Potential for reduced water consumption in biorefining of lignocellulosic biomass to bioethanol and biogas.

Increasing ethanol demand and public concerns about environmental protection promote the production of lignocellulosic bioethanol. Compared to that of starch- and sugar-based bioethanol production, the production of lignocellulosic bioethanol is water-intensive. A large amount of water is consumed during pretreatment, detoxification, saccharification, and fermentation. Water is a limited resource, and very high water consumption limits the industrial production of lignocellulosic bioethanol and decreases its environmental feasibility. In this review, we focused on the potential for reducing water consumption during the production of lignocellulosic bioethanol by performing pretreatment and fermentation at high solid loading, omitting water washing after pretreatment, and recycling wastewater by integrating bioethanol production and anaerobic digestion. In addition, the feasibility of these approaches and their research progress were discussed. This comprehensive review is expected to draw attention to water competition between bioethanol production and human use.

Effect of organic loading rate on thermophilic methane fermentation of stillage eluted from ethanol fermentation of waste paper and kitchen waste.

Thermophilic methane fermentation was a valid approach for handling the stillage eluted from ethanol fermentation of waste paper and kitchen waste. The wide organic loading rate (OLR) range (2-14 g VTS/(L d)) for stable performance and relatively high energy recovery efficiency (79.0%) were achieved, and OLR of 8 g VTS/(L d) was optimum for achievement of highest biogas evolution and VTS removal efficiency. Microbial community analysis revealed that hydrolysis of cellulose was the critical step for methane production from the stillage.

Production of ethanol from a mixture of waste paper and kitchen waste via a process of successive liquefaction, presaccharification, and simultaneous saccharification and fermentation.

Efficient ethanol production from waste paper requires the addition of expensive nutrients. To reduce the production cost of ethanol from waste paper, a study on how to produce ethanol efficiently by adding kitchen waste (potentially as a carbon source, nutrient source, and acidity regulator) to waste paper was performed and a process of successive liquefaction, presaccharification, and simultaneous saccharification and fermentation (L+PSSF) was developed. The individual saccharification performances of waste paper and kitchen waste were not influenced by their mixture. Liquefaction of kitchen waste at 90°C prior to presaccharification and simultaneous saccharification and fermentation (PSSF) was essential for efficient ethanol fermentation. Ethanol at concentrations of 46.6 or 43.6g/l was obtained at the laboratory scale after fermentation for 96h, even without pH adjustment and/or the addition of extra nutrients. Similarly, ethanol at a concentration of 45.5g/l was obtained at the pilot scale after fermentation for 48h. The ethanol concentration of L+PSSF of the mixture of waste paper and kitchen waste was comparable to that of PSSF of waste paper with added nutrients (yeast extract and peptone) and pH adjustment using H2SO4, indicating that kitchen waste is not only a carbon source but also an excellent nutrient source and acidity regulator for fermentation of the mixture of waste paper and kitchen waste.

Tyramine and ß-phenylethylamine, from fermented food products, as agonists for the human trace amine-associated receptor 1 (hTAAR1) in the stomach.

The aromatic amines tyramine and ß-phenylethylamine are abundant in fermented foods. Recently, a family of human trace amine-associated receptors (hTAARs) was discovered that responds to these compounds. This study examined the expression of hTAAR genes in five human organs. Among them, the stomach expressed hTAAR1 and hTAAR9. Interestingly, more hTAAR1 was expressed in the pylorus than in the other stomach regions. The CRE-SEAP reporter assay revealed that only hTAAR1 functioned as a Gs-coupled receptor in response to tyramine and ß-phenylethylamine stimulation. The ß-phenylethylamine-mediated hTAAR1 activity could be potentiated using 3-isobutyl-1-methylxanthine. These data suggest that tyramine and ß-phenylethylamine in fermented foods act at hTAAR1 as agonists in the pylorus of stomach.

Efficient production of ethanol from waste paper and the biochemical methane potential of stillage eluted from ethanol fermentation.

