Efficient production of butyric acid from lignocellulosic biomass by revealing the mechanisms of Clostridium tyrobutyricum tolerance to phenolic inhibitors.

Phenolic compounds (PCs) generated during pretreatment of lignocellulosic biomass severely hinder the biorefinery by Clostridia. As a hyperbutyrate-producing strain, Clostridium tyrobutyricum has excellent tolerance to PCs, but its tolerance mechanism is poorly understood. In this study, a comprehensive transcriptome analysis was applied to elucidate the response of C. tyrobutyricum to four typical PCs. The findings revealed that the expression levels of genes associated with PC reduction, HSPs, and membrane transport were significantly altered under PC stress. Due to PCs being reduced to low-toxicity alcohols/acids by C. tyrobutyricum, enhancing the reduction of PCs by overexpressing reductase genes could enhance the strain's tolerance to PCs. Under 1.0 g/L p-coumaric acid stress, compared with the wild-type strain, ATCC 25755/sdr1 exhibited a 31.2 % increase in butyrate production and a 38.5 % increase in productivity. These insights contribute to the construction of PC-tolerant Clostridia, which holds promise for improving biofuel and chemical production from lignocellulosic biomass.

First Report of Alternaria alternata Causing Leaf Spot on Coffea arabica in China.

Yunnan Province is the major region for coffee (Coffea arabica) cultivation in China, contributing to over 98% of the national yield and total production value (Ma et al. 2022). In May 2023, brown spot symptoms were observed only on the leaves of coffee plants in a field located in Baoshan City (98°52'37.988400"E, 24°58'17.673600"N), Yunnan Province. Notably, brown and irregularly shaped spots initially started on the leaf bases. The spots enlarged and developed concentric rings with dark brown margins, which are often surrounded by yellow halos. Finally, the necrotic spots spread across the entire leaf and caused the leaf to curl and fall off. The incidence of the disease was approximately 3% of the coffee plants (n = 600). The symptomatic leaves collected from 10 plants were sectioned (5 × 5 mm), subjected to surface sterilization with 70% ethanol for 40 s, rinsed with sterile distilled water, air-dried, and transferred to potato dextrose agar (PDA). Fungi with grayish-white, cotton-like aerial mycelia grew after 7 days at 28°C. The older mycelia of these isolates displayed dark gray pigmentation. Single conidia were cultivated on PDA, and 15 morphologically similar monosporic isolates were ultimately obtained. Microscopic observation revealed that these isolates produced branched, septate, transparent and amber mycelium. Brown, elliptical or pear-shaped conidia with 2 to 4 transversal septa and 0 to 3 longitudinal septa, measuring 9.6 to 33.3 long × 6.0 to 15.0 µm wide (n = 30), were observed on potato carrot agar (PCA). Molecular identification of multiple genes, such as ITS (Schoch et al. 2012), RPB2 (O'Donnell et al. 2010) and GAPDH (Berbee et al. 1999), indicated consistent 100% identity among these isolates. Sequences of the representative isolates CFSY1-CFSY5 were deposited in GenBank (acc. nos.: OR351112, PP188577, PP188578, PP294863, PP294864, OR509742, PP215341-PP215344, OR509740 and PP239378-PP239381), revealing 98.35% - 100% homology with distinct Alternaria alternata strains previously deposited in GenBank (acc. nos.: PP110780, MN649031 and OR485338). The multigene phylogenetic analysis positioned isolates CFSY1-CFSY5 within a distinct cluster, alongside diverse A. alternata isolates. Based on morphological and molecular characterizations, the pathogen was identified as A. alternata. To verify its pathogenicity, a conidial suspension (1×106 conidia/mL) of isolate CFSY1 was sprayed on six leaves of three healthy one-year-old C. arabica seedlings. Subsequently, the inoculated seedlings were covered with plastic bags and placed in a growth chamber under controlled conditions (a 14 h daylight period and a 10 h dark period at 28°C). The experiment was repeated three times. After 20 days, typical brown spot symptoms analogous to those originally observed in the field appeared on the leaves in all inoculated plants. Reisolation, morphology identification and DNA sequencing substantiated Koch's postulates. In contrast, control plants treated with sterilized water remained asymptomatic, and no pathogen was reisolated from them. Significantly, A. alternata has been previously reported as the causal agent for leaf spot disease in a diverse variety of woody plant species in China, including Prunus avium (Ahmad et al. 2020), Magnolia grandiflora (Liu et al. 2019) and citrus (Wang et al. 2010). This study represents the first report of brown leaf spot caused by A. alternata specifically on C. arabica in China, enriching the contents of fungal pathogens under Chinese coffee cultivation conditions.

