Comprehensive comparative study on n-caproate production by Clostridium kluyveri: batch vs. continuous operation modes.

Recently, there has been notable interest in researching and industrially producing medium-chain carboxylic acids (MCCAs) like n-caproate and n-caprylate via chain elongation process. This study presents a comprehensive assessment of the behavior and MCCA production profiles of Clostridium kluyveri in batch and continuous modes, at different ethanol:acetate molar ratios (1.5:1, 3.5:1 and 5.5:1). The highest n-caproate concentration, 12.9 ± 0.67 g/L (92.9 ± 1.39 % MCCA selectivity), was achieved in batch mode at a 3.5:1 ratio. Interestingly, higher ratios favored batch mode selectivity over continuous mode when this was equal or higher to 3.5:1. Steady state operation yielded the highest n-caproate (9.5 ± 0.13 g/L) and n-caprylate (0.35 ± 0.020 g/L) concentrations at the 3.5:1 ratio. Increased ethanol:acetate ratios led to a higher excessive ethanol oxidation (EEO) in both operational modes, potentially limiting n-caproate production and selectivity, especially at the 5.5:1 ratio. Overall, this study reports the efficient MCCA production of both batch and continuous modes by C. kluyveri.

Kinetics-based development of two-stage continuous fermentation of 1,3-propanediol from crude glycerol by Clostridium butyricum.

BACKGROUND: Glycerol, as a by-product, mainly derives from the conversion of many crops to biodiesel, ethanol, and fatty ester. Its bioconversion to 1,3-propanediol (1,3-PDO) is an environmentally friendly method. Continuous fermentation has many striking merits over fed-batch and batch fermentation, such as high product concentration with easy feeding operation, long-term high productivity without frequent seed culture, and energy-intensive sterilization. However, it is usually difficult to harvest high product concentrations. RESULTS: In this study, a three-stage continuous fermentation was firstly designed to produce 1,3-PDO from crude glycerol by Clostridium butyricum, in which the first stage fermentation was responsible for providing the excellent cells in a robust growth state, the second stage focused on promoting 1,3-PDO production, and the third stage aimed to further boost the 1,3-PDO concentration and reduce the residual glycerol concentration as much as possible. Through the three-stage continuous fermentation, 80.05 g/L 1,3-PDO as the maximum concentration was produced while maintaining residual glycerol of 5.87 g/L, achieving a yield of 0.48 g/g and a productivity of 3.67 g/(L·h). Based on the 14 sets of experimental data from the first stage, a kinetic model was developed to describe the intricate relationships among the concentrations of 1,3-PDO, substrate, biomass, and butyrate. Subsequently, this kinetic model was used to optimize and predict the highest 1,3-PDO productivity of 11.26 g/(L·h) in the first stage fermentation, while the glycerol feeding concentration and dilution rate were determined to be 92 g/L and 0.341 h-1, separately. Additionally, to achieve a target 1,3-PDO production of 80 g/L without the third stage fermentation, the predicted minimum volume ratio of the second fermenter to the first one was 11.9. The kinetics-based two-stage continuous fermentation was experimentally verified well with the predicted results. CONCLUSION: A novel three-stage continuous fermentation and a kinetic model were reported. Then a simpler two-stage continuous fermentation was developed based on the optimization of the kinetic model. This kinetics-based development of two-stage continuous fermentation could achieve high-level production of 1,3-PDO. Meanwhile, it provides a reference for other bio-chemicals production by applying kinetics to optimize multi-stage continuous fermentation.

Contracting the Host Range of Bacteriophage T7 Using a Continuous Evolution System.

Bacteriophage T7 is an intracellular virus that recognizes its host via tail and tail fiber proteins known as receptor-binding proteins (RBPs). The RBPs attach to a specific lipopolysaccharide (LPS) displayed on the host. While there are various reports of phage host range expansion resulting from mutations in the RBP encoding genes, there is little evidence for contraction of host range. Notably, most experimental systems have not monitored changes in host range in the presence of several hosts simultaneously. Here, we use a continuous evolution system to show that T7 phages grown in the presence of five restrictive strains and one permissive host, each with a different LPS, gradually cease to recognize the restrictive strains. Remarkably, this result was obtained in experiments with six different permissive hosts. The altered specificity is due to mutations in the RBPs as determined by gene sequencing. The results of using this system demonstrate a major role for RBPs in restricting the range of futile infections, and this process can be harnessed to reduce the host range in applications such as recognition and elimination of a specific bacterial serotype by bacteriophages.

Continuous fermentation using high cell density cell recycle system for L-lactic acid production.

For complete utilization of high glucose at ∼100 g/L, a high cell density (HCD) continuous fermentation system was established using Lb. delbrueckii NCIM 2025 for the bioproduction of lactic acid (LA). An integrated membrane cell recycling system coupled with the continuous bioreactor, aided to achieve the highest 34.77 g/L h LA productivity and 0.94-0.98 g/g yield. ∼34 times higher productivity was observed (in comparison to batch fermentation conducted in this study), when the continuous operations were carried out at the maximum dilution rate and wet cell weight i.e. 0.36 h-1 and 230 g/L, respectively. These results show the potential of this method for large-scale lactic acid production because it not only produces high titers but also ensures that glucose is used effectively. The method's superior performance in comparison to earlier studies suggests it as an affordable and sustainable alternative for the production of LA.

