Bioenergy from dairy manure: technologies, challenges and opportunities.

Dairy manure (DM) is a kind of cheap cellulosic biomass resource which includes lignocellulose and mineral nutrients. Random stacks not only leads damage to the environment, but also results in waste of natural resources. The traditional ways to use DM include returning it to the soil or acting as a fertilizer, which could reduce environmental pollution to some extent. However, the resource utilization rate is not high and socio-economic performance is not utilized. To expand the application of DM, more and more attention has been paid to explore its potential as bioenergy or bio-chemicals production. This article presented a comprehensive review of different types of bioenergy production from DM and provided a general overview for bioenergy production. Importantly, this paper discussed potentials of DM as candidate feedstocks not only for biogas, bioethanol, biohydrogen, microbial fuel cell, lactic acid, and fumaric acid production by microbial technology, but also for bio-oil and biochar production through apyrolysis process. Additionally, the use of manure for replacing freshwater or nutrients for algae cultivation and cellulase production were also discussed. Overall, DM could be a novel suitable material for future biorefinery. Importantly, considerable efforts and further extensive research on overcoming technical bottlenecks like pretreatment, the effective release of fermentable sugars, the absence of robust organisms for fermentation, energy balance, and life cycle assessment should be needed to develop a comprehensive biorefinery model.

Biochar-mediated enhanced ethanol fermentation (BMEEF) in Zymomonas mobilis under furfural and acetic acid stress.

BACKGROUND: Pretreatment of lignocellulosic biomass generates different types of inhibitors (e.g., furfural and acetic acid), which could remarkably inhibit subsequent ethanol fermentation. Here, biochar as an additive in the fermentation broth was first applied to enhance ethanol production by Z. mobilis wild-type strain ZM4 in the presence of typical inhibitors. RESULTS: This study showed that the biochar-mediated tolerance to furfural and acetic acid for the strain Z. mobilis ZM4 was the highest reported level, resulting in much higher ethanol productivity under stress conditions than that in non-treated conditions. Further analysis showed that adsorptive detoxification was not the controlling factor for enhanced ethanol production under stress conditions, attributed to its low removal of furfural (< 20%) and incapability of acetic acid removal. When biochar was filtered from the biochar-treated inhibitor-containing broth, it still showed enhanced ethanol production. Furthermore, Z. mobilis immobilized on biochar was also observed. Thus, biochar extracts in the fermentation broth and cell immobilization on biochar might be the controlling factors for enhanced ethanol production under stress conditions. CONCLUSIONS: These results indicate that biochar-mediated enhanced ethanol fermentation (BMEEF) might be a promising strategy for ethanol production from lignocellulosic biomass.

Replacing process water and nitrogen sources with biogas slurry during cellulosic ethanol production.

BACKGROUND: Environmental issues, such as the fossil energy crisis, have resulted in increased public attention to use bioethanol as an alternative renewable energy. For ethanol production, water and nutrient consumption has become increasingly important factors being considered by the bioethanol industry as reducing the consumption of these resources would decrease the overall cost of ethanol production. Biogas slurry contains not only large amounts of wastewater, but also the nutrients required for microbial growth, e.g., nitrogen, ammonia, phosphate, and potassium. Therefore, biogas slurry is an attractive potential resource for bioethanol production that could serve as an alternative to process water and nitrogen sources. RESULTS: In this study, we propose a method that replaces the process water and nitrogen sources needed for cellulosic ethanol production by Zymomonas mobilis with biogas slurry. To test the efficacy of these methods, corn straw degradation following pretreatment with diluted NaOH and enzymatic hydrolysis in the absence of fresh water was evaluated. Then, ethanol fermentation using the ethanologenic bacterial strain Z. mobilis ZMT2 was conducted without supplementing with additional nitrogen sources. After pretreatment with 1.34% NaOH (w/v) diluted in 100% biogas slurry and continuous enzymatic hydrolysis for 144 h, 29.19 g/L glucose and 12.76 g/L xylose were generated from 30 g dry corn straw. The maximum ethanol concentration acquired was 13.75 g/L, which was a yield of 72.63% ethanol from the hydrolysate medium. Nearly 94.87% of the ammonia nitrogen was depleted and no nitrate nitrogen remained after ethanol fermentation. The use of biogas slurry as an alternative to process water and nitrogen sources may decrease the cost of cellulosic ethanol production by 10.0-20.0%. By combining pretreatment with NaOH diluted in biogas slurry, enzymatic hydrolysis, and ethanol fermentation, 56.3 kg of ethanol was produced by Z. mobilis ZMT-2 through fermentation of 1000 kg of dried corn straw. CONCLUSIONS: In this study, biogas slurry replaced process water and nitrogen sources during cellulosic ethanol production. The results suggest that biogas slurry is a potential alternative to water when pretreating corn straw and, thus, has important potential applications in cellulosic ethanol production from corn straw. This study not only provides a novel method for utilizing biogas slurry, but also demonstrates a means of reducing the overall cost of cellulosic ethanol.

