Xylitol production from non-detoxified corncob hemicellulose acid hydrolysate by Candida tropicalis

The interest on use of lignocellulose for producing chemicals is increasing as these feedstocks are low cost, renewable and widespread sources of sugars. Corncob is an attractive raw material for xylitol production due to its high content of xylan. In this study, hemicellulose hydrolysate from corncobs without detoxification was used for xylitol production by Candida tropicalis CCTCC M2012462. Compared with prepared xylose medium, xylitol production with dilute acid hydrolysate medium does not seem to influence specific xylose reductase activity. The decrease in xylitol productivity with dilute acid hydrolysate medium is a result of a lower biomass concentration and lag-phase time. It appears that biomass growth rate is essential for xylitol production. In xylitol fermentation with a low initial inhibitors concentration and substrate feeding strategy, a maximal xylitol concentration of 38.8 g l⁻¹ was obtained after 84 h of fermentation, giving a yield of 0.7 g g⁻¹ xylose and a productivity of 0.46 g l⁻¹ h⁻¹.     

d- and l-lactic acid production from fresh sweet potato through simultaneous saccharification and fermentation

The aim of this study was to develop a bioprocess for l- and d-lactic acid production from raw sweet potato through simultaneous saccharification and fermentation by Lactobacillus paracasei and Lactobacillus coryniformis, respectively. The effects of enzyme and nitrogen source concentrations as well as of the ratio of raw material to medium were investigated. At dried material concentrations of 136.36–219.51 g L⁻¹, yields of 90.13–91.17% (w/w) and productivities of 3.41–3.83 g L⁻¹ h⁻¹ were obtained with lactic acid concentrations as high as 198.32 g L⁻¹ for l-lactic acid production. In addition, d-lactic acid was produced with yields of 90.11–84.92% (w/w) and productivities of 2.55–3.11 g L⁻¹ h⁻¹ with a maximum concentration of 186.40 g L⁻¹ at the same concentrations of dried material. The simple and efficient process described in this study will benefit the tuber and root-based lactic acid industries without requiring alterations in plant equipment.     

Bench-scale fermentation of Trichoderma viride on wastewater sludge: Rheology,lytic enzymes and biocontrol activity

Conidiation and lytic enzyme production by Trichoderma viride at different solids concentration of pre-treated municipal wastewater sludge was examined in a 15-L fermenter. The maximum conidia concentration (5.94 × 10⁷ CFU mL⁻¹ at 96 h) was obtained at 30 g L⁻¹ suspended solids. The maximum lytic enzyme activities were achieved around 12–30 h of fermentation. Bioassay against a fungal phytopathogen, Fusarium sp. showed maximum activity in the sample drawn around 96 h of fermentation at 30 g L⁻¹ suspended solids concentration. Entomotoxicity against spruce budworm larvae showed maximum value ≈17290 SBU μL⁻¹ at 30 g L⁻¹ suspended solids concentration at the end of fermentation (96 h). Plant bioassay showed dual action of T. viride, i.e., disease prevention and growth promotion. The rheological analyses of fermentation sludges showed the pseudoplastic behaviour. In order to maintain required dissolved oxygen concentration ≥30%, the agitation and aeration requirements significantly increased at 35 g L⁻¹ compared to 30 and 25 g L⁻¹. The oxygen uptake rate and volumetric oxygen mass transfer coefficient, k_La at 35 g L⁻¹ did not increase in comparison to 30 g L⁻¹ due to rheological complexity of the broth during fermentation. Thus, the successful fermentation operation of the biocontrol fungus T. viride is a rational indication of its potential for mass-scale production for agriculture and forest sector as a biocontrol agent.     

