science Foundation of China (Grant No. 21576279, 20906076) and the KRIBB Research Initiative Program (KGM2211531).

1. Preparation of Bamboo Hydrolysate
<ol>
	<li>Add 5 g of bamboo powder to 40 mL of an NaOH solution to achieve a 10% (g/g) final concentration in a 250 mL flask. Use a series of NaOH solutions ranging from 0.05 M to 0.50 M in increments of 0.05 M.
	<ol>
		<li>Incubate the mixture for 60 min at 60 &#176;C or 100 &#176;C in a water bath. Incubate at 121 &#176;C in an autoclave.</li>
	</ol>
	</li>
	<li>After incubation, leave the mixture to stand for 4 h at room temperature. Then, remove the supernatant. Add 100 mL of fresh water and mix.
	<ol>
		<li>Repeat this washing 5-6 times, until the pH of the supernatant reaches 6.8. Use the obtained solid for the next step of enzymatic hydrolysis.</li>
	</ol>
	</li>
	<li>For hydrolysis, adjust 50 mL of the above mixture to pH 5.0 using 98% H2SO4. Then, add 0.5 mL of 200 filter paper activity (FPU)/mL cellulase. 
	NOTE: The final ratio of cellulase to bamboo should be 20 FPU per 1 g of bamboo.</li>
	<li>Incubate the mixture at 50 &#176;C for 36 h in a shaker. 
	NOTE: After hydrolysis, some solid will remain in the mixture.</li>
	<li>Obtain a clear hydrolysate by centrifuging at 7,690 x g for 5 min.</li>
	<li>Quantify the glucose, xylose, and other chemicals with a high-pressure liquid chromatograph system equipped with a refractive index detector and a photodiode array detector. Use an HPX-87H column (300 mm x 7.8 mm) and a mobile phase of 5 mM H2SO4 solution at a 0.8 mL/min flow rate12.</li>
	<li>For a large volume of hydrolysate preparation, mix 2 kg of bamboo powder and 18 L of 0.25 M NaOH in a 30-L stainless-steel tank.
	<ol>
		<li>Incubate the tank at 121 &#176;C in an autoclave for 60 min. After incubation, wash the mixture with 40 L of water in a 60 L plastic tank. Repeat this washing 5 times.</li>
		<li>Use water to adjust the total washed bamboo to a volume of 20 L. Adjust the pH of the mixture to 5.0 using 98% H2SO4.</li>
		<li>Enzymatically hydrolyze the mixture in a modified shaking water bath, equipped with a mechanical agitator to ensure that the mixture is well distributed. Add 200 mL of 200 FPU/mL cellulase and incubate the mixture at 50 &#176;C for 36 h.</li>
		<li>After hydrolysis, centrifuge the mixture at 4,700 x g for 10 min.</li>
	</ol>
	</li>
</ol>
2. 2,3-Butanediol Production by Wildtype K. pneumoniae
<ol>
	<li>Use 3 L of bamboo hydrolysate as the solvent for medium preparation.</li>
	<li>Prepare a fermentation medium containing 4 g/L corn steep liquor, 2 g/L (NH4)2SO4, 6 g/L K2HPO4, 3 g/L KH2PO4, and 0.2 g/L MgSO4. Adjust the pH of the medium to neutral using 2.5 M NaOH. Use a 5-L bioreactor containing 3 L of autoclaved fermentation medium for fermentation.</li>
	<li>Use K. pneumoniae cells stored in a -80 &#176;C low-temperature refrigerator.</li>
	<li>For the seed culture, incubate a 250 mL flask containing 50 mL of Luria-Bertani (LB) medium overnight at 37 &#176;C, with shaking at 200 rpm. Use LB medium containing 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl. 
	NOTE: The cell density of the seed culture should reach OD 2 (optical density at 600 nm).</li>