Engineered Zymomonas mobilis for salt tolerance using EZ-Tn5-based transposon insertion mutagenesis system.

BACKGROUND: The cell growth and ethanol yield of Zymomonas mobilis may be detrimentally affected by salt stress frequently present in some biomass-based fermentation systems, leading to a decrease in the rate of sugar conversion to ethanol or other bioproducts. To address this problem, improving the salt tolerance of Z. mobilis is a desirable way. However, limited progress has been made in development of Z. mobilis with higher salt tolerance for some technical challenges in the past decades. Recently, transposon insertion mutant system has been widely used as a novel genetic tool in many organisms to develop mutant strains. In this study, Tn5-based transposon insertion mutagenesis system firstly used for construction of higher salt tolerance strain in Z. mobilis. RESULTS: Approximately 200 Z. mobilis ZM4 mutants were generated by using Tn5-based transposon mutagenesis system. The mutant strain ZMT2 with improved salt tolerance phenotype was obtained by screening on RM agar plates with additional 1 % NaCl. Strain ZMT2 was confirmed to exhibit better fermentation performance under NaCl stress than wild type of strain ZM4. The transposon insertion was located in ZMO1122 (himA) by genome walking. Discruption of himA gene showed that himA may play an important role in response to salt tolerance in Z. mobils. CONCLUSIONS: The mutant strain ZMT2 with a transposon insertion in himA gene of the genome showed obviously higher sugar conversion rate to ethonal under up to 2 % NaCl stress than did the wild ZM4 strain. Besides, ZMT2 exhibited shared fermentative capabilities with wild ZM4 strain under no or low NaCl stress. This report firstly showed that himA played a role in responding to NaCl stress. Furthermore, the result indicated that Tn5-based transposon mutagenesis system was a feasible tool not only for genetic engineering in Z. mobilis strain improvement, but also in tapping resistent genes.

Complete genome sequence of strain Lentibacillus amyloliquefaciens LAM0015(T) isolated from saline sediment.

The type strain Lentibacillus amyloliquefaciens LAM0015(T) with considerably highly NaCl tolerance is a member of halophiles. Here we report its genome sequence, the first to publish complete genome sequence of the Lentibacillus genus. It contains 3,858,520bp with an average GC content of 42.12%, encoding multiple valuable proteins academically and industrially. The genome sequence of strain LAM0015(T) provides basic information for further elucidation of halophilic mechanism and wider exploitation of functional genes.

Adaptive laboratory evolution of ethanologenic Zymomonas mobilis strain tolerant to furfural and acetic acid inhibitors.