Effect of Zinc nanoparticles on oxidative stress-related genes and antioxidant enzymes activity in the brain of Oreochromis niloticus and Tilapia zillii

This study was carried out to determine the median lethal concentrations (LC₅₀) of Zinc nanoparticles (ZnNPs) on Oreochromis niloticus and Tilapia zillii. The biochemical and molecular potential effects of ZnNPs (500 and 2000 μg L⁻¹) on the antioxidant system in the brain tissue of O. niloticus and T. zillii were investigated. Four hundred fish were used for acute and sub-acute studies. ZnNP LC₅₀ concentrations were investigated in O. niloticus and T. zillii. The effect of 500 and 2000 μg L⁻¹ ZnNPs on brain antioxidants of O. niloticus and T. zillii was investigated. The result indicated that 69 h LC₅₀ was 5.5 ± 0.6 and 5.6 ± 0.4 for O. nilotica and T. zillii, respectively. Fish exposed to 500 μg L⁻¹ ZnNPs showed a significant increase in reduced glutathione (GSH), total glutathione (tGSH) levels, superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) activity and gene expression. On the contrary, malondialdehyde (MDA) levels significantly decreased. Meanwhile, fish exposed to 2000 μg L⁻¹ ZnNPs showed a significant decrease of GSH, tGSH levels, SOD, CAT, GR, GPx and GST activity and gene expression. On the contrary, MDA levels significantly increased. It was concluded that, the 96 h LC₅₀ of ZnNPs was 5.5 ± 0.6 and 5.6 ± 0.4 for O. nilotica and T. zillii, respectively. ZnNPs in exposure concentrations of 2000 μg/L induced a deleterious effect on the brain antioxidant system of O. nilotica and T. zillii. In contrast, ZnNPs in exposure concentrations of 500 μg L⁻¹ produced an inductive effect on the brain antioxidant system of O. nilotica and T. zillii.     

Production of specific-molecular-weight hyaluronan by metabolically engineered Bacillus subtilis 168

Low-molecular-weight hyaluronan (LMW-HA) has attracted much attention because of its many potential applications. Here, we efficiently produced specific LMW-HAs from sucrose in Bacillus subtilis. By coexpressing the identified committed genes (tuaD, gtaB, glmU, glmM, and glmS) and downregulating the glycolytic pathway, HA production was significantly increased from 1.01 g L⁻¹ to 3.16 g L⁻¹, with a molecular weight range of 1.40×10⁶–1.83×10⁶ Da. When leech hyaluronidase was actively expressed after N-terminal engineering (1.62×10⁶ U mL⁻¹), the production of HA was substantially increased from 5.96 g L⁻¹ to 19.38 g L⁻¹. The level of hyaluronidase was rationally regulated with a ribosome-binding site engineering strategy, allowing the production of LMW-HAs with a molecular weight range of 2.20×10³–1.42×10⁶ Da. Our results confirm that this strategy for the controllable expression of hyaluronidase, together with the optimization of the HA synthetic pathway, effectively produces specific LMW-HAs, and could also be used to produce other LMW polysaccharides.     

2,3-Butanediol production using Klebsiella oxytoca ATCC 8724: Evaluation of biomass derived sugars and fed-batch fermentation process

For this study, 2,3-butanediol (BD) fermentation from pure and biomass-derived sugar were optimized in shake-flask and 5-L bioreactor levels using Klebsiella oxytoca ATCC 8724. The results showed that 70 g/L of single sugar (glucose or xylose) and 90 g/L of mixed-sugar (glucose:xylose = 2:1) were optimum concentrations for efficient 2,3-BD fermentation. At optimum sugar concentrations, 2,3-BD productivities were 1.03, 0.64 and 0.50 gL⁻¹ h⁻¹, and yields were 0.43, 0.36 and 0.35 g/g in glucose, xylose and mixed-sugar medium, respectively. The lack of simultaneous utilization of glucose and xylose led to the lowest productivity in the mixed-sugar medium. Detoxification of biomass hydrolyzates was necessary for efficient 2,3-BD fermentation when sugar concentrations in the medium was 90 g/L or higher, but not with sugar concentrations of 30 g/L or less. A fed-batch fermentation using glucose medium led to an increase 2,3-BD titer to 79.4 g/L and yields 0.47 g/g, while productivity decreased to 0.79 gL⁻¹ h⁻¹. However, the fed-batch process was inefficient using mixed-sugar and biomass hydrolyzates because of poor xylose utilization. These results indicated that appropriate biomass processing technologies must be developed to generate separate glucose and xylose streams to produce high 2,3-BD titer from biomass-derived sugar using a fed-batch process.     