	<li>Inoculate one flask of 50 mL of the seed culture in the bioreactor. Maintain the culture at pH 6.0 and 37 &#176;C. Use an air supplementation rate of 2 L/min and an agitation rate of 250 rpm, creating a microaerobic condition.</li>
	<li>Collect 5 mL samples every 2 h during the fermentation and analyze them with high-pressure liquid chromatography to determine the chemical concentrations in the broth4.
	<ol>
		<li>Monitor the alkali added to the bioreactor online using MFCS/DA. 
		NOTE: Organic acids are produced in the fermentation process, and NaOH is added to keep the culture pH stable. The volume of alkali added represents the amount of acid produced. When the alkali-added line plateaus, the process is finished, either because of carbon source exhaustion or cell death.</li>
	</ol>
	</li>
</ol>
3. R-acetoin Production by the budC Mutant of K. pneumoniae
<ol>
	<li>Prepare a medium for acetoin production containing 4 g/L corn steep liquor, 2 g/L (NH4)2SO4, 3 g/L sodium acetate, 0.4 g/L KCl, and 0.1 g/L MgSO4.</li>
	<li>Use K. pneumoniae-&#916;budC for R-acetoin production. Repeat steps 2.4-2.5. 
	NOTE: budC encodes 2,3-butanediol dehydrogenase. K. pneumoniae-&#916;budC loses the 2,3-butanediol dehydrogenase activity and produces R-acetoin instead of 2,3-butanediol.</li>
	<li>Maintain the culture at pH 6.0 and 37 &#176;C. Use an air supplementation rate of 4 L/min and an agitation rate of 450 rpm, an aerobic condition. 
	NOTE: The oxygen supplementation is higher than that of 2,3-butanediol production.</li>
	<li>Assay the samples as in step 2.6.</li>
</ol>
4. 2-Ketogluconic Acid Production by the budA Mutant of K. pneumoniae
<ol>
	<li>For 2-ketogluconic acid production, use the same medium as for R-acetoin production.</li>
	<li>Use K. pneumoniae-&#916;budA for 2-ketogluconic acid production. Repeat steps 2.4-2.5. 
	NOTE: budA encodes &#945;-acetolactate decarboxylase. K. pneumoniae-&#916;budA loses the &#945;-acetolactate decarboxylase activity and produces 2-ketogluconic acid, using glucose as a substrate. 
	NOTE: 2-ketogluconic acid synthesis is an acidic condition-dependent process. A two-stage fermentation has been developed for 2-ketogluconic acid production10; in the first stage, the seed culture is inoculated in the bioreactor.</li>
	<li>Maintain the culture at pH 7.0 and 37 &#176;C. Use an air supplementation rate of 4 L/min and an agitation rate of 500 rpm. 
	NOTE: In these conditions, cells grow very fast (the cell density reaches OD 7 in about 4 h). Then, the fermentation progresses to the second stage, during which the culture pH decreases to 5.0. No acid is added; the organic acids produced in the culture naturally lead to the pH decrease. In these acidic conditions, cell growth stops, but 2-ketogluconic acid is synthesized and accumulates in the broth.</li>
	<li>Assay the samples as in step 2.6. Using the hydrolysate of bamboo as the carbon source. 
	NOTE: The glucose in the medium is converted to 2-ketogluconic acid, and the xylose is converted to xylonic acid. Thus, a mixture of 2-ketogluconic acid and xylonic acid is obtained.</li>
</ol>