Waste paper can serve as a feedstock for ethanol production due to being rich in cellulose and not requiring energy-intensive thermophysical pretreatment. In this study, an efficient process was developed to convert waste paper to ethanol. To accelerate enzymatic saccharification, pH of waste paper slurry was adjusted to 4.5-5.0 with H2SO4. Presaccharification and simultaneous saccharification and fermentation (PSSF) with enzyme loading of 40 FPU/g waste paper achieved an ethanol yield of 91.8% and productivity of 0.53g/(Lh) with an ethanol concentration of 32g/L. Fed-batch PSSF was used to decrease enzyme loading to 13 FPU/g waste paper by feeding two separate batches of waste paper slurry. Feeding with 20% w/w waste paper slurry increased ethanol concentration to 41.8g/L while ethanol yield decreased to 83.8%. To improve the ethanol yield, presaccharification was done prior to feeding and resulted in a higher ethanol concentration of 45.3g/L, a yield of 90.8%, and productivity of 0.54g/(Lh). Ethanol fermentation recovered 33.2% of the energy in waste paper as ethanol. The biochemical methane potential of the stillage eluted from ethanol fermentation was 270.5mL/g VTS and 73.0% of the energy in the stillage was recovered as methane. Integrating ethanol fermentation with methane fermentation, recovered a total of 80.4% of the energy in waste paper as ethanol and methane.

Dynamics of the microbial community during continuous methane fermentation in continuously stirred tank reactors.

Methane fermentation is an attractive technology for the treatment of organic wastes and wastewaters. However, the process is difficult to control, and treatment rates and digestion efficiency require further optimization. Understanding the microbiology mechanisms of methane fermentation is of fundamental importance to improving this process. In this review, we summarize the dynamics of microbial communities in methane fermentation chemostats that are operated using completely stirred tank reactors (CSTRs). Each chemostat was supplied with one substrate as the sole carbon source. The substrates include acetate, propionate, butyrate, long-chain fatty acids, glycerol, protein, glucose, and starch. These carbon sources are general substrates and intermediates of methane fermentation. The factors that affect the structure of the microbial community are discussed. The carbon source, the final product, and the operation conditions appear to be the main factors that affect methane fermentation and determine the structure of the microbial community. Understanding the structure of the microbial community during methane fermentation will guide the design and operation of practical wastewater treatments.

Digestion performance and microbial community in full-scale methane fermentation of stillage from sweet potato-shochu production.

Sweet potato shochu is a traditional Japanese spirit produced mainly in the South Kyushu area in Japan. The amount of stillage reaches approximately 8 x 10(5) tons per year. Wastewater mainly containing stillage from the production of sweet potato-shochu was treated thermophilically in a full-scale treatment plant using fixed-bed reactors (8 reactors x 283 m3). Following the addition of Ni2+ and Co2+, the reactors have been stably operated for six years at a high chemical oxygen demand (COD) loading rate of 14 kg/(m3 x day). Analysis of coenzyme content and microbial communities indicated that similar microbial communities were present in the liquid phase and on the fiber carriers installed in reactors. Bacteria in the phyla Firmicutes as well as Bacteroidetes were dominant bacteria, and Methanosarcina thermophila as well as Methanothermobacter crinale were dominant methanogens in the reactors. This study reveals that stillage from sweet potato-shochu production can be treated effectively in a full-scale fixed-bed reactor under thermophilic conditions with the help of Ni2+ and Co2+. The high diversity of bacterial community and the coexistence of both aceticlastic and hydrogenotrophic methanogens contributed to the excellent fermentation performance.

Production of bio-fuel ethanol from distilled grain waste eluted from Chinese spirit making process.

Distilled grain waste eluted from Chinese spirit making is rich in carbohydrates, and could potentially serve as feedstock for the production of bio-fuel ethanol. Our study evaluated two types of saccharification methods that convert distilled grain waste to monosaccharides: enzymatic saccharification and concentrated H2SO4 saccharification. Results showed that enzymatic saccharification performed unsatisfactorily because of inefficient removal of lignin during pretreatment. Concentrated H2SO4 saccharification led to a total sugar recovery efficiency of 79.0 %, and to considerably higher sugar concentrations than enzymatic saccharification. The process of ethanol production from distilled grain waste based on concentrated H2SO4 saccharification was then studied. The process mainly consisted of concentrated H2SO4 saccharification, solid-liquid separation, decoloration, sugar-acid separation, oligosaccharide hydrolysis, and continuous ethanol fermentation. An improved simulated moving bed system was employed to separate sugars from acid after concentrated H2SO4 saccharification, by which 95.8 % of glucose and 85.8 % of xylose went into the sugar-rich fraction, while 83.3 % of H2SO4 went into the acid-rich fraction. A flocculating yeast strain, Saccharomyces cerevisiae KF-7, was used for continuous ethanol fermentation, which produced an ethanol yield of 91.9-98.9 %, based on glucose concentration.