Glucose and xylose stimulation of a β-glucosidase from the thermophilic fungus Humicola insolens: A kinetic and biophysical study

β-Glucosidases activated by glucose and xylose are uncommon yet intriguing enzymes that may enhance cellulose saccharification efficiency, and are of interest for application in bioethanol production processes. The molecular mechanisms of activation are completely unknown, and the aim of this study was the kinetic and biophysical characterization of the stimulation of a β-glucosidase from Humicola insolens by glucose and xylose. The effects of the monosaccharides were concentration dependent, where in a stimulatory range (0.1–50 mmol L⁻¹), the activity increased up to 2-fold; in a stimulatory-inhibitory range (50–450 mmol L⁻¹ glucose or 50–730 mmol L⁻¹ xylose), the enzyme continued to be stimulated, but the activity was lower than maximal. Above 450 mmol L⁻¹ glucose or 730 mmol L⁻¹ xylose, increasing inhibition occurred. Dynamic light scattering confirmed that the enzyme is monomeric (54 kDa) and kinetic, intrinsic tryptophan fluorescence emission and far ultraviolet circular dichroism analyses indicated that the enzyme possesses a catalytic site (CS) and a modulator binding site (MS). Glucose or xylose binding to the MS induces conformational changes that stimulate the catalytic activity at the CS. Glucose and xylose may compete with the substrate for the CS while the substrate competes with the monosaccharides for binding to the MS. The stimulation of the enzymatic activity by glucose and xylose, which compete for the same sites on the enzyme molecule, is not synergistic. These data reveal allosteric interactions between the MS and the CS in H. insolens β-glucosidase that result in fine modulation of the catalytic activity by the monosaccharides. A kinetic model was developed that accurately described the experimental data for enzyme stimulation by glucose and/or xylose. Understanding the regulatory mechanisms of the enzyme activity, with the aid of kinetic models, may be useful for the application of the enzyme in cellulose hydrolysis processes.     

Optimization of biomass production with enhanced glucan and dietary fibres content by Pleurotus ostreatus ATHUM 4438 under submerged culture

This work was aimed at optimizing biomass production by the edible basidiomycete Pleurotus ostreatus ATHUM 4438 in a submerged process with enhanced glucan and dietary fibres content. β-Glucan from Pleurotus sp. (pleuran) has been used as food supplements due to its immunosuppressive activity. Like other dietary fibre components, oyster mushroom polysaccharides can stimulate the growth of colon microorganisms (probiotics), i.e. act as prebiotics. We used the FF MicroPlate for substrate utilization and growth monitoring. The pattern of substrate catabolism forms a substrate assimilation fingerprint which is useful in selecting media components for media optimization of maximum biomass production. Different carbon sources (95) were used and then 8 of them were tested in shake flask cultures. The effect of various organic and complex nitrogen sources on biomass production was also examined and response surface methodology based on central composite design was applied to explore the optimal medium composition. When the optimized culture medium was tested in a 20-L stirred tank bioreactor, using 57 g L⁻¹ xylose and 37 g L⁻¹ corn steep liquor, high yields (39.2 g L⁻¹) of dry biomass was obtained. The yield coefficients for total glucan and dietary fibres on mycelial biomass formed were 140 ± 4 and 625 ± 9 mg g⁻¹ mycelium dry weight, respectively.     