<ol>
	<li>Zeng, A. P., Biebl, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bulk+chemicals+from+biotechnology:+the+case+of+1,+3-propanediol+production+and+the+new+trends.">Bulk chemicals from biotechnology: the case of 1, 3-propanediol production and the new trends.</a> Adv Biochem Eng Biotechnol. 74, 239-259 (2002).</li><li>Wei, D., Wang, M., Jiang, B., Shi, J., Hao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+dihydroxyacetone+kinases+I+and+II+in+the+dha+regulon+of+Klebsiella+pneumoniae.">Role of dihydroxyacetone kinases I and II in the dha regulon of Klebsiella pneumoniae.</a> J Biotechnol. 177, 13-19 (2014).</li><li>Celi&#324;ska, E., Grajek, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotechnological+production+of+2,3-butanediol-current+state+and+prospects.">Biotechnological production of 2,3-butanediol-current state and prospects.</a> Biotechnol Adv. 27 (6), 715-725 (2009).</li><li>Chen, C., Wei, D., Shi, J., Wang, M., Hao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+2,+3-butanediol+stereoisomer+formation+in+Klebsiella+pneumoniae.">Mechanism of 2, 3-butanediol stereoisomer formation in Klebsiella pneumoniae.</a> Appl Microbiol Biotechnol. 98 (10), 4603-4613 (2014).</li><li>Wei, D., Wang, M., Shi, J., Hao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Red+recombinase+assisted+gene+replacement+in+Klebsiella+pneumoniae.">Red recombinase assisted gene replacement in Klebsiella pneumoniae.</a> J Ind Microbiol Biotechnol. 39 (8), 1219-1226 (2012).</li><li>Wei, D., Sun, J., Shi, J., Liu, P., Hao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+strategy+to+improve+efficiency+for+gene+replacement+in+Klebsiella+pneumoniae.">New strategy to improve efficiency for gene replacement in Klebsiella pneumoniae.</a> J Ind Microbiol Biotechnol. 40 (5), 523-527 (2013).</li><li>Chen, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+RecBCD+in+Klebsiella+pneumoniae+by+Gam+and+its+effect+on+the+efficiency+of+gene+replacement.">Inhibition of RecBCD in Klebsiella pneumoniae by Gam and its effect on the efficiency of gene replacement.</a> J Basic Microbiol. 56 (2), 120-126 (2016).</li><li>Wang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=R-acetoin+accumulation+and+dissimilation+in+Klebsiella+pneumoniae.">R-acetoin accumulation and dissimilation in Klebsiella pneumoniae.</a> J Ind Microbiol Biotechnol. 42 (8), 1105-1115 (2015).</li><li>Wei, D., Xu, J., Sun, J., Shi, J., Hao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2-Ketogluconic+acid+production+by+Klebsiella+pneumoniae+CGMCC+1.6366.">2-Ketogluconic acid production by Klebsiella pneumoniae CGMCC 1.6366.</a> J Ind Microbiol Biotechnol. 40 (6), 561-570 (2013).</li><li>Sun, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-stage+fermentation+for+2-ketogluconic+acid+production+by+Klebsiella+pneumoniae.">Two-stage fermentation for 2-ketogluconic acid production by Klebsiella pneumoniae.</a> J Microbiol Biotechnol. 24 (6), 781-787 (2014).</li><li>Wang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gluconic+acid+production+by+gad+mutant+of+Klebsiella+pneumoniae.">Gluconic acid production by gad mutant of Klebsiella pneumoniae.</a> World J Microbiol Biotechnol. 32 (8), 1-11 (2016).</li><li>Wang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+xylonic+acid+by+Klebsiella+pneumoniae.">Production of xylonic acid by Klebsiella pneumoniae.</a> Appl Microbiol Biotechnol. 100 (23), 10055-10063 (2016).</li><li>Kumar, P., Barrett, D. 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The authors have nothing to disclose.