Development of a continuous fermentation process for the production of recombinant uricase enzyme by Pichia pastoris.

Recent studies in the biopharmaceutical industry have shown an increase in the productivity and production efficiency of recombinant proteins by continuous culture. In this research, a new upstream fermentation process was developed for the production of recombinant uricase in the methylotrophic yeast Pichia pastoris. Expression of recombinant protein in this system is under the control of the AOX1 promoter and therefore requires methanol as an inducing agent and carbon/energy source. Considering the biphasic growth characteristics of conventional fed-batch fermentation, physical separation of the growth and induction stages for better control of the continuous fermentation process resulted in higher dry-cell weight (DCW) and enhanced recombinant urate oxidase activity. The DCW and recombinant uricase activity enzyme for fed-batch fermentation were 79 g/L and 6.8 u/mL. During the continuous process, in the growth fermenter at a constant dilution rate of 0.025 h-1 , DCW increased to 88.39 g/L. In the induction fermenter, at methanol feeding rates of 30, 60, and 80 mL/h, a recombinant uricase activity was 4.13, 7.2, and 0 u/mL, respectively. The optimum methanol feeding regime in continuous fermentation resulted in a 4.5-fold improvement in productivity compared with fed-batch fermentation from 0.04 u/mL/h (0.0017 mg/mL/h) to 0.18 u/mL/h (0.0078 mg/mL/h).

Evaluating the potential of semi-continuous itaconic acid fermentation by Aspergillus terreus: operational profile and experiences.

Itaconic acid is an important bio-based chemical. The present study aims to evaluate the applicability of semi-continuous fermentation technique for itaconic acid production by Aspergillus terreus. The fermentation is planned to be connected with bipolar membrane electrodialysis unit for acid recovery. This process allows the reuse of residual glucose from the effluent. Our particular attention was focused on the effect of glucose concentration. Two different glucose supplementation strategies were tested: constant glucose concentration in the refilling medium and adjusted glucose concentration in order to maintain a continuously high - 120 g/L - glucose concentration in the fermentor. The itaconic acid titre, yield and productivity for the 24 h time periods between draining/refilling interventions were investigated. The constantly high glucose concentration in the fermentor resulted in doubled biomass formation. The average itaconic acid titre was 32.9 ± 2.7 g/L. The producing strain formed numerous spores during semi-continuous fermentation that germinated continuously. Yield and volumetric productivity showed a periodic pattern during the procedure.

Engineering Saccharomyces cerevisiae for improved biofilm formation and ethanol production in continuous fermentation.

BACKGROUND: Biofilm-immobilized continuous fermentation has the potential to enhance cellular environmental tolerance, maintain cell activity and improve production efficiency. RESULTS: In this study, different biofilm-forming genes (FLO5, FLO8 and FLO10) were integrated into the genome of S. cerevisiae for overexpression, while FLO5 and FLO10 gave the best results. The biofilm formation of the engineered strains 1308-FLO5 and 1308-FLO10 was improved by 31.3% and 58.7% compared to that of the WT strain, respectively. The counts of cells adhering onto the biofilm carrier were increased. Compared to free-cell fermentation, the average ethanol production of 1308, 1308-FLO5 and 1308-FLO10 was increased by 17.4%, 20.8% and 19.1% in the biofilm-immobilized continuous fermentation, respectively. Due to good adhering ability, the fermentation broth turbidity of 1308-FLO5 and 1308-FLO10 was decreased by 22.3% and 59.1% in the biofilm-immobilized fermentation, respectively. Subsequently, for biofilm-immobilized fermentation coupled with membrane separation, the engineered strain significantly reduced the pollution of cells onto the membrane and the membrane separation flux was increased by 36.3%. CONCLUSIONS: In conclusion, enhanced biofilm-forming capability of S. cerevisiae could offer multiple benefits in ethanol fermentation.

High cell density continuous fermentation for L-lactic acid production from cane molasses.

Commercial production of lactic acid (LA) utilizes mostly glucose or lactose coupled with yeast extract (YE) as a supplement. With sugars, nitrogen, and vitamin supplementation being most of the LA production costs, the use of inexpensive molasses, a by-product of the sugar industry, can provide considerable cost savings. There are just a few publications on the production of LA from molasses; consequently, the present investigation was conducted using molasses supplemented with yeast extract. The research was done in a continuous-flow, high-cell-density (HCD) bioreactor with an external membrane microfiltration device for cell recycling. The system, run at 1 L with Lactobacillus delbrueckii NCIM 2025, produced a LA yield of 0.95-0.98 g/g from ∼100 g sugars/L when supplemented with 1 g/L YE. Dilution rates in the range of 0.04-0.36 h-1 resulted in volumetric lactic acid productivities in the range of 4.3-27.6 g/L h, which compares favorably with the highest values recorded in literature, for glucose in the presence of YE, which was as high as 30 g/L. The utilization of cane molasses has a significant impact on the economics of lactic acid production, as measured by a comparison of costs with commercial glucose.