First Report of Fusarium oxysporum Causing Leaf Wilt on Dinteranthus vanzylii (Green Stone Plant) in China.

Dinteranthus vanzylii is a low-growing species in the family Aizoaceae, native to southern Africa, with a pair of thick grey leaves covered with dark red spots and stripes. This stone-like succulent grows near the ground, which may protect it from water evaporation and herbivores. Dinteranthus vanzylii has become popular in China due to its attractive appearance and easy indoor cultivation. In September 2021, 7% of D. vanzylii (approximately 140 pots) showed leaf wilt symptoms in a commercial greenhouse located in Ningde (119°35'39.696″E, 27°23'30.556″N), Fujian Province, China. The diseased plants were shrivelling and eventually underwent necrosis. Their leaf tissues were rotting and carpeted with white mycelium. The leaf tissues of 10 symptomatic plants were cut into 0.5 cm2 pieces, surface-sterilized and placed on PDA medium. According to the colony morphology after 7 days of culture, 20 fungal isolates with abundant whitish aerial mycelium were divided into two types: 8 isolates produced lilac pigment whereas 12 did not. Both produced unicellular ovoid microconidia, sickled-shaped macroconidia with 3 - 4 septa and single or paired smooth, thick-walled chlamydospores on carnation leaf agar (CLA). Molecular identification based on DNA sequences from EF1-α (O'Donnell et al. 1998), RPB1 and RPB2 (O'Donnell et al. 2010) revealed 100% identity among isolates within each group; however, there were several base differences between two types. Sequences of representative isolates KMDV1 and KMDV2 were deposited in GenBank (acc. nos.: OP910243, OP910244, OR030448, OR030449, OR030450 and OR030451), which showed 99.10% - 99.74% identity with different F. oxysporum strains (GenBank acc. nos.: KU738441, LN828039, MN457050, MN457049, ON316742 and ON316741). Phylogenetic tree inferred from the concatenated EF1-α, RPB1 and RPB2 revealed that these isolates clustered with F. oxysporum. Thus, these isolates were identified as F. oxysporum. Using a root-drenching method, 10 one-year-old healthy D. vanzylii were inoculated in conidial suspensions (1*106 conidia/mL) of isolates KMDV1 and KMDV2 for 60 min, respectively. They were transplanted into pots with sterilized soil and incubated in a plant-growth chamber at 25°C and 60% relative humidity. Control plants were treated with sterilized water. The pathogenicity test was repeated three times. All plants inoculated with each isolate developed leaf wilt symptoms after 15 days and were dead after 20 - 30 days. However, no symptoms were observed in the control plants. Fusarium oxysporum was reisolated and confirmed based on morphology and EF1-α sequence analysis. No pathogens were isolated from the control plants. This is the first report of F. oxysporum causing leaf wilt disease on D. vanzylii in China. To date, several diseases have been reported on members of the Aizoaceae. For instance, collar and stem rot on Lampranthus sp. caused by Pythium aphanidermatum (Garibaldi et al. 2009), wilt on Lampranthus sp. and Tetragonia tetragonioides caused by Verticillium dahliae (Garibaldi et al. 2010; Garibaldi et al. 2013), and leaf spot on Sesuvium portulacastrum caused by Gibbago trianthemae (Chen et al., 2022). Our research could provide insight into fungal diseases on members of the Aizoaceae and contribute to their cultivation and management.

Transcriptome analysis reveals reasons for the low tolerance of Clostridium tyrobutyricum to furan derivatives.