K. pneumoniae belongs to the genus Klebsiella in the family Enterobacteriaceae. K. pneumoniae is distributed widely in natural environments such as soil, vegetation, and water14. The wildtype K. pneumoniae strain used in this research was isolated from soil and is used for 1,3-propanediol production15. K. pneumoniae and mutants of this species produce many chemicals under different conditions.
<p class="jove_content"...

Klebsiella pneumoniae is a bacterium that grows well under both aerobic and anaerobic conditions. K. pneumoniae is an important industrial microorganism used to produce many chemicals. 1,3-propanediol is a valuable chemical that is mainly used as a monomer to synthesize polytrimethylene terephthalate. Polytrimethylene terephthalate is a biodegradable polyester that exhibits better properties than those of 1,2-propanediol, butanediol, or ethylene glycol1. 1,3-propanediol is produced by K. pneumoniae using glycerol as a substrate in oxygen-limited conditions2. 2,3-butanediol and its derivatives have applications in the field of plastics, solvent production, and synthetic rubber and have the potential to be used as biofuel3. With glucose as the substrate, 2,3-butanediol is the main metabolite of the wildtype strain4. 2,3-butanediol is synthesized from pyruvate. First, two molecules of pyruvate condense to yield &#945;-acetolactate; this reaction is catalyzed by &#945;-acetolactate synthase. &#945;-acetolactate is then converted to acetoin by &#945;-acetolactate decarboxylase. R-acetoin can be further reduced to 2,3-butanediol when catalyzed by butanediol dehydrogenase. An efficient gene replacement method suitable for K. pneumoniae has been explored, and many mutants have been constructed5,6,7. A budC mutant, which lost its 2,3-butanediol dehydrogenase activity, accumulates high levels of acetoin in culture broth. Acetoin is used as an additive to enhance the flavor of food8. When budA, which encodes &#945;-acetolactate decarboxylase, is mutated, 2-ketogluconic acid accumulates in the broth. 2-ketogluconic acid is used for the synthesis of erythorbic acid (isoascorbic acid), an antioxidant used in the food industry9. 2-ketogluconic acid is an intermediate of the glucose oxidation pathway; in this pathway, located in the periplasmic space, glucose is oxidized to gluconic acid and then further oxidized to 2-ketogluconic acid. Gluconic acid and 2-ketogluconic acid produced in the periplasm can be transported to the cytoplasm for further metabolism. The accumulation of 2-ketogluconic acid is dependent upon acidic conditions, and higher air supplementation favors 2-ketogluconic acid production10. Gluconate dehydrogenase, encoded by gad, catalyzes the conversion of gluconic acid to 2-ketolguconic acid. The gad mutant of K. pneumoniae produced high levels of gluconic acid instead of 2-ketogluconic acid, and this process is also dependent upon acidic conditions. Gluconic acid is a bulk organic acid and is used as an additive to increase the properties of cement11. Glucose oxidation to gluconic acid is catalyzed by glucose dehydrogenase. Xylose is also a suitable substrate of glucose dehydrogenase. When xylose is used as a substrate, K. pneumoniae produces xylonic acid12.
Chemical production using biomass as a feedstock is a hot topic in biotechnology13. The main components of biomass are cellulose, hemicellulose, and lignin. However, these macromolecular compounds cannot be directly catabolized by most microorganisms (including K. pneumoniae). Cellulose and hemicelluloses in the biomass must be hydrolyzed to glucose and xylose and can then be used by microorganisms. The presence of lignin in lignocelluloses creates a protective barrier that prevents biomass hydrolyzation by enzymes. Thus, a pretreatment process that removes lignin and hemicelluloses and reduces the crystallinity of cellulose is always performed during biomass utilization by microorganisms. Many pretreatment methods have been developed: acid, alkaline, ammonia, and steam pretreatments are common.
Bamboo is abundant in tropical and subtropical regions and is an important biomass resource. Here, the preparation of bamboo hydrolysate and chemical production using bamboo hydrolysate are presented

<table><tbody><tr><td>autoclave</td><td>SANYO</td><td>3780</td><td></td></tr><tr><td>bioreactor</td><td>Sartorius stedim biotech</td><td>Bostat Aplus</td><td></td></tr><tr><td>agitator</td><td>IKA</td><td>RW 20</td><td></td></tr><tr><td>water bath shaker</td><td>Zhicheng</td><td>ZWY-110X50</td><td></td></tr><tr><td>high performance liquid chromatograph system</td><td>Shimadzu Corp</td><td>20AVP</td><td></td></tr><tr><td>centrifuge</td><td>Hitachi</td><td>CR22G III</td><td></td></tr><tr><td>Bamboo powder</td><td>purchased from Zhejiang Province, China</td><td></td><td>mesh number, 50</td></tr><tr><td>cellulase</td><td>Youtell Biochemical, Shandong, China</td><td></td><td>200 PFU/mL</td></tr></tbody></table>

production of chemicals by klebsiella pneumoniae using bamboo hydrolysate as feedstock

Bamboo is an important biomass, and bamboo hydrolysate is used by Klebsiella pneumoniae as a feedstock for chemical production. Here, bamboo powder was pretreated with NaOH and washed to a neutral pH. Cellulase was added to the pretreated bamboo powder to generate the hydrolysate, which contained 30 g/L glucose and 15 g/L xylose and was used as the carbon source to prepare a medium for chemical production. When cultured in microaerobic conditions, 12.7 g/L 2,3-butanediol was produced by wildtype K. pneumoniae. In aerobic conditions, 13.0 g/L R-acetoin was produced by the budC mutant of K. pneumoniae. A mixture of 25.5 g/L 2-ketogluconic acid and 13.6 g/L xylonic acid was produced by the budA mutant of K. pneumoniae in a two-stage, pH-controlled fermentation with high air supplementation. In the first stage of fermentation, the culture was maintained at a neutral pH; after cell growth, the fermentation proceeded to the second stage, during which the culture was allowed to become acidic.

