Different traits for cold tolerance of extremely thermophilic Calditerricola strains isolated from mesothermal municipal sewage sludge and its hyperthermal compost.

Extreme thermophiles Calditerricola satsumensis DD2 and D3 were isolated from mesothermal municipal sludge, a material used for hyperthermal composting. To understand the ecologically anomalous findings, their behavior at various temperatures, membrane fatty acid composition, and draft genome sequences were compared with those of C. satsumensis YMO81T and Calditerricola yamamurae YMO722T, already isolated from hyperthermal compost. All four strains grew between 56 and 83 °C. However, strains DD2 and D3 were stable for ≥48 h at a wide range of temperatures (20-75 °C), while strains YMO81T and YMO722T were highly labile at lower temperatures. The former strains maintained their colony-forming ability for >180 days at 20 °C, while the latter strains lost it within 1 d. All four strains showed similar composition of membrane fatty acid, which were not affected by 20 °C treatment. Comparative draft genome analyses showed that 13 candidate genes were present only in strains DD2 and D3, and the specific expression of six gene homologs was confirmed. A DNA chaperone, site-specific recombinase XerD homolog, had tetra adenine sequence at its upper gene region, and was up-regulated by 20 °C treatment in DD2 and D3, suggesting a possible role in the cold tolerance of sludge-derived strains. In addition, the lack of another possible DNA chaperone, a homolog of the ATP-dependent DNA helicase, in the compost-derived strains may accelerate their sensitivity to cold shock. In conclusion, we speculate that the specific phenotypic and genotypic characteristics of sludge-derived strains are responsible for their unusual ecological distribution at ambient temperatures.

Cyclic pairwise interaction representing a rock-paper-scissors game maintains the population of the vulnerable yeast Saccharomyces cerevisiae within a multispecies sourdough microbiome.

IMPORTANCE: Traditionally, multispecies consisting of lactic acid bacteria and yeasts collaboratively engage sourdough fermentation, which determines the quality of the resulting baked goods. Nonetheless, the successive transfer of these microbial communities can result in undesirable community dynamics that prevent the formation of high-quality sourdough bread. Thus, a mechanistic understanding of the community dynamics is fundamental to engineer sourdough complex fermentation. This study describes the population dynamics of five species of lactic acid bacteria-yeast communities in vitro using a generalized Lotka-Volterra model that examines interspecies interactions. A vulnerable yeast species was maintained within up to five species community dynamics by obtaining support with a cyclic interspecies interaction. Metaphorically, it involves a rock-paper-scissors game between two lactic acid bacteria species. Application of the generalized Lotka-Volterra model to real food microbiomes including sourdoughs will increase the reliability of the model prediction and help identify key microbial interactions that drive microbiome dynamics.

Dilution rates and their transition modes influence organic acid productivity and bacterial community structure on continuous meta-fermentation using complex microorganisms.

We investigated the effect of dilution rates (D) (0.05, 0.15, and 0.4 h-1) and its transition mode strategies (constant, up, and down modes) on organic acid productivity and bacterial community structure on continuous meta-fermentation using complex microorganisms. The number of bacterial species decreased with increasing D in the constant mode while up and down modes maintained high and low values, respectively, regardless of the changing D values. Caldibacillus hisashii was the predominant species in all modes at all D values, while other bacterial species, including Anaerosalibacter bizertensis and Clostridium cochlearium were predominant in only certain modes and D values. The highest total organic acid productivity of 3.16 g L-1 h-1 was obtained with 82.2% lactic acid selectivity at D = 0.4 h⁻1 in constant mode. Heterofermentation occurred in the up mode, while the down mode exhibited the maximum butyric acid productivity of 0.348 g L-1 h-1 with 43.8% selectivity at D = 0.05 h-1. The constant, up, and down modes showed the distinct main products of lactic, acetic and formic, and butyric acids, respectively. In this study, we proposed a new parameter of species-specific productivity (SSP) to estimate which species and how much a bacterium quantitatively contributes to the targeted organic acid productivity in continuous meta-fermentation. SSP was determined based on the abundance of functional genes encoding key enzymes from the results of 16S amplicon analysis. In conclusion, D values and their transition modes affect productivity by changing the bacterial community structure, and are a significant factor in establishing a highly productive process in continuous meta-fermentation.

Distribution of bacterial community structures on human scalp hair shaft in relation to scalp sites.