Potent L-lactic acid assimilation of the fermentative and heterothallic haploid yeast Saccharomyces cerevisiae NAM34-4C.

We screened an industrial thermotolerant Saccharomyces cerevisiae strain, KF7, as a potent lactic-acid-assimilating yeast. Heterothallic haploid strains KF7-5C and KF7-4B were obtained from the tetrads of the homothallic yeast strain KF7. The inefficient sporulation and poor spore viability of the haploid strains were improved by two strategies. The first strategy was as follows: (i) the KF7-5C was crossed with the laboratory strain SH6710; (ii) the progenies were backcrossed with KF7-5C three times; and (iii) the progenies were inbred three times to maintain a genetic background close to that of KF7. The NAM12 diploid between the cross of the resultant two strains, NAM11-9C and NAM11-13A, showed efficient sporulation and exhibited excellent growth in YPD medium (pH 3.5) at 35°C with 1.4-h generation time, indicating thermotolerance and acid tolerance. The second strategy was successive intrastrain crosses. The resultant two strains, KFG4-6B and KFG4-4B, showed excellent mating capacity. A spontaneous mutant of KFG4-6B, KFG4-6BD, showed a high growth rate with a generation time of 1.1 h in YPD medium (pH 3.0) at 35°C. The KFG4-6BD strain produced ascospores, which were crossed with NAM11-2C and its progeny to produce tetrads. These tetrads were crossed with KFG4-4B to produce NAM26-14A and NAM26-15A. The latter strain had a generation time of 1.6 h at 35°C in pH 2.5, thus exhibiting further thermotolerance and acid tolerance. A progeny from a cross of NAM26-14A and NAM26-15A yielded the strain NAM34-4C, which showed potent lactic acid assimilation and high transformation efficiency, better than those of a standard laboratory strain.

Efficient production of bioethanol from corn stover by pretreatment with a combination of sulfuric acid and sodium hydroxide.

Corn stover is the most abundant agricultural residue in China and a valuable reservoir for bioethanol production. In this study, we proposed a process for producing bioethanol from corn stover; the pretreatment prior to presaccharification, followed by simultaneous saccharification and fermentation (SSF) by using a flocculating Saccharomyces cerevisiae strain, was optimized. Pretreatment with acid-alkali combination (1% H2SO4, 150 °C, 10 min, followed by 1% NaOH, 80°C, 60 min) resulted in efficient lignin removal and excellent recovery of xylose and glucose. A glucose recovery efficiency of 92.3% was obtained by enzymatic saccharification, when the pretreated solid load was 15%. SSF was carried out at 35 °C for 36 hr after presaccharification at 50 °C for 24 hr, and an ethanol yield of 88.2% was achieved at a solid load of 15% and an enzyme dosage of 15 FPU/g pretreated corn stover.

Pretreatment followed by anaerobic digestion of secondary sludge for reduction of sewage sludge volume.

The influence of two pretreatment methods, thermal treatment and low-pressure wet oxidation, on the sludge digestion efficiency was examined. Batch thermophilic anaerobic digestion was used to evaluate the effectiveness of the pretreatment methods in terms of volatile suspended solids (VSS) digestion efficiency and gas production. The results showed that the gas production was not proportional to the VSS degradation efficiency of either thermal treatment or low-pressure wet oxidation. Low-pressure wet oxidation treatment at 150 °C along with 40% of the theoretical oxygen required to oxidize organic carbon gave the highest gas production and the VSS digestion efficiency of 77% at a VSS loading rate of 8 g l(-1) d(-1). The digestion efficiency was about 30% higher than that of thermophilic anaerobic digestion without sludge pretreatment. Sewage sludge could be treated effectively at a high VSS digestion efficiency with this pretreatment followed by thermophilic anaerobic digestion.

Reduction in environmental impact of sulfuric acid hydrolysis of bamboo for production of fuel ethanol.