In this protocol, the bamboo was pretreated using alkali. The optimal incubation parameters-a temperature of 121 &#176;C and 0.25 M NaOH-were determined in Figures 1 and 2. The pretreated bamboo was enzymatically hydrolyzed, and the glucose and xylose concentrations obtained in the hydrolysate were measured. Higher temperatures favored sugar production, so 121 &#176;C was selected as the optimal temperature. The glucose and xylose produced in the hydrolys...

Watch this Scientific Journal Video about Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock at JoVE.com

Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock

Bamboo powder was pretreated with NaOH and enzymatically hydrolyzed. The hydrolysate of bamboo was used as the feedstock for 2,3-butanediol, R-acetoin, 2-ketogluconic acid, and xylonic acid production by Klebsiella pneumoniae.

Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock

Bamboo is an important biomass, and bamboo hydrolysate is used by Klebsiella pneumoniae as a feedstock for chemical production. Here, bamboo powder was ...

production-chemicals-klebsiella-pneumoniae-using-bamboo-hydrolysate

Research

JoVE Journal

Bioengineering

8.9K Views.  Chinese Academy of Sciences.  Bamboe poeder werd voorbehandeld met NaOH en enzymatisch gehydrolyseerd. Het hydrolysaat van bamboe werd gebruikt als voedingsstof voor 2,3-butaandiol, R- acetoïne, 2-ketogluconzuur en xylonzuurproductie door Klebsiella pneumoniae . 

Video: Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock

Methods for Facilitating Microbial Growth on Pulp Mill Waste Streams and Characterization of the Biodegradation Potential of Cultured Microbes

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high quality paper. Black liquor is a pulping waste generated by the kraft process that has potential for downstream bioconversion. However, the recalcitrant nature of the lignocellulose resources, its chemical derivatives that constitute the majority of available organic carbon within black liquor, and its basic pH present challenges to microbial biodegradation of this waste material. Methods for the collection and modification of black liquor for microbial growth are aimed at utilization of this pulp waste to convert the lignin, organic acids, and polysaccharide degradation byproducts into valuable chemicals. The lignocellulose extraction techniques presented provide a reproducible method for preparation of lignocellulose growth substrates for understanding metabolic capacities of cultured microorganisms. Use of gas chromatography-mass spectrometry enables the identification and quantification of the fermentation products resulting from the growth of microorganisms on pulping waste. These methods when used together can facilitate the determination of the metabolic activity of microorganisms with potential to produce fermentation products that would provide greater value to the pulping system and reduce effluent waste, thereby increasing potential paper milling profits and offering additional uses for black liquor.

Industrial wastes can be collected and modified to analyze microbial growth. Lignocellulose extraction techniques provide components to analyze specific biodegradation ability. Gas chromatography-mass spectrometry identifies fermentation products of microorganisms grown on pulping waste. These methods determine the metabolic capacity of microorganisms to degrade pulping waste.

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high ...

Environment

Utilizing the Ethylene-releasing Compound, 2-Chloroethylphosphonic Acid, as a Tool to Study Ethylene Response in Bacteria

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ripening. Exogenous ethylene applied during early plant development triggers the triple response phenotype; a shorter and thicker hypocotyl with an exaggerated apical hook. Despite the intimate relationship between plants and bacteria, the effect of exogenous ethylene on bacteria has been greatly overlooked. This is partly due to the difficulty of controlling gaseous ethylene within the laboratory without specialized equipment. 2-Chloroethylphosphonic acid (CEPA) is a compound that decomposes into ethylene, chlorine, and phosphate in a 1:1:1:1 molar ratio when dissolved in an aqueous medium of pH 3.5 or greater. Here we describe the use of CEPA to produce in situ ethylene for the investigation of ethylene response in bacteria using the fruit-associated, cellulose-producing bacterium Komagataeibacter xylinus as a model organism. The protocols described herein include both the verification of ethylene production from CEPA via the Arabidopsis thaliana triple response assay and the effects of exogenous ethylene on K. xylinus cellulose production, pellicle properties and colonial morphology. These protocols can be adapted to examine the effect of ethylene on other microbes using appropriate growth media and phenotype analyses. The use of CEPA provides researchers with a simple and efficient alternative to pure ethylene gas for the routine determination of bacterial ethylene response.