Lignocellulosic biomass is considered the most abundant and renewable feedstock for biobased butyric acid production. However, the furan derivatives (FAs, mainly furfural and 5-hydroxymethylfurfural) generated from the pretreatment of lignocellulose severely inhibit the growth of Clostridium tyrobutyricum, which is the best strain for producing butyric acid. The tolerance mechanism of C. tyrobutyricum to FAs has not been investigated thus far. Here, the response of C. tyrobutyricum ATCC 25755 to FA challenge was first evaluated by using comprehensive transcriptional analysis. The results indicated that the genes related to membrane transport, heat shock proteins, and transcriptional regulation were upregulated under FA stress. However, the expression of almost all genes encoding reductases was not changed, and only the ad gene CTK_RS02625 and the bud gene CTK_RS07810 showed a significant increase of ~ 1.05-fold. Then, the enzyme activity assays indicated that BUD could catalyze the reduction of FAs with relatively low activity and that AD could not participate in the conversion of FAs, indicating that the inability to rapidly convert FAs to their low-toxicity alcohols may be the main reason for the low FA tolerance of C. tyrobutyricum. This research provides insights into the development of FA-tolerant strains, thereby enhancing the bioconversion of lignocellulosic biomass to butyric acid. KEY POINTS: • The response of C. tyrobutyricum to FAs was evaluated for the first time. • Genes encoding membrane transporters and heat shock proteins were triggered by FAs. • A lack of effective FA reductases leads to low FA tolerance in C. tyrobutyricum.

First Report of Fusarium oxysporum Causing Stem Rot on Mammillaria humboldtii in China.

Mammillaria humboldtii found in Mexico is a short-globose ornamental cactus species of the Cactaceae family, which has gained increasing popularity in China. It is characterized by tuberculate stems, dimorphic areoles, small pink flowers and pitted seed cell walls. The populations of wild M. humboldtii are critically endangered and are now of international conservation concern. In July 2021, stem rot symptoms were observed on M. humboldtii in a commercial greenhouse located in Zhangzhou (117°39'44.0064″E, 24°28'3.7236″N), Fujian Province (southern China). The typical symptoms were water-soaking, rotting and wilting on the stem, eventually leading to necrosis of the plants within 20 to 30 days. The vascular system of infected stems and roots showed a reddish-brown discolouration. The disease affected approximately 10% of 1000 plants. Fungi were isolated from the diseased stems of 26 samples, which were chopped into small pieces (5 × 5 mm), surface-sterilized with 75% ethanol for 40 s, and placed onto potato dextrose agar (PDA). After seven days of dark culture at 28°C, morphologically similar fungal isolates with whitish aerial mycelium and purple pigment were observed. On carnation leaf agar (CLA), isolates produced sickle and slightly curved macroconidia with three to four septa, measuring 12.8 to 27.9 × 1.9 to 3.8 µm (n = 15), and unicellular, ovoid to elliptical microconidia measuring 3.8 to 7.7 × 1.4 to 2.5 µm (n = 30). Smooth walled chlamydospores were terminal or intercalary, single or in pairs, measuring 9.2 to 13.1 µm (n = 15) in diameter. For molecular identification, the internal transcribed spacer (ITS) region of rDNA (Schoch et al. 2012), translation elongation factor-1α (EF1-α) (Maryani et al. 2019) and gene coding endopolygalacturonase 1 (PG1) (Hirano et al. 2006) of the representative isolate FJMH7 were amplified, purified and sequenced. BLASTn analysis of the ITS, EF1-α and pg1 sequences (GenBank accession numbers: ON832660, ON843495 and ON843496) showed 100%, 99.70% and 98.96% identity with F. oxysporum (GenBank accession numbers: KX611626, OM801797 and KF437345), respectively. Phylogenetic analysis based on the the concatenated ITS and EF1-α sequences and pg1 genes placed isolate FJMH7 with F. oxysporum reference strains in the phylogenetic trees. Based on morphological identification and sequence analysis, this isolate was identified as F. oxysporum. For the pathogenicity assay, six 6-month-old healthy plants of Mammillaria humboldtii were inoculated by dipping roots in a conidial suspension (106 conidia/mL) of isolate FJMH7 cultured in Bilai's medium for three days. Six noninoculated plants treated with Bilai's medium served as a control. Plants were transplanted into pots filled with sterilized soils and placed in a glasshouse at 25°C. After 15 days, all the inoculated plants exhibited rot symptoms on stems, which were similar to those observed in the commercial greenhouses. All inoculated plants were dead 30 days after inoculation. Control plants did not show any symptoms. F. oxysporum was reisolated and confirmed based on morphology and sequencing. No fungi were reisolated from control plants. To fulfil Koch's postulates, the pathogenicity assay was repeated twice with the same results. To date, F. oxysporum isolates have been reported on golden barrel cactus (Echinocactus grusonii) (Polizzi et al. 2004), night-blooming cereus (Hylocereus undatus) (Wright et al. 2007), apple cactus (Cereus peruvianus monstruosus) (Garibaldi et al. 2011), Schlumbergera truncate (Lops et al. 2013), Astrophytum ornatum (Quezada-Salinas et al. 2017) and Nopalea cochenillifera (Santiago et al. 2018). To our knowledge, this is the first report of F. oxysporum on M. humboldtii in China, indicating that this pathogen could cause wilt and rot disease on different cactus hosts.