Furfural and acetic acid from lignocellulosic hydrolysates are the prevalent inhibitors to Zymomonas mobilis during cellulosic ethanol production. Developing a strain tolerant to furfural or acetic acid inhibitors is difficul by using rational engineering strategies due to poor understanding of their underlying molecular mechanisms. In this study, strategy of adaptive laboratory evolution (ALE) was used for development of a furfural and acetic acid-tolerant strain. After three round evolution, four evolved mutants (ZMA7-2, ZMA7-3, ZMF3-2, and ZMF3-3) that showed higher growth capacity were successfully obtained via ALE method. Based on the results of profiling of cell growth, glucose utilization, ethanol yield, and activity of key enzymes, two desired strains, ZMA7-2 and ZMF3-3, were achieved, which showed higher tolerance under 7 g/l acetic acid and 3 g/l furfural stress condition. Especially, it is the first report of Z. mobilis strain that could tolerate higher furfural. The best strain, Z. mobilis ZMF3-3, has showed 94.84% theoretical ethanol yield under 3-g/l furfural stress condition, and the theoretical ethanol yield of ZM4 is only 9.89%. Our study also demonstrated that ALE method might also be used as a powerful metabolic engineering tool for metabolic engineering in Z. mobilis. Furthermore, the two best strains could be used as novel host for further metabolic engineering in cellulosic ethanol or future biorefinery. Importantly, the two strains may also be used as novel-tolerant model organisms for the genetic mechanism on the "omics" level, which will provide some useful information for inverse metabolic engineering.

Improving furfural tolerance of Zymomonas mobilis by rewiring a sigma factor RpoD protein.

Furfural from lignocellulosic hydrolysates is the key inhibitor for bio-ethanol fermentation. In this study, we report a strategy of improving the furfural tolerance in Zymomonas mobilis on the transcriptional level by engineering its global transcription sigma factor (σ(70), RpoD) protein. Three furfural tolerance RpoD mutants (ZM4-MF1, ZM4-MF2, and ZM4-MF3) were identified from error-prone PCR libraries. The best furfural-tolerance strain ZM4-MF2 reached to the maximal cell density (OD600) about 2.0 after approximately 30 h, while control strain ZM4-rpoD reached its highest cell density of about 1.3 under the same conditions. ZM4-MF2 also consumed glucose faster and yield higher ethanol; expression levels and key Entner-Doudoroff (ED) pathway enzymatic activities were also compared to control strain under furfural stress condition. Our results suggest that global transcription machinery engineering could potentially be used to improve stress tolerance and ethanol production in Z. mobilis.

Zymomonas mobilis: a novel platform for future biorefineries.

Biosynthesis of liquid fuels and biomass-based building block chemicals from microorganisms have been regarded as a competitive alternative route to traditional. Zymomonas mobilis possesses a number of desirable characteristics for its special Entner-Doudoroff pathway, which makes it an ideal platform for both metabolic engineering and commercial-scale production of desirable bio-products as the same as Escherichia coli and Saccharomyces cerevisiae based on consideration of future biomass biorefinery. Z. mobilis has been studied extensively on both fundamental and applied level, which will provide a basis for industrial biotechnology in the future. Furthermore, metabolic engineering of Z. mobilis for enhancing bio-ethanol production from biomass resources has been significantly promoted by different methods (i.e. mutagenesis, adaptive laboratory evolution, specific gene knock-out, and metabolic engineering). In addition, the feasibility of representative metabolites, i.e. sorbitol, bionic acid, levan, succinic acid, isobutanol, and isobutanol produced by Z. mobilis and the strategies for strain improvements are also discussed or highlighted in this paper. Moreover, this review will present some guidelines for future developments in the bio-based chemical production using Z. mobilis as a novel industrial platform for future biofineries.

Bamboo: a new source of carbohydrate for biorefinery.

Bamboo is perennial woody grass, which distributed widely in the world and belonged to the Gramineae family and Bambuseae subfamily. It may be consider as a candidate lignocellulosic substrate for bio-ethanol production for its environmental benefits and higher annual biomass yield. The conversion of bamboo into bio-ethanol, bio-methane, natural food, flavonoids, and functional xylo-oligosaccharides production were reviewed in this paper. Future prospects for research include pretreatment, enzymatic hydrolysis and fermentation will also be performed to improve the whole process of ethanol production more economical. And revealing the molecular regulation mechanism of the fast growth of bamboo will provide chance for improving bamboo or other energy plants biomass yield through genetic engineering.