The protocols outlined herein facilitate the convenient investigation of bacterial ethylene responses by utilizing 2-chloroethylphosphonic acid (CEPA). Ethylene is produced in situ through the decomposition of CEPA in an aqueous bacterial growth medium, circumventing the requirement for pure ethylene gas.

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ...

Biochemistry

Evaluation of Integrated Anaerobic Digestion and Hydrothermal Carbonization for Bioenergy Production

Lignocellulosic biomass is one of the most abundant yet underutilized renewable energy resources. Both anaerobic digestion (AD) and hydrothermal carbonization (HTC) are promising technologies for bioenergy production from biomass in terms of biogas and HTC biochar, respectively. In this study, the combination of AD and HTC is proposed to increase overall bioenergy production. Wheat straw was anaerobically digested in a novel upflow anaerobic solid state reactor (UASS) in both mesophilic (37 &#176;C) and thermophilic (55 &#176;C) conditions. Wet digested from thermophilic AD was hydrothermally carbonized at 230 &#176;C for 6 hr for HTC biochar production. At thermophilic temperature, the UASS system yields an average of 165 LCH4/kgVS&#160;(VS: volatile solids) and 121 L CH4/kgVS&#160;at mesophilic AD over the continuous operation of 200 days. Meanwhile, 43.4 g of HTC biochar with 29.6 MJ/kgdry_biochar was obtained from HTC of 1 kg digestate (dry basis) from mesophilic AD. The combination of AD and HTC, in this particular set of experiment yield 13.2 MJ of energy per 1 kg of dry wheat straw, which is at least 20% higher than HTC alone and 60.2% higher than AD only.

A novel Upflow Anaerobic Solid State (UASS) reactor was used for biogas production from fibrous feedstock. Digestate from UASS reactor was hydrothermally carbonized into HTC biochar in a pressurized batch reactor. The integration of the two bioenergy concepts was applied in this study to increase overall bioenergy production.

Lignocellulosic biomass is one of the most abundant yet underutilized renewable energy resources. Both anaerobic digestion (AD) and hydrothermal ...

A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)9 addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.
A three-step pathway for alkane degradation was implemented in E. coli to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native &beta;-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2, rubA3, rubA4and rubB) of the alkane hydroxylase system from Gordonia sp. TF68,21 were transformed into E. coli. For the conversion of long-chain alkanes (C15-C36), theladA gene from Geobacillus thermodenitrificans was implemented. For the required further steps of the degradation process, ADH and ALDH (originating from G. thermodenitrificans) were introduced10,11. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.
To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.
The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFD&alpha; and &beta;, both from Pyrococcus horikoshii OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFD&alpha; and &beta; improve the protein folding process e.g. under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n-hexane in the culture medium were observed.
Summarizing, the results indicate that the toolkit enables E. coli to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.

A sustainable auto regulating bacterial system for the remediation of oil pollutions was designed using standard interchangeable DNA parts (BioBricks). An engineered E. coli strain was used to degrade alkanes via &beta;-oxidation in toxic aqueous environments. The respective enzymes from different species showed alkane degradation activity. Additionally, an increased tolerance to n-hexane was achieved by introducing genes from alkane-tolerant bacteria.

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The ...

Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst immobilization for improved production yield and sustained synthesis. Described herewith is the experimental procedure for the development of such a system consisting of two E. coli strains that express functionally complementary enzymes. Together, these two enzymes can function co-operatively to mediate the regeneration of expensive cofactors for improving the product yield of the bioreaction. In addition, the method of synthesizing an immobilized form of the coupled biocatalytic system by encapsulation of whole cells in calcium alginate beads is reported. As an example, we present the improved biosynthesis of L-xylulose from L-arabinitol by coupling E. coli cells expressing the enzymes L-arabinitol dehydrogenase or NADH oxidase. Under optimal conditions and using an initial concentration of 150 mM L-arabinitol, the maximal L-xylulose yield reached 96%, which is higher than those reported in the literature. The immobilized form of the coupled whole-cell biocatalysts demonstrated good operational stability, maintaining 65% of the yield obtained in the first cycle after 7 cycles of successive re-use, while the free cell system almost completely lost the catalytic activity. Therefore, the methods reported here provides two strategies that could help improve the industrial production of L-xylulose, as well as other value-added compounds requiring the use of cofactors in general.