The Effects of Different Processing Methods on the Levels of Biogenic Amines in Zijuan Tea.

This study aimed to evaluate the effects of processing methods on the content of biogenic amines in Zijuan tea by using derivatization and hot trichloroacetic acid extraction with HPLC-UV. The results showed that the most abundant biogenic amine in the original leaves was butylamine, followed by ethylamine, methylamine, 1,7-diaminoheptane, histamine, tyramine, and 2-phenethylamine. However, during the process of producing green tea, white tea, and black tea, the content of ethylamine increased sharply, which directly led to their total contents of biogenic amines increasing by 184.4%, 169.3%, and 178.7% compared with that of the original leaves, respectively. Unexpectedly, the contents of methylamine, ethylamine, butylamine, and tyramine in dark tea were significantly reduced compared with those of the original leaves. Accordingly, the total content of biogenic amines in dark tea was only 161.19 µg/g, a reduction of 47.2% compared with that of the original leaves, indicating that the pile-fermentation process could significantly degrade the biogenic amines present in dark tea.

Butyric acid production from spent coffee grounds by engineered Clostridium tyrobutyricum overexpressing galactose catabolism genes.

Clostridium tyrobutyricum cannot utilize galactose, which is abundant in lignocellulose and red algae, as a carbon source for butyric acid production. Hence, when using galactose-rich coffee ground hydrolysate as the substrate, the fermentation performance of C. tyrobutyricum is poor. In this work, a recombinant strain, C. tyrobutyricum ATCC 25755/ketp, overexpressing galactose catabolism genes (galK, galE, galT, and galP) from Clostridium acetobutylicum ATCC 824 was constructed for the co-utilization of glucose and galactose. Batch fermentation in the bioreactor showed that ATCC 25755/ketp could efficiently utilize galactose without glucose-induced carbon catabolite repression and consume nearly 100% of the galactose present in the spent coffee ground hydrolysate. Correspondingly, the butyric acid concentration and productivity of ATCC 25755/ketp reached 34.3 g/L and 0.36 g/L·h, respectively, an increase of 78.6% and 56.5% compared with the wild-type strain, indicating its potential for butyric acid production from hydrolysates of inexpensive and galactose-rich biomass.

Metabolic engineering of Clostridium tyrobutyricum for enhanced butyric acid production from undetoxified corncob acid hydrolysate.