Bacterial community structure on the human skin is specific to each individual and varies among different body sites. In this study, we investigated differences in bacterial community structure among 5 hair sampling sites and among 12 individuals. Significant differences were found between individuals in terms of alpha diversity and relative abundance of major bacterial phyla and genera, whereas no differences were found between hair sampling sites. The principal coordinate analysis plots of within-individual group tended to converge individually, whereas those of within-hair sampling site group did not cluster. In addition, weighted UniFrac analysis showed that the individual-based category was a statistically significant category but not the scalp hair sampling site-based category. These results suggest that the distribution of bacterial community structures on scalp hair shafts within individuals was relatively steady, even when the scalp hair sampling site was different.

Scalp bacterial species influence SIRT1 and TERT expression in keratinocytes.

Scalp bacteria on the human scalp and scalp hair comprise distinct community structures for sites and individuals. To evaluate their effect on human keratinocyte cellular activity, including that of the hair follicular keratinocytes, the expression of several longevity genes was examined using HaCaT cells. A screening system that uses enhanced green fluorescent protein (EGFP) fluorescence was established to identify scalp bacteria that enhance silent mating type information regulation 2 homolog-1 (SIRT1) promoter activity in transformed HaCaT cells (SIRT1p-EGFP). The results of quantitative polymerase chain reaction revealed that several predominant scalp bacteria enhanced (Cutibacterium acnes and Pseudomonas lini) and repressed (Staphylococcus epidermidis) the expressions of SIRT1 and telomerase reverse transcriptase (TERT) genes in HaCaT cells. These results suggest that the predominant scalp bacteria are related to the health of the scalp and hair, including repair of the damaged scalp and hair growth, by regulating gene expression in keratinocytes.

Clarification of the Dynamic Autothermal Thermophilic Aerobic Digestion Process Using Metagenomic Analysis.

This study details a unique process of autothermal thermophilic aerobic digestion (ATAD) of human excreta useful in producing nitrogen-rich and pathogen-free organic fertilizer. The process was divided into initial, middle, and final phases, based on changes in temperature, dissolved oxygen (DO), and bacterial community structure. The aim of this study was to determine bacterial factors that would affect liquid fertilizer production in the process, using shotgun metagenomic analysis of each phase. Although the abundances of all 28 gene categories include 4 categories in SEED subsystems level 1 were similar to those in another type of wastewater treatment system, the abundances of 4 gene categories changed remarkably. Among them, a decrease in the abundance of the phage-related gene category and the presence of antibacterial substances in secondary metabolism may explain the change in bacterial community structure from the material to the initial phase. Increases in the abundances of two gene categories, phage-related and secondary metabolism, coincided with a decrease in alpha diversity from the material to the initial phase. A potential increase in the abundance of genes in the category of sporulation from the middle to the final phase was correlated with deterioration of growth conditions and stabilization processes. In addition, prompt consumption of short-chain fatty acids in the initial phase and unusually stable ammonia accumulation throughout the process could be explained by the presence/absence of related metabolic genes. In conclusion, the relationships between bacterial function and unique characteristics of ATAD were revealed; our findings support the enhancement of liquid fertilizer production from wastewater. IMPORTANCE Metagenome analysis was performed to determine the microbial dynamics of the unique autothermal thermophilic aerobic digestion process of human excreta, which includes initial, middle, and final phases. In this study, we revealed the details of functional genes related to physicochemical and bacterial characteristics in the ATAD process. Four gene categories showed increases and decreases during the digestion process. In addition, the unusual stable accumulation of ammonia and prompt consumption of short-chain fatty acids were explained by the absence or presence of related metabolic genes. In addition to revealing the relationships between bacteria and physicochemical properties, the results of this research may support improving wastewater management systems worldwide by using the ATAD process in liquid fertilizer production systems.

Application of autothermal thermophilic aerobic digestion as a sustainable recycling process of organic liquid waste: Recent advances and prospects.

Autothermal thermophilic aerobic digestion (ATAD) has been used to stabilize organic waste since the 1960s and is considered sustainable technology. ATAD has several advantages, including high biodegradation efficiency, pathogen inactivation, and ease of operation. Although ATAD research has a long history, the number of studies on ATAD is much lower than those on similar aerobic processes, particularly composting. Previous review articles addressed the origin, design, operational experiences, metabolism, and the microorganisms at the thermophilic stage of ATAD. This article reviews the digestion systems, applications, and characteristics of ATAD; compares system performance and microbial community structure of ATAD with those of other biological processes such as composting, activated sludge, and anaerobic digestion; and discusses the physicochemical properties and factors of ATAD. The challenges, opportunities, and prospects for the application of ATAD are also discussed. This review suggests that ATAD is feasible for treating organic liquid waste (1-6% total solid content) in small-sized towns and can help establish a sustainable society.