Presented is the protocol for co-immobilizing whole-cell biocatalysts for cofactor regeneration and improved reusability, using the production of L-xylulose as an example. The cofactor regeneration is achieved by coupling two Escherichia coli strains expressing functionally complementary enzymes; the whole-cell biocatalyst immobilization is achieved by cell encapsulation in calcium alginate beads.

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst ...

Chemistry

A Novel Method for the Pentosan Analysis Present in Jute Biomass and Its Conversion into Sugar Monomers Using Acidic Ionic Liquid

Recently, ionic liquids (ILs) are used for biomass valorization into valuable chemicals because of their remarkable properties such as thermal stability, lower vapor pressure, non-flammability, higher heat capacity, and tunable solubility and acidity. Here, we demonstrate a method for the synthesis of C5 sugars (xylose and arabinose) from the pentosan present in jute biomass in a one-pot process by utilizing a catalytic amount of Br&#248;nsted acidic 1-methyl-3-(3-sulfopropyl)-imidazolium hydrogen sulfate IL. The acidic IL is synthesized in the lab and characterized using NMR spectroscopic techniques for understanding its purity. The various properties of BAIL are measured such as acid strength, thermal and hydrothermal stability, which showed that the catalyst is stable at a higher temperature (250 &#176;C) and possesses very high acid strength (Ho 1.57). The acidic IL converts over 90% of pentosan into sugars and furfural. Hence, the presenting method in this study can also be employed for the evaluation of pentosan concentration in other kinds of lignocellulosic biomass.

We present a protocol for the synthesis of C5 sugars (xylose and arabinose) from a renewable non-edible lignocellulosic biomass (i.e., jute) with the presence of Br&#248;nsted acidic ionic liquids (BAILs) as the catalyst in water. The BAILs catalyst exhibited better catalytic performance than conventional mineral acid catalysts (H2SO4 and HCl).

Recently, ionic liquids (ILs) are used for biomass valorization into valuable chemicals because of their remarkable properties such as thermal stability, ...

Enhanced Oil Recovery using a Combination of Biosurfactants

Biosurfactants are surface-active compounds capable of reducing the surface tension between two phases of different polarities. Biosurfactants have been emerging as promising alternatives to chemical surfactants due to less toxicity, high biodegradability, environmental compatibility and tolerance to extreme environmental conditions. Here, we illustrate the methods used for screening of microbes capable of producing biosurfactants. The biosurfactant producing microbes were identified using drop collapse, oil spreading, and emulsion index assays. Biosurfactant production was validated by determining the reduction in surface tension of the media due to growth of the microbial members. We also describe the methods involved in characterization and identification of biosurfactants. Thin layer chromatography of the extracted biosurfactant followed by differential staining of the plates was performed to determine the nature of the biosurfactant. LCMS, 1H NMR, and FT-IR were used to chemically identify the biosurfactant. We further illustrate the methods to evaluate the application of the combination of produced biosurfactants for enhancing residual oil recovery in a simulated sand pack column.

We illustrate the methods involved in screening and identification of the biosurfactant producing microbes. Methods for chromatographic characterization and chemical identification of the biosurfactants, determining the industrial applicability of the biosurfactant&#160;in enhancing residual oil recovery are also presented.

Biosurfactants are surface-active compounds capable of reducing the surface tension between two phases of different polarities. Biosurfactants have been ...

Microalgal Cultivation for Biomethane Obtention from Swine Waste Biogas

Biogas Purification through the use of a Microalgae-Bacterial System in Semi-Industrial High Rate Algal Ponds

In recent years, a number of technologies have emerged to purify biogas into biomethane. This purification entails a reduction in the concentration of polluting gases such as carbon dioxide and hydrogen sulfide to increase the content of methane. In this study, we used a microalgal cultivation technology to treat and purify biogas produced from organic waste from the swine industry to obtain ready-to-use biomethane. For cultivation and purification, two 22.2 m3 open-pond photobioreactors coupled with an absorption-desorption column system were set up in San Juan de los Lagos, Mexico. Several recirculation liquid/biogas ratios (L/G) were tested to obtain the highest removal efficiencies; other parameters, such as pH, dissolved oxygen (DO), temperature, and biomass growth, were measured. The most efficient L/Gs were 1.6 and 2.5, resulting in a treated biogas effluent with a composition of 6.8%vol and 6.6%vol in CO2, respectively, and removal efficiencies for H2S up to 98.9%, as well as maintaining O2 contamination values of less than 2%vol. We found that pH greatly determines CO2 removal, more so than L/G, during cultivation because of its participation in the photosynthetic process of microalgae and its ability to vary pH when solubilized due to its acidic nature. DO, and temperature oscillated as expected from the light-dark natural cycles of photosynthesis and the time of day, respectively. Biomass growth varied with CO2 and nutrient feeding as well as reactor harvesting; however, the trend remained primed for growth.