Fuel ethanol can be produced from bamboo by concentrated sulfuric acid hydrolysis followed by continuous ethanol fermentation. To reduce the environmental impact of this process, treatment of the stillage, reuse of the sulfuric acid and reduction of the process water used were studied. The total organic carbon (TOC) concentration of stillage decreased from 29,688 to 269 mg/l by thermophilic methane fermentation followed by aerobic treatment. Washing the solid residue from acid hydrolysis with effluent from the biological treatment increased the sugar recovery from 69.3% to 79.3%. Sulfuric acid recovered during the acid-sugar separation process was condensed and reused for hydrolysis, resulting in a sugar recovery efficiency of 76.8%, compared to 80.1% when fresh sulfuric acid was used. After acetate removal, the condensate could be reused as elution water in the acid-sugar separation process. As much as 86.3% of the process water and 77.6% of the sulfuric acid could be recycled.

A secreted placental alkaline phosphatase-based reporter assay system for screening of compounds acting at an octopamine receptor stably expressed in a mammalian cell line.

Octopamine receptors are attractive insecticide targets. To screen compounds acting at octopamine receptors simply and rapidly, we constructed a chemiluminescent reporter gene assay system that detects secreted placental alkaline phosphatase transcriptionally regulated by the cAMP response element for a silkworm octopamine receptor. This system proved useful in high-throughput screening to develop octopamine receptor-specific insecticides.

Rac1 and Stathmin but Not EB1 Are Required for Invasion of Breast Cancer Cells in Response to IGF-I.

Cell migration is considered necessary for the invasion that accompanies the directional formation of the cellular protrusions termed lamellipodia. In invasive breast cancer MDA-MB-231 cells, lamellipodia formation is preceded by translocation of the actin cytoskeletal regulatory protein WAVE2 to the leading edge. WAVE2 translocation and lamellipodia formation require many signaling molecules, including PI3K, Rac1, Pak1, IRSp53, stathmin, and EB1, but whether these molecules are necessary for invasion remains unclear. In noninvasive breast cancer MCF7 cells, no lamellipodia were induced by IGF-I, whereas in MDA-MB-231 cells, Rac1, stathmin, and EB1 were overexpressed. Depletion of Rac1 or stathmin by small interfering RNA abrogated the IGF-I-induced invasion of MDA-MB-231 cells; however, depletion of EB1 did not, indicating the necessity of Rac1 and stathmin but not EB1 for invasion. The signaling pathway leading to cell invasion may not be identical but shares some common molecules, leading to cell migration through lamellipodia formation.

Development of an efficient process for the treatment of residual sludge discharged from an anaerobic digester in a sewage treatment plant.

In order to reduce the discharge of residual sludge from an anaerobic digester, pre-treatment methods including low-pressure wet-oxidation, Fenton oxidation, alkali treatment, ozone oxidation, mechanical destruction and enzymatic treatment were evaluated and compared. VSS removal efficiencies of greater than 50% were achieved in cases of low-pressure wet-oxidation, Fenton oxidation and alkali treatment. Residual sludge from an anaerobic digester was pre-treated and subjected to thermophilic anaerobic digestion. As a result, the process of low-pressure wet-oxidation followed by anaerobic digestion achieved the highest VSS removal efficiency of 83%. The total efficiency of VSS removal of sewage sludge consisting of primary and surplus sludge would be approximately 92%, assuming that the VSS removal efficiency of sewage sludge is 50% in the anaerobic digester of the sewage treatment plant.

Synthesis of chitosan-caffeic acid derivatives and evaluation of their antioxidant activities.

In this study, the antioxidant activities of different molecular weights (M(w)) and grafting ratios of chitosan-caffeic acid derivatives were investigated. The grafting process was achieved using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) as covalent connector under different conditions such as molecular-weight of chitosan, molar ratio of chitosan and caffeic acid, reaction temperature, pH, and reaction time. The half-inhibition concentrations (IC50) of products were calculated by reduction of the 1,1-diphenyl picryl hydrazyl in the radical-scavenging assay and reduction of the Fe³+/ferricyanide complex to the ferrous form in reducing power assay. The EDAC showed maximum activity at 3-h, pH 5.0 and room temperature conditions, except high-molecular-weight chitosan in pH 2.0. The products were water-soluble in all pH and showed lower viscosity than native chitosan. The highest grafting ratio of caffeic acid was observed at 15% in low-molecular-weight chitosan. After 5% grafting of caffeic acid into chitosan, the grafting efficiency was increased by decreasing molecular-weight of chitosan at the same conditions. Caffeic acid has main role in the antioxidant activity of products. The maximum IC50 of radical-scavenging activity (0.064 mg/ml) was observed at the highest caffeic acid containing derivative. Water-soluble chitosan and caffeic acid derivatives were obtained by this study without activity loss.