Over expression of GroESL in Cupriavidus necator for heterotrophic and autotrophic isopropanol production

We previously reported a metabolic engineering strategy to develop an isopropanol producing strain of Cupriavidus necator leading to production of 3.4 g L⁻¹ isopropanol. In order to reach higher titers, isopropanol toxicity to the cells has to be considered. A toxic effect of isopropanol on the growth of C. necator has been indeed observed above a critical value of 15 g L⁻¹. GroESL chaperones were first searched and identified in the genome of C. necator. Native groEL and groES genes from C. necator were over-expressed in a strain deleted for PHA synthesis. We demonstrated that over-expressing groESL genes led to a better tolerance of the strain towards exogenous isopropanol. GroESL genes were then over-expressed within the best engineered isopropanol producing strain. A final isopropanol concentration of 9.8 g L⁻¹ was achieved in fed-batch culture on fructose as the sole carbon source (equivalent to 16 g L⁻¹ after taking into account evaporation). Cell viability was slightly improved by the chaperone over-expression, particularly at the end of the fermentation when the isopropanol concentration was the highest. Moreover, the strain over-expressing the chaperones showed higher enzyme activity levels of the 2 heterologous enzymes (acetoacetate carboxylase and alcohol dehydrogenase) of the isopropanol synthetic operon, translating to a higher specific production rate of isopropanol at the expense of the specific production rate of acetone. Over-expressing the native chaperones led to a 9–18% increase in the isopropanol yield on fructose.     

Simultaneous saccharification and fermentation of kraft pulp by recombinant Escherichia coli for phenyllactic acid production

Simultaneous saccharification and fermentation (SSF) of renewable cellulose for the production of 3-phenyllactic acid (PhLA) by recombinant Escherichia coli was investigated. Kraft pulp recovered from biomass fractionation processes was used as a model cellulosic feedstock and was hydrolyzed using 10–50 filter paper unit (FPU) g⁻¹ kraft pulp of a commercial cellulase mixture, which increased the glucose yield from 21% to 72% in an enzyme dose-dependent manner. PhLA fermentation of the hydrolyzed kraft pulp by a recombinant E. coli strain expressing phenylpyruvate reductase from Wickerhamia fluorescens TK1 produced 1.9 mM PhLA. The PhLA yield obtained using separate hydrolysis and fermentation was enhanced from 5.8% to 42% by process integration into SSF of kraft pulp (20 g L⁻¹) in a complex medium (pH 7.0) at 37 °C. The PhLA yield was negatively correlated with the initial glucose concentration, with a five-fold higher PhLA yield observed in culture medium containing 10 g L⁻¹ glucose compared to 100 g L⁻¹. Taken together, these results suggest that the PhLA yield from cellulose in kraft pulp can be improved by SSF under glucose-limited conditions.     

Metabolic engineering of Bacillus sp. for diacetyl production

Diacetyl, a highly valuable product that is extensively used as an ingredient of food, tobacco, and daily chemicals such as perfumes, can be produced from the nonenzymatic oxidative decarboxylation of α-acetolactate during bacterial fermentation and converted to acetoin and 2,3-butanediol by 2,3-butanediol dehydrogenase. In the present study, Bacillus sp. DL01, which gives high acetoin production, was metabolically engineered to improve diacetyl production. After the deletion of α-acetolactate decarboxylase (ALDC)-encoding gene (alsD) by homologous recombination, the engineered strain, named Bacillus sp. DL01-ΔalsD, lost ALDC activity and produced 1.53 g/L diacetyl without acetoin and 2,3-butanediol accumulation. The channeling of carbon flux into diacetyl biosynthetic pathway was amplified by an overexpressed α-acetolactate synthase (ALS)-encoding gene (alsS) in Bacillus sp. DL01-ΔalsD-alsS, which produced 4.02 g/L α-acetolactate and 1.94 g/L diacetyl, and the conversion from α-acetolactate to diacetyl was increased by 1-fold after 20 mM Fe³⁺ was added to the fermentation medium. A titer of 8.69 g/L diacetyl, the highest reported diacetyl production, was achieved by fed-batch fermentation in optimal conditions using the metabolically engineered strain of Bacillus sp. DL01-ΔalsD-alsS. These results are of great importance as a new method for the efficient production of diacetyl by food-safe bacteria.     