Author Spotlight: Scaling Microalgal Biotechnology for Enhanced Biomethane Production 

Air pollution impacts the quality of life of all organisms. Here, we describe the use of microalgae biotechnology for the treatment of biogas (simultaneous removal of carbon dioxide and hydrogen sulfide) and the production of biomethane through semi-industrial open high-rate algal ponds and subsequent analysis of treatment efficiency, pH, dissolved oxygen, and microalgae growth.

In recent years, a number of technologies have emerged to purify biogas into biomethane. This purification entails a reduction in the concentration of ...

Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme saccharification processes release sugars that can be fermented by yeast. Traditional industrial yeasts do not ferment xylose (comprising up to 40% of plant sugars) and are not able to function in concentrated hydrolyzates. Concentrated hydrolyzates are needed to support economical ethanol recovery, but they are laden with toxic byproducts generated during pretreatment. While detoxification methods can render hydrolyzates fermentable, they are costly and generate waste disposal liabilities. Here, adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of the native Scheffersomyces stipitis strain NRRL Y-7124 that are able to efficiently convert hydrolyzates to economically recoverable ethanol despite adverse culture conditions. Improved individuals are enriched in an evolving population using multiple selection pressures reliant on natural genetic diversity of the S. stipitis population and mutations induced by exposures to two diverse hydrolyzates, ethanol or UV radiation. Final evolution cultures are dilution plated to harvest predominant isolates, while intermediate populations, frozen in glycerol at various stages of evolution, are enriched on selective media using appropriate stress gradients to recover most promising isolates through dilution plating. Isolates are screened on various hydrolyzate types and ranked using a novel procedure involving dimensionless relative performance index (RPI) transformations of the xylose uptake rate and ethanol yield data. Using the RPI statistical parameter, an overall relative performance average is calculated to rank isolates based on multiple factors, including culture conditions (varying in nutrients and inhibitors) and kinetic characteristics. Through application of these techniques, derivatives of the parent strain had the following improved features in enzyme saccharified hydrolyzates at pH 5-6: reduced initial lag phase preceding growth, reduced diauxic lag during glucose-xylose transition, significantly enhanced fermentation rates, improved ethanol tolerance and accumulation to 40 g/L.

Adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of Scheffersomyces stipitis strain NRRL Y-7124 that are able to rapidly consume hexose and pentose mixed sugars in enzyme saccharified undetoxified hydrolyzates and to accumulate over 40 g/L ethanol.

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme ...

Bacterial Cellulose Spheres that Encapsulate Solid Materials

Bacterial cellulose (BC) spheres have been increasingly researched since the popularization of BC as a novel material. This protocol presents an affordable and simple method for BC sphere production. In addition to producing these spheres, an encapsulation method for solid particles has also been identified. To produce BC spheres, water, black tea, sugar, vinegar, and bacterial culture are combined in a baffled flask and the contents are agitated. After determining the proper culture conditions for BC sphere formation, their ability to encapsulate solid particles was tested using biochar, polymer beads, and mine waste. Spheres were characterized using ImageJ software and thermal gravimetric analysis (TGA). Results indicate that spheres with 7.5 mm diameters can be made in 7 days. Adding various particles increases the average size range of the BC capsules. The spheres encapsulated 10 - 20% of their dry mass. This method shows low-cost sphere production and encapsulation that is possible with easily obtainable materials. BC spheres may be used in the future as a contaminant removal aid, controlled release fertilizer coating, or soil amendment.

This protocol presents an easy, inexpensive method of forming bacterial cellulose (BC) spheres. This biomaterial can function as an encapsulation medium for solid materials, including biochar, polymer spheres, and mine waste.

Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock

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