Nonmuscle myosin IIA is required for lamellipodia formation through binding to WAVE2 and phosphatidylinositol 3,4,5-triphosphate.

Investigation of the mechanism underlying cell membrane-targeted WAVE2 capture by phosphatidylinositol 3,4,5-triphosphate (PIP(3)) through IRSp53 revealed an unidentified 250-kDa protein (p250) bound to PIP(3). We identified p250 as nonmuscle myosin IIA heavy chain (MYH9) by mass spectrometry and immunoblot analysis using anti-MYH9 antibody. After stimulation with insulin-like growth factor I (IGF-I), MYH9 colocalized with PIP(3) in lamellipodia at the leading edge of cells. Depletion of MYH9 expression by small interfering RNA (siRNA) and inhibition of myosin II activity by blebbistatin abrogated the formation of actin filament (F-actin) arcs and lamellipodia induced by IGF-I. MYH9 was constitutively associated with WAVE2, which was dependent on myosin II activity, and the MYH9-WAVE2 complex colocalized to PIP(3) at the leading edge after IGF-I stimulation. These results indicate that MYH9 is required for lamellipodia formation since it provides contractile forces and tension for the F-actin network to form convex arcs at the leading edge through constitutive binding to WAVE2 and colocalization with PIP(3) in response to IGF-I.

Functional replacement of yeast flavocytochrome b(2) with bacterial L-lactate dehydrogenase.

Using yeast genetic complementation, we show here that Rhodobacter sphaeroidesl-lactate dehydrogenase can functionally replace flavocytochrome b(2) in Saccharomyces cerevisiae, only when a matrix-targeting signal of flavocytochrome b(2) is fused with the bacterial enzyme. The recombinant l-lactate dehydrogenase may add alternative route of mitochondrial electron transport other than flavocytochrome b(2).

Production of fuel ethanol and methane from garbage by high-efficiency two-stage fermentation process.

A two-stage fermentation process, consisting of a simultaneous saccharification and fermentation (SSF) stage and a dry methane fermentation stage, was developed to utilize garbage for the production of fuel ethanol and methane. Garbage from families, canteens and concessionaires was used for the study. Saccharification method was studied and the results indicated that the liquefaction pretreatment and the combination of cellulase and glucoamylase was effective for polysaccharide hydrolysis of family garbage with a high content of holocellulose and that SSF was suitable for ethanol fermentation of garbage. Ethanol productivity could be markedly increased from 1.7 to 7.0 g/l/h by repeated-batch SSF of family garbage. A high ethanol productivity of 17.7 g/l/h was achieved when canteen garbage was used. The stillage after distillation was treated by dry methane fermentation and the results indicated that the stillage was almost fully digested and that about 850 ml of biogas was recovered from 1 g of volatile total solid (VTS). Approximately 85% of the energy of the garbage was converted to fuels, ethanol and methane by this process.

BetaPIX and GIT1 regulate HGF-induced lamellipodia formation and WAVE2 transport.

Formation of lamellipodia is the first step during cell migration, and involves actin reassembly at the leading edge of migrating cells through the membrane transport of WAVE2. However, the factors that regulate WAVE2 transport to the cell periphery for initiating lamellipodia formation have not been elucidated. We report here that in human breast cancer MDA-MB-231 cells, the hepatocyte growth factor (HGF) induced the association between the constitutive complex of betaPIX and GIT1 with WAVE2, which was concomitant with the induction of lamellipodia formation and WAVE2 transport. Although depletion of betaPIX by RNA interference abrogated the HGF-induced WAVE2 transport and lamellipodia formation, GIT1 depletion caused HGF-independent WAVE2 transport and lamellipodia formation. Collectively, we suggest that betaPIX releases cells from the GIT1-mediated suppression of HGF-independent responses and recruits GIT1 to WAVE2, thereby facilitating HGF-induced WAVE2 transport and lamellipodia formation.