Poly-β-hydroxybutyrate/ectoine co-production by ectoine-excreting strain Halomonas salina

The ectoine-excreting bacterial strain of Halomonas salina was employed in the co-production of poly-β-hydroxybutyrate (PHB) and ectoine (Ect) during a fermentation process (PHB/Ect co-production). An efficient PHB/Ect co-production process was carried out at low NaCl concentration (30 g L⁻¹). It was established using ¹H Nuclear Magnetic Resonance spectroscopy that H. salina produces PHB. The effects of the NaCl concentration, the initial C/N ratio, the phosphate concentration and mixed carbon sources were investigated with respect to PHB/Ect co-production. The PHB/Ect co-production system comprised growing and non-growing cell phases and was developed with NaCl concentration of 30 g L⁻¹. The optimal conditions for PHB/Ect co-production by the ectoine-excreting strain of H. salina were 30 g L⁻¹ NaCl, with an initial C/N ratio of 15, an initial phosphate concentration of 12 g L⁻¹ and mixed carbon sources of 55 g L⁻¹ glucose and 25 g L⁻¹ monosodium glutamate. Using a PHB/Ect co-production system with growing and non-growing cell phases prevents the inhibition of PHB synthesis by high concentration of NaCl and significantly reduces ectoine degradation. PHB and ectoine concentrations as high as 35.3 g L⁻¹ and 8.6 g L⁻¹, respectively, were achieved. The efficient co-production of PHB and ectoine at a low NaCl concentration has been realised.     

Direct bioconversion of d-xylose to 1,2,4-butanetriol in an engineered Escherichia coli

The compound 1,2,4-butanetriol (BT) is a valuable chemical used in the production of plasticizers, polymers, cationic lipids and other medical applications, and is conventionally produced via hydrogenation of malate. In this report, BT is biosynthesized by an engineered Escherichia coli from d-xylose. The pathway: d-xylose → d-xylonate → 2-keto-3-deoxy-d-xylonate → 3,4-dihydroxybutanal → BT, was constructed in E. coli by recruiting a xylose dehydrogenase and a keto acid decarboxylase from Caulobacter crescentus and Pseudomonas putida, respectively. Authentic BT was detected from cultures of the engineered strain. Further improvement on the strain was performed by blocking the native d-xylose and d-xylonate metabolic pathways which involves disruption of xylAB, yjhH and yagE genes in the host chromosome. The final construct produced 0.88 g L⁻¹ BT from 10 g L⁻¹ d-xylose with a molar yield of 12.82%. By far, this is the first report on the direct production of BT from d-xylose by a single microbial host. This may serve as a starting point for further metabolic engineering works to increase the titer of BT toward industrial scale viability.     

Continuous succinic acid production by Actinobacillus succinogenes in a biofilm reactor: Steady-state metabolic flux variation

Continuous anaerobic fermentations were performed in a novel external-recycle, biofilm reactor using d-glucose and CO₂ as carbon substrates. Succinic acid (SA) yields were found to be an increasing function of glucose consumption with the succinic acid to acetic acid ratio increasing from 2.4 g g⁻¹ at a glucose consumption of 10 g L⁻¹, to 5.7 g g⁻¹ at a glucose consumption of 50 g L⁻¹. The formic acid to acetic acid ratio decreased from an equimolar value (0.77 g g⁻¹) at a glucose consumption of 10 g L⁻¹ to a value close to zero at 50 g L⁻¹. The highest SA yield on glucose and highest SA titre obtained were 0.91 g g⁻¹ and 48.5 g L⁻¹ respectively. Metabolic flux analysis based on the established C₃ and C₄ metabolic pathways of Actinobacillus succinogenes revealed that the increase in the succinate to acetate ratio could not be attributed to the decrease in formic acid and that an additional source of NADH was present. The fraction of unaccounted NADH increased with glucose consumption, suggesting that additional reducing power is present in the medium or is provided by the activation of an alternative metabolic pathway.     