Resistance to furan derivatives and phenolic compounds plays an important role in the use of lignocellulosic biomass for biological production of chemicals and fuels. This study confirmed that expression of short-chain dehydrogenase/reductase (SDR) from Clostridium beijerinckii NCIMB 8052 significantly improved the tolerance of C. tyrobutyricum to furfural due to the enhanced activity for furfural reduction. And on this basis, co-expression of SDR and heat shock chaperones GroESL could simultaneously enhance the tolerance of C. tyrobutyricum to furan derivatives and phenolic compounds, which were the main inhibitors presented in dilute-acid lignocellulosic hydrolysates. Consequently, the recombinant strain ATCC 25755/sdr+groESL exhibited good performance in butyric acid production with corncob acid hydrolysate as the substrate. Batch fermentation in bioreactor showed that the butyrate produced by ATCC 25755/sdr+groESL was 32.8 g/L, increased by 28.1% as compared with the wild-type strain. Meanwhile, the butyrate productivity increased from 0.19 g/L·h to 0.29 g/L·h.

The significance of proline on lignocellulose-derived inhibitors tolerance in Clostridium acetobutylicum ATCC 824.

When lignocellulosic biomass was used for acetone-butanol-ethanol (ABE) fermentation, several lignocellulose-derived inhibitors, which are toxic to Clostridium acetobutylicum, were generated during acid hydrolysis process and seriously hindered the industrialization of lignocellulosic butanol. In this study, an engineered strain 824(proABC) with significantly improved tolerance to multiple lignocellulose-derived inhibitors (formic acid and phenolic compounds) was constructed by strengthening the proline biosynthesis. The engineered strain exhibited more effective synthesis ability of proline and scavenging ability of reactive oxygen species (ROS). Consequently, the butanol produced by 824(proABC) was 1-, 2.4- or 3.4-fold higher than that of the wild type strain when using the undetoxified hydrolysate of soybean straw, rice straw or corn straw as the substrate, respectively. Therefore, enhancing the proline biosynthesis can be used as an effective strategy to improve the tolerance of C. acetobutylicum to multiple lignocellulose-derived inhibitors, and 824(proABC) has great potential to produce butanol from undetoxified lignocellulosic hydrolysates.

Improving the fermentation performance of Clostridium acetobutylicum ATCC 824 by strengthening the VB1 biosynthesis pathway.

Vitamin B1 (VB1) is an essential coenzyme for carbohydrate metabolism and involved in energy generation in most organisms. In this study, we found that insufficient biosynthesis of VB1 in Clostridium acetobutylicum ATCC 824 is a major limiting factor for efficient acetone-butanol-ethanol (ABE) fermentation. In order to improve the fermentation performance of C. acetobutylicum ATCC 824, the VB1 biosynthesis pathway was strengthened by overexpressing the thiC, thiG, and thiE genes. The engineered strain 824(thiCGE) showed enhanced VB1 and energy synthesis, resulting in better growth, faster sugar consumption, higher solvents production, and lower acids formation than the wild-type strain in both VB1 free and normal P2 medium (1 mg/L). Compared with the wild-type strain, 824(thiCGE) produced 13.0 ± 0.1% or 12.7 ± 1.2% more butanol in VB1 free P2 medium when glucose or xylose was used as the substrate, respectively. When mixed sugar (glucose:xylose = 2:1) was used as the substrate in VB1 free P2 medium, the xylose consumption rate and butanol titer of 824(thiCGE) were 45.8 ± 1.9% and 20.4 ± 0.3% higher than those of the wild-type strain. All these results demonstrated that this metabolic engineering strategy could provide a new and effective way to improve the cellular performance of solventogenic clostridia. In addition, it may have some potential application value in ABE fermentation using simple medium and/or lignocellulosic biomass.

Metabolic engineering of Clostridium tyrobutyricum for enhanced butyric acid production with high butyrate/acetate ratio.