Host factors that shape the bacterial community structure on scalp hair shaft.

In this study, we performed 16S rRNA amplicon sequencing analysis of scalp hair shaft from 109 volunteers, who were surveyed using a questionnaire about daily scalp hair care, and employed multiple statistical analyses to elucidate the factors that contribute to the formation of bacterial community structures on scalp hair shaft. Scalp hair microbiota were found to be specific for each individual. Their microbiota were clearly divided into two clusters. Genus level richness of Pseudomonas (Ps) and Cutibacterium (Cu) contributed to the clusters. The clusters around Pseudomonas and Cutibacterium were named Ps-type and Cu-type, respectively. The host gender influenced the bacterial cell numbers of the major genera that included Cutibacterium, Lawsonella, Moraxella, and Staphylococcus on scalp hair shaft. In addition to host intrinsic factors, extrinsic factors such as hair styling and colouring affected the bacterial cell numbers of the major genera. These factors and chemical treatments, such as bleaching and perming, also affected the Ps-type to Cu-type ratios. These results suggest that bacterial community structures on scalp hair shaft are influenced by both intrinsic and extrinsic factors.

Meta-fermentation system with a mixed culture for the production of optically pure l-lactic acid can be reconstructed using the minimum isolates with a simplified pH control strategy.

Meta-l-lactic acid fermentation from non-treated kitchen refuse was reconstructed using a combination of isolated bacterial strains under several pH control strategies. The meta-fermentation system was successfully reconstructed using a combination of Weizmannia coagulans MN-07, Caldibacillus thermoamylovorans OM55-6, and Caldibacillus hisashii N-11 strains. Additionally, a simplified constant pH control strategy was employed, which decreased fermentation time and increased production. The optimum pH (6.5) for the reconstructed meta-fermentation was favorable for the respective pure cultures of the three selected strains. The l-lactic acid production performance of the reconstructed meta-fermentation system was as follows: concentration, 24.5 g L-1 ; optical purity, 100%; productivity, 0.341 g L-1 h-1 ; yield, 1.06 g g-1 . These results indicated that constant pH control was effective in the reconstructed meta-fermentation with the best performance of l-lactic acid production at pH optimal for the selected bacterial growth, while the switching from swing pH control would suppress the activities of unfavorable bacterial species in un-isolated meta-fermentation.

Lab-scale autothermal thermophilic aerobic digestion can maintain and remove nitrogen by controlling shear stress and oxygen supply system.

Autothermal thermophilic aerobic digestion (ATAD) is used to treat human excreta hygienically. We previously reported a unique full-scale ATAD, showing distinctive bacterial community transitions and producing high-nitrogen-content liquid fertilizer; nevertheless, the mechanism remains unclear. One hypothesis involves using a gas-inducing (GI) agitator. We designed a lab-scale GI system and compared it with a disk-turbine (DT) agitator system by mimicking the temperature shift of full-scale ATAD. The agitation system and its agitation speed greatly affected physicochemical properties and bacterial community structure. GI system at 1000 rpm (GI1000; high total carbon removal efficiency, 88.3%), with few nitrifying and denitrifying bacteria, maintained a high ammoniacal nitrogen concentration and had more shared operational taxonomic units related to Acinetobacter sp., Arcobacter sp., and Longimicrobium sp. with the full-scale ATAD compared with the GI system at 490 rpm and DT system at 1000 rpm (DT1000). Furthermore, DT1000, with a high abundance of nitrifying and denitrifying bacteria such as Alcaligenes aquatilis and Pseudomonas caeni, removed 94.7% total nitrogen with 71.9% total carbon removal efficiency. These results suggested that shear stress and oxygen supply system would change the bacterial community structure, thus affected ATAD performances. Consequently, it is possible that ATAD can be applied for not only production of highly nitrogen-containing liquid fertilizer but also extremely nitrogen removal of wastewater.

Transcriptome profile of carbon catabolite repression in an efficient l-(+)-lactic acid-producing bacterium Enterococcus mundtii QU25 grown in media with combinations of cellobiose, xylose, and glucose.