Galactose-limited fed-batch cultivation of Escherichia coli for the production of lacto-N-tetraose

Lacto-N-tetraose (Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc) is one of the most abundant oligosaccharide structures in human milk. We recently described the synthesis of lacto-N-tetraose by a whole-cell biotransformation with recombinant Escherichia coli cells. However, only about 5% of the lactose was converted into lacto-N-tetraose by this approach. The major product obtained was the intermediate lacto-N-triose II (GlcNAc(β1-3)Gal(β1-4)Glc).In order to improve the bioconversion of lactose to lacto-N-tetraose, we have investigated the influence of the carbon source on the formation of lacto-N-tetraose and on the intracellular availability of the glycosyltransferase substrates, UDP-N-acetylglucosamine and UDP-galactose. By growth of the recombinant E. coli cells on D-galactose, the yield of lacto-N-tetraose (810.8 mg L⁻¹ culture) was 3.6-times higher compared to cultivation on D-glucose.Using fed-batch cultivation with galactose as sole energy and carbon source, a large-scale synthesis of lacto-N-tetraose was demonstrated. During the 26 h feeding phase the growth rate (μ = 0.05) was maintained by an exponential galactose feed. In total, 16 g L⁻¹ lactose were fed and resulted in final yields of 12.72 ± 0.21 g L⁻¹ lacto-N-tetraose and 13.70 ± 0.10 g L⁻¹ lacto-N-triose II. In total, 173 g of lacto-N-tetraose were produced with a space-time yield of 0.37 g L⁻¹ h⁻¹.     

Efficient phototrophic production of a high-value sesquiterpenoid from the eukaryotic microalga Chlamydomonas reinhardtii

The heterologous expression of terpene synthases in microbial hosts has opened numerous possibilities for bioproduction of desirable metabolites. Photosynthetic microbial hosts present a sustainable alternative to traditional fermentative systems, using freely available (sun)light and carbon dioxide as inputs for bio-production. Here, we report the expression of a patchoulol synthase from Pogostemon cablin Benth in the model green microalga Chlamydomonas reinhardtii. The sesquiterpenoid patchoulol was produced from the alga and was used as a marker of sesquiterpenoid production capacity. A novel strategy for gene loading was employed and patchoulol was produced up to 922±242 µg g⁻¹ CDW in six days. We additionally investigated the effect of carbon source on sesquiterpenoid productivity from C. reinhardtii in scale-up batch cultivations. It was determined that up to 1.03 mg L⁻¹ sesquiterpenoid products could be produced in completely photoautotrophic conditions and that the alga exhibited altered sesquiterpenoid production metabolism related to carbon source.     

Comparison of salinity tolerance of three Atriplex spp. in well-watered and drying soils