Butyric acid fermentation by Clostridium couples with the synthesis of acetic acid. But the presence of acetic acid reduces butyric acid yield and increases separation and purification costs of butyric acid. Hence, enhancing the butyrate/acetate ratio is important for economical butyric acid production. This study indicated that enhancing the acetyl-CoA to butyrate flux by overexpression of both the butyryl-CoA/acetate CoA transferase (cat1) and crotonase (crt) genes in C. tyrobutyricum could significantly reduce acetic acid concentration. Fed-batch fermentation of ATCC 25755/cat1 + crt resulted in increased butyrate/acetate ratio of 15.76 g/g, which was 2.24-fold higher than that of the wild-type strain. Furthermore, in order to simultaneously increase the butyrate/acetate ratio, butyric acid concentration and productivity, the recombinant strain ATCC 25755/ppcc (co-expression of 6-phosphofructokinase (pfkA) gene, pyruvate kinase (pykA) gene, cat1, and crt) was constructed. Consequently, ATCC 25755/ppcc produced more butyric acid (46.8 vs. 35.0 g/L) with a higher productivity (0.83 vs. 0.49 g/L·h) and butyrate/acetate ratio (13.22 vs. 7.22 g/g) as compared with the wild-type strain in batch fermentation using high glucose concentration (120 g/L). This study demonstrates that enhancing the acetyl-CoA to butyrate flux is an effective way to reduce acetic acid production and increase butyrate/acetate ratio.

Butyric acid production from lignocellulosic biomass hydrolysates by engineered Clostridium tyrobutyricum overexpressing Class I heat shock protein GroESL.

Lignocellulosic biomass is the most abundant and renewable substrate for biological fermentation, but the inhibitors present in the lignocellulosic hydrolysates could severely inhibit the cell growth and productivity of industrial strains. This study confirmed that overexpressing of native groESL in Clostridium tyrobutyricum could significantly improve its tolerance to lignocellulosic hydrolysate-derived inhibitors, especially for phenolic compounds. Consequently, ATCC 25755/groESL showed a better performance in butyric acid fermentation with hydrolysates of corn cob, corn straw, rice straw, wheat straw, soybean hull and soybean straw, respectively. When corn straw and rice straw hydrolysates, which showed strong toxicity to C. tyrobutyricum, were used as the substrates, 29.6 g/L and 30.1 g/L butyric acid were obtained in batch fermentation, increased by 26.5% and 19.4% as compared with the wild-type strain, respectively. And more importantly, the butyric acid productivity reached 0.31 g/L·h (vs. 0.20-0.21 g/L·h for the wild-type strain) due to the shortened lag phase.

Improving cellular robustness and butanol titers of Clostridium acetobutylicum ATCC824 by introducing heat shock proteins from an extremophilic bacterium.

In recent years, increasing concerns over environment, energy and climate have renewed interest in biotechnological production of butanol. However, growth inhibition by fermentation products and inhibitory components from raw biomass has hindered the development of acetone-butanol-ethanol (ABE) fermentation. Improving the cellular robustness of Clostridium acetobutylicum is of great importance for efficient ABE production. In this study, we attempted to improve the robustness and butanol titers of C. acetobutylicum ATCC824 by overexpressing GroESL and DnaK from the extremely radioresistant bacterium Deinococcus wulumuqiensis R12 and from C. acetobutylicum ATCC824 itself. Three recombinant strains were obtained and designated 824(dnaK R12), 824(groESL R12) and 824(groESL824). These three recombinants were found to have significantly improved tolerances to stresses including butanol, furfural, oxidation and acid. Meanwhile, the butanol titers increased to 13.0g/L, 11.2g/L and 10.7g/L, which were 49.4%, 28.7% and 23.0% higher than that from the wild-type strain (8.7g/L), respectively. For 824(dnaK R12), the production of acetic and butyric acids decreased by 97.1% (1.4g/L vs. 0.04g/L) and 100% (0.3g/L vs. 0g/L), respectively, compared with the wild-type strain. Overexpressing GroESL and DnaK from D. wulumuqiensis R12 also resulted in better growth and ABE production than the wild-type strain on fermentation in the presence of 2.5g/L furfural. Strain 824(groESL R12) was superior to 824(groESL 824) in diverse types of stress-tolerance and butanol titer, indicating that GroESL from the extremophilic bacterium could perform its function more efficiently in the heterologous host than native GroESL. Our study provides evidence that extremophilic bacteria can be excellent resources for engineering C. acetobutylicum to improve its robustness and butanol titer.