Enterococcus mundtii QU25, a non-dairy lactic acid bacterium of the phylum Firmicutes, is capable of simultaneously fermenting cellobiose and xylose, and is described as a promising strain for the industrial production of optically pure l-lactic acid (≥ 99.9%) via homo-fermentation of lignocellulosic hydrolysates. Generally, Firmicutes bacteria show preferential consumption of sugar (usually glucose), termed carbon catabolite repression (CCR), while hampering the catabolism of other sugars. In our previous study, QU25 exhibited apparent CCR in a glucose-xylose mixture phenotypically, and transcriptional repression of the xylose operon encoding initial xylose metabolism genes, likely occurred in a CcpA-dependent manner. QU25 did not exhibit CCR phenotypically in a cellobiose-xylose mixture. The aim of the current study is to elucidate the transcriptional change associated with the simultaneous utilization of cellobiose and xylose. To this end, we performed RNA-seq analysis in the exponential growth phase of E. mundtii QU25 cells grown in glucose, cellobiose, and/or xylose as either sole or co-carbon sources. Our transcriptomic data showed that the xylose operon was weakly repressed in cells grown in a cellobiose-xylose mixture compared with that in cells grown in a glucose-xylose mixture. Furthermore, the gene expression of talC, the sole gene encoding transaldolase, is expected to be repressed by CcpA-mediated CCR. QU25 metabolized xylose without using transaldolase, which is necessary for homolactic fermentation from pentoses using the pentose-phosphate pathway. Hence, the metabolism of xylose in the presence of cellobiose by QU25 may have been due to 1) sufficient amounts of proteins encoded by the xylose operon genes for xylose metabolism despite of the slight repression of the operon, and 2) bypassing of the pentose-phosphate pathway without the TalC activity. Accordingly, we have determined the targets of genetic modification in QU25 to metabolize cellobiose, xylose and glucose simultaneously for application of the lactic fermentation from lignocellulosic hydrolysates.

Relationship between the bacterial community structures on human hair and scalp.

In this study, we investigated and compared characteristics of the bacterial community structures on hair (scalp hair) and scalp in 18 individuals. Significant differences were found between the sites, in terms of cell density, alpha and beta diversity, and relative abundance of the phyla, Firmicutes and Proteobacteria, whereas no difference was found in relative abundance of the phylum Actinobacteria. Bacteria of the genus Cutibacterium showed similar relative abundance at both sites, whereas those of genus Pseudomonas were highly abundant on hair, and those of genus Staphylococcus were significantly lesser in abundance on hair than on scalp. Statistical correlations between the sites were high for the individual relative abundance of five major operational taxonomic units (OTUs). This suggests that the bacterial community structure on hair is composed of hair-specific genus, Pseudomonas, and skin-derived genera, Cutibacterium and Staphylococcus, and is distinguishable from other human skin microbiomes.

Non-carbon loss long-term continuous lactic acid production from mixed sugars using thermophilic Enterococcus faecium QU 50.

In this study, a non-sterile (open) continuous fermentation (OCF) process with no-carbon loss was developed to improve lactic acid (LA) productivity and operational stability from the co-utilization of lignocellulose-derived sugars by thermophilic Enterococcus faecium QU 50. The effects of different sugar mixtures on LA production were firstly investigated in conventional OCF at 50°C, pH 6.5 and a dilution rate of 0.20 hr-1 . The xylose consumption ratio was greatly lower than that of glucose in fermentations with glucose/xylose mixtures, indicating apparent carbon catabolite repression (CCR). However, CCR could be efficiently eliminated by feeding solutions containing the cellobiose/xylose mixture. In OCF at a dilution rate ca. 0.10 hr-1 , strain QU 50 produced 42.6 g L-1 of l-LA with a yield of 0.912 g g-1 -consumed sugars, LA yield of 0.655 g g-1 based on mixed sugar-loaded, and a productivity of 4.31 g L-1 hr-1 from simulated energy cane hydrolyzate. In OCF with high cell density by cell recycling, simultaneous and complete co-utilization of sugars was achieved with stable LA production at 60.1 ± 3.25 g L-1 with LA yield of 0.944 g g-1 -consumed sugar and LA productivity of 6.49 ± 0.357 g L-1 hr-1 . Besides this, a dramatic increase in LA yield of 0.927 g g-1 based on mixed sugar-loaded with prolonged operational stability for at least 500 hr (>20 days) was established. This robust system demonstrates an initial green step with a no-carbon loss under energy-saving toward the feasibility of sustainable LA production from lignocellulosic sugars.