Members of the Chenopodiaceae are well adapted to both salt and drought stress and can serve as model species to understand the mechanisms of tolerance in plants. We grew Atriplex hortensis (ATHO), A. canescens (ATCA), and A. lentiformis (ATLE) along a NaCL salinity gradient under non-water-limited conditions and in drying soils in greenhouse experiments. The species differed in photosynthetic carbon fixation pathway, capacity for sodium uptake, and habitat preferences. Under non-water-limited conditions, ATLE (C4) maintained high growth rates up to 30 g L⁻¹ NaCl. ATHO (C3) had lower growth than ATLE at high salinities, while ATCA (C4) grew more slowly than either ATLE or ATHO and showed no net growth above 20 g L⁻¹ NaCl. ATHO and ATLE accumulated twice as much sodium in their shoots as ATCA, but all three species had increasing sodium levels at higher salinities. Potassium, magnesium and calcium levels were relatively constant over the salinity gradient. All three species showed marked accumulation of chloride across the salinity gradient, whereas nitrate, phosphorous and sulfate decreased with salinity. The effect of drought was simulated by growing plants in sealed pots with an initial charge of water plus NaCl, and allowing them to grow to the end point at which they no longer were able to extract water from the soil solution. Drought and salinity were not additive stress factors for Atriplex spp. in this experiment. NaCl increased their ability to extract water from the soil solution compared to fresh water controls. ATLE showed increased shoot dry matter production and increased water use efficiency (WUE) as initial salinity levels increased from 0 to 30 g L⁻¹ NaCl, whereas dry matter production and WUE peaked at 5 g L⁻¹ for ATHO and ATCA. Final soil moisture salinities tolerated by species were 85 g L⁻¹, 55 g L⁻¹ and 160 g L⁻¹ NaCl for ATHO, ATCA and ATLE, respectively. C4 photosynthesis and sodium accumulation in shoots were associated with high drought and salt tolerance.     

A new bioprocess for the production of prebiotic lactosucrose by an immobilized β-galactosidase

A new bioprocess for the synthesis of lactosucrose was studied using a covalently immobilized β-galactosidase on macrospheres of chitosan. The effects of temperature and pH on the production of lactosucrose and other oligosaccharides were evaluated. At 30 °C and pH 7.0, the maximum concentration of lactosucrose reached to 79 g L⁻¹. The change of the reaction conditions allowed to modify the qualitative profile of the final products without quantitative change in the total of oligosaccharides produced. At pH 7 and 30 °C, products profile was 79 g L⁻¹ of lactosucrose, 37 g L⁻¹ of galactooligosaccharides and 250 g L⁻¹ of total oligosaccharides, while at pH 5 and 64 °C the concentrations for the same compounds were 40, 62 and 250 g L⁻¹, respectively. The immobilization increased the thermal stability up to 260-fold. Using 300 g L⁻¹ of sucrose and 300 g L⁻¹ of lactose, and 8.5 mg of chitosan mL⁻¹, 30 cycles of reuse were performed and the biocatalyst kept the maximal lactosucrose synthesis. These results fulfill some important aspects for the enzyme immobilization and oligosaccharides synthesis: the simplicity of the protocols, the high operational stability of the enzyme and the possibility of driving the final products.     

Volatile fatty acids accumulation and rhamnolipid generation in situ from waste activated sludge fermentation stimulated by external rhamnolipid addition

The solubilization and acidification of waste activated sludge (WAS) were apparently enhanced by external rhamnolipid (RL) addition. The maximum solute carbohydrate concentrations increased linearly from 48 ± 5 mg COD L⁻¹ in the un-pretreated WAS (blank) to 566 ± 19 mg COD L⁻¹, and protein increased from 1050 ± 8 to 3493 ± 16 mg COD L⁻¹ at RL dosage of 0.10 g g⁻¹ TSS. The highest VFAs concentration peaked at 3840 mg COD L⁻¹ at RL dosage of 0.04 g g⁻¹ TSS, which was 4.24-fold higher than the blank test. RL was generated in situ during WAS fermentation when external RL was added. It was detected that RL concentration was increased from initial 880 ± 92 mg L⁻¹ to 1312 ± 7 mg L⁻¹ at the end of 96 h with RL dosage of 0.04 g g⁻¹ TSS, which was increased to 1.49-fold. Meanwhile, methane production was notably reduced to a quite low level of 2.0 mL CH₄ g⁻¹ VSS, showing effective inhibition of methanogens by RL (58.8 mL CH₄ g⁻¹ VSS in the blank). In addition, the activity of hydrolytic enzymes (protease and α-glucosidase) was enhanced accordingly. VFAs accumulation and RL generation in situ demonstrated that the additional RL substantially performed enhanced biological effects for waste activated sludge fermentation.