Enhanced butyric acid tolerance and production by Class I heat shock protein-overproducing Clostridium tyrobutyricum ATCC 25755.

The response of Clostridium tyrobutyricum to butyric acid stress involves various stress-related genes, and therefore overexpression of stress-related genes can improve butyric acid tolerance and yield. Class I heat shock proteins (HSPs) play an important role in the process of protecting bacteria from sudden changes of extracellular stress by assisting protein folding correctly. The results of quantitative real-time PCR indicated that the Class I HSGs grpE, dnaK, dnaJ, groEL, groES, and htpG were significantly upregulated under butyric acid stress, especially the dnaK and groE operons. Overexpression of groESL and htpG could significantly improve the tolerance of C. tyrobutyricum to butyric acid, while overexpression of dnaK and dnaJ showed negative effects on butyric acid tolerance. Acid production was also significantly promoted by increased GroESL expression levels; the final butyric acid and acetic acid concentrations were 28.2 and 38% higher for C. tyrobutyricum ATCC 25755/groESL than for the wild-type strain. In addition, when fed-batch fermentation was carried out using cell immobilization in a fibrous-bed bioreactor, the butyric acid yield produced by C. tyrobutyricum ATCC 25755/groESL reached 52.2 g/L, much higher than that for the control. The improved butyric acid yield is probably attributable to the high GroES and GroEL levels, which can stabilize the biosynthetic machinery of C. tyrobutyricum under extracellular butyric acid stress.

Enhanced isopropanol and n-butanol production by supplying exogenous acetic acid via co-culturing two clostridium strains from cassava bagasse hydrolysate.

The focus of this study was to produce isopropanol and butanol (IB) from dilute sulfuric acid treated cassava bagasse hydrolysate (SACBH), and improve IB production by co-culturing Clostridium beijerinckii (C. beijerinckii) with Clostridium tyrobutyricum (C. tyrobutyricum) in an immobilized-cell fermentation system. Concentrated SACBH could be converted to solvents efficiently by immobilized pure culture of C. beijerinckii. Considerable solvent concentrations of 6.19 g/L isopropanol and 12.32 g/L butanol were obtained from batch fermentation, and the total solvent yield and volumetric productivity were 0.42 g/g and 0.30 g/L/h, respectively. Furthermore, the concentrations of isopropanol and butanol increased to 7.63 and 13.26 g/L, respectively, under the immobilized co-culture conditions when concentrated SACBH was used as the carbon source. The concentrations of isopropanol and butanol from the immobilized co-culture fermentation were, respectively, 42.62 and 25.45 % higher than the production resulting from pure culture fermentation. The total solvent yield and volumetric productivity increased to 0.51 g/g and 0.44 g/L/h when co-culture conditions were utilized. Our results indicated that SACBH could be used as an economically favorable carbon source or substrate for IB production using immobilized fermentation. Additionally, IB production could be significantly improved by co-culture immobilization, which provides extracellular acetic acid to C. beijerinckii from C. tyrobutyricum. This study provided a technically feasible and cost-efficient way for IB production using cassava bagasse, which may be suitable for industrial solvent production.

Improved welan gum production by Alcaligenes sp. ATCC31555 from pretreated cane molasses.

Welan gum production by Alcaligenes sp. ATCC31555 from cane molasses was studied in batch fermentation to reduce production costs and enhance gum production. The pretreatment of cane molasses, agitation speed and the addition of supplements were investigated to optimize the process. Sulfuric acid hydrolysis was found to be the optimal pretreatment, resulting in a maximum gum concentration of 33.5 g/L, which is 50.0% higher than those obtained from the molasses' mother liquor. Agitation at 600 rpm at 30°C and addition of 10% n-dodecane following fermentation for 36 h increased the maximum gum production up to 41.0 ± 1.41 g/L, which is 49.1% higher than the greatest welan gum concentration in the literature so far. The welan gum product showed an acceptable molecular weight, similar rheological properties and better thermal stability to that obtained from glucose. These results indicate that cane molasses may be a suitable and inexpensive substrate for cost-effective industrial-scale welan gum production.