Methane production from food waste via mesophilic anaerobic digestion with ethanol pre-fermentation: Methanogenic pathway and microbial community analyses.

To investigate the methanogenic pathway and microbial community in a mesophilic anaerobic digestion (AD) system with food waste (FW) ethanol pre-fermentation (EP), two semi-continuous AD systems were operated by feeding FW with (PSR) and without EP (control). In this study, δ13C-ethanol was supplemented as solo substrate for AD sludge when the reactors operation stabilized to analyze the methanogenic pathways. The results suggested that approximately 59.3% of methane was produced from acetotrophic methanogens, while 40.7% was formed by hydrogenotrophic methanogens in the PSR group. On the other hand, compared with control, methane produced via CO2 reduction pathway was increased by 4.70%. Meanwhile, the composition variations of the microbial community in AD supported the above conclusion, since the relative abundances of Clostridium and Methanobacterium were enhanced by 7.6% and 10.2%, respectively in PSR reactor. These results provided a theoretical basis for AD applications and biogas yield improvements with EP process.

Smart fermentation engineering for butanol production: designed biomass and consolidated bioprocessing systems.

There is a renewed interest in acetone-butanol-ethanol (ABE) fermentation from renewable substrates for the sustainable and environment-friendly production of biofuel and platform chemicals. However, the ABE fermentation is associated with several challenges due to the presence of heterogeneous components in the renewable substrates and the intrinsic characteristics of ABE fermentation process. Hence, there is a need to select optimal substrates and modify their characteristics suitable for the ABE fermentation process or microbial strain. This "designed biomass" can be used to establish the consolidated bioprocessing systems. As there are very few reports on designed biomass, the main objectives of this review are to summarize the main challenges associated with ABE fermentation from renewable substrates and to introduce feasible strategies for designing the substrates through pretreatment and hydrolysis technologies as well as through the establishment of consolidated bioprocessing systems. This review offers new insights on improving the efficiency of ABE fermentation from designed renewable substrates.

Mode and Structure of the Bacterial Community on Human Scalp Hair.

Bacterial communities on various parts of the human body are distinct. We were the first to report the existence of a stable bacterial community on human scalp hair and demonstrated that an analysis of its structure by terminal restriction fragment length polymorphism (T-RFLP) is helpful for individual discrimination. However, the ecology of the bacterial community on human scalp hair has not yet been elucidated in detail. We herein investigated the mode, quantity, and phylogeny of bacterial communities on the human hair shaft and root and showed the results obtained from one representative individual. Direct SEM observations of hair, without a pretreatment, confirmed the ubiquitous presence of bacteria-like coccoids and rods on the shaft and root of hair from the human scalp, with 105-106 cells cm-2 of hair and 107 cells cm-2 of hair, respectively. These values corresponded to the 16S rRNA gene copy numbers obtained by qPCR. These numbers were not significantly affected by detergent washing. These results represented those obtained from many individuals with different hair lengths, ages, and gender. The major OTUs on the human scalp hair shaft and root were the same and included two species of Pseudomonas (phylum Proteobacteria), Cutibacterium and Lawsonella (phylum Actinobacteria), and Staphylococcus (phylum Firmicutes). These results suggest that major bacteria on the human hair shaft are indigenous and derived from the hair root.

Dynamics of Microbial Populations Responsible for Biodegradation during the Full-Scale Treatment of Palm Oil Mill Effluent.

Despite efforts to address the composition of the microbial community during the anaerobic treatment of palm oil mill effluent (POME), its composition in relation to biodegradation in the full-scale treatment system has not yet been extensively examined. Therefore, a thorough analysis of bacterial and archaeal communities was performed in the present study using MiSeq sequencing at the different stages of the POME treatment, which comprised anaerobic as well as facultative anaerobic and aerobic processes, including the mixed raw effluent (MRE), mixing pond, holding tank, and final discharge phases. Based on the results obtained, the following biodegradation processes were suggested to occur at the different treatment stages: (1) Lactobacillaceae (35.9%) dominated the first stage, which contributed to high lactic acid production; (2) the higher population of Clostridiaceae in the mixing pond (47.7%) and Prevotellaceae in the holding tank (49.7%) promoted acetic acid production; (3) the aceticlastic methanogen Methanosaetaceae (0.6-0.8%) played a role in acetic acid degradation in the open digester and closed reactor for methane generation; (4) Syntrophomonas (21.5-29.2%) appeared to be involved in the degradation of fatty acids and acetic acid by syntrophic cooperation with the hydrogenotrophic methanogen, Methanobacteriaceae (0.6-1.3%); and (5) the phenols and alcohols detected in the early phases, but not in the final discharge phase, indicated the successful degradation of lignocellulosic materials. The present results contribute to a better understanding of the biodegradation mechanisms involved in the different stages of the full-scale treatment of POME.

Semi-hydrolysate of paper pulp without pretreatment enables a consolidated fermentation system with in situ product recovery for the production of butanol.

Utilization of lignocellulosic biomasses for biobutanol fermentation usually requires costly processes of pretreatment and enzymatic hydrolysis. In this study, paper pulp (93.2% glucan) was used as a starting biomass material to produce biobutanol. We conducted enzymatic semi-hydrolysis of paper pulp without pretreatment and with low enzyme loading, which produced high concentrations of cellobiose (13.9 g L-1) and glucose (21.3 g L-1). In addition, efficient fermentation of the semi-hydrolysate was achieved similar to that with the use of commercial sugars without inhibitors. Finally, we designed a novel non-isothermal simultaneous saccharification and fermentation with in situ butanol recovery, which was composed of a repeated semi-hydrolysis process and successive butanol-extractive fermentation process under the respective optimal conditions. The consolidated system improved butanol production, butanol yields, and butanol productivities and enabled repeated use of medium when compared with other integrated hydrolysis and fermentation processes.

Novel biobutanol fermentation at a large extractant volume ratio using immobilized Clostridium saccharoperbutylacetonicum N1-4.

Product inhibition by butanol and acetone is a known drawback in acetone-butanol-ethanol (ABE) fermentation. Extractive fermentation improves butanol production by several ABE-producing Clostridium spp., but only low volume ratios (<4) of extractant to broth (Ve/Vb) have been studied. Here, a novel extractive fermentation process was developed using Clostridium saccharoperbutylacetonicum N1-4 and a large Ve/Vb ratio. A mixture of oleyl alcohol-tributyrin (1:1 (v/v)) yielded high distribution coefficients for both butanol (3.14) and acetone (0.660). Although a fed-batch culture using free cells and the oleyl alcohol-tributyrin mixture at a Ve/Vb ratio of 5 had a lag phase of >24 h, it produced a higher concentration of total butanol (i.e., butanol produced in all the phases per broth volume used) of 24.2 g/L-broth after 96 h compared with 14.4 g/L-broth at a Ve/Vb ratio of 1, resulting in a low butanol concentration in the aqueous phase. The use of cells immobilized with calcium alginate beads shortened the lag phase to <12 h. Butanol production was achieved not only in a 3-phase mode (extractant, beads, and tryptone-yeast extract-acetate (TYA) medium) but also in a 2-phase mode (extractant and beads containing TYA medium, without an aqueous phase) at a Ve/Vb ratio of 5, resulting butanol concentrations of 30.9 g/L-broth and 27.7 g/L-broth, respectively. The 3-phases fed-batch extractive fermentation at a Ve/Vb ratio of 10 showed a better performance compared with published reports: a total butanol concentration of 64.6 g/L-broth and a butanol yield to consumed sugar of 0.378 C-mol/C-mol.

Semi-hydrolysis with low enzyme loading leads to highly effective butanol fermentation.

To improve butanol fermentation efficiencies, semi-hydrolysate with low enzyme loading using H2SO4 pretreated rice straw was designed, which preferably produced cellobiose with xylose (instead of glucose). Fermentation of semi-hydrolysates avoided carbon catabolite repression (CCR) and produced higher butanol yield to enzyme loading (0.0290 g U-1), a newly proposed parameter, than the conventional glucose-oriented hydrolysate (0.00197 g U-1). Further, overall butanol productivity was improved from 0.0628 g L-1 h-1 to 0.265 g L-1 h-1 during fermentation of undetoxified semi-hydrolysate by using high cell density. A novel simultaneously repeated hydrolysis and fermentation (SRHF) was constructed by recycling of enzymes and cells, which further improved butanol yield to enzyme loading by 183% and overall butanol productivity by 6.04%. Thus, semi-hydrolysate with SRHF is a smartly designed biomass for efficient butanol fermentation of lignocellulosic materials.