High-rate continuous n-butanol production by Clostridium acetobutylicum from glucose and butyric acid in a single-pass fibrous-bed bioreactor.

Biobutanol produced in acetone-butanol-ethanol (ABE) fermentation at batch mode cannot compete with chemically derived butanol because of the low reactor productivity. Continuous fermentation can dramatically enhance productivity and lower capital and operating costs, but are rarely used in industrial fermentation because of increased risks of culture degeneration, cell washout, and contamination. In this study, cells of the asporogenous Clostridium acetobutylicum ATCC55025 were immobilized in a single-pass fibrous-bed bioreactor (FBB) for continuous production of butanol from glucose and butyrate at various dilution rates. Butyric acid in the feed medium helped maintaining cells in the solventogenic phase for stable continuous butanol production. At a dilution rate of 1.88 h-1 , butanol was produced at 9.55 g/L, with a yield of 0.24 g/g and productivity of 16.8 g/L/h, which was the highest productivity ever achieved for biobutanol fermentation and an 80-fold improvement over the conventional ABE fermentation. The extremely high productivity was attributed to the high density of viable cells (~100 g/L at >70% viability) immobilized in the fibrous matrix, which also enabled the cells to better tolerate butanol and butyric acid. The FBB was stable for continuous operation for an extended period of over 1 month.

Control of redox potential in a novel continuous bioelectrochemical system led to remarkable metabolic and energetic responses of Clostridium pasteurianum grown on glycerol.

BACKGROUND: Electro-fermentation (EF) is an emerging tool for bioprocess intensification. Benefits are especially expected for bioprocesses in which the cells are enabled to exchange electrons with electrode surfaces directly. It has also been demonstrated that the use of electrical energy in BES can increase bioprocess performance by indirect secondary effects. In this case, the electricity is used to alter process parameters and indirectly activate desired pathways. In many bioprocesses, oxidation-reduction potential (ORP) is a crucial process parameter. While C. pasteurianum fermentation of glycerol has been shown to be significantly influenced electrochemically, the underlying mechanisms are not clear. To this end, we developed a system for the electrochemical control of ORP in continuous culture to quantitatively study the effects of ORP alteration on C. pasteurianum by metabolic flux analysis (MFA), targeted metabolomics, sensitivity and regulation analysis. RESULTS: In the ORP range of -462 mV to -250 mV, the developed algorithm enabled a stable anodic electrochemical control of ORP at desired set-points and a fixed dilution rate of 0.1 h-1. An overall increase of 57% in the molar yield for 1,3-propanediol was observed by an ORP increase from -462 to -250 mV. MFA suggests that C. pasteurianum possesses and uses cellular energy generation mechanisms in addition to substrate-level phosphorylation. The sensitivity analysis showed that ORP exerted its strongest impact on the reaction of pyruvate-ferredoxin-oxidoreductase. The regulation analysis revealed that this influence is mainly of a direct nature. Hence, the observed metabolic shifts are primarily caused by direct inhibition of the enzyme upon electrochemical production of oxygen. A similar effect was observed for the enzyme pyruvate-formate-lyase at elevated ORP levels. CONCLUSIONS: The results show that electrochemical ORP alteration is a suitable tool to steer the metabolism of C. pasteurianum and increase product yield for 1,3-propanediol in continuous culture. The approach might also be useful for application with further anaerobic or anoxic bioprocesses. However, to maximize the technique's efficiency, it is essential to understand the chemistry behind the ORP change and how the microbial system responds to it by transmitted or direct effects.

Enhancing biohydrogen gas production in anaerobic system via comparative chemical pre-treatment on palm oil mill effluent (POME).

Biological hydrogen production using palm oil mill effluent (POME) as a carbon source through dark fermentation process has been suggested to be a promising bioenergy potential and enacts as alternative renewable energy source. Results have indicated that among various 1.5% (w/v) chemical pre-treatments (sodium hydroxide, NaOH; hydrochloric acid, HCl; sulphuric acid, H2SO4; phosphoric acid, H3PO4 and nitric acid, HNO3) on POME, using H3PO4 would generate maximum biohydrogen production of 0.193 mmol/L/h, which corresponded to a yield of 1.51 mol H2/mol TCconsumed with an initial total soluble carbohydrate concentration of 23.52 g/L. Among H3PO4 concentrations (1%-10%), the soluble carbohydrate content and the biohydrogen produced was highest and increased by 1.70-fold and 2.35-fold respectively at 2.5% (w/v), as compared to untreated POME. The batch fermentation maximum hydrogen production rate and yield of 0.208 mmol/L/h and 1.69 mol H2/mol TCconsumed were achieved at optimum pre-treatment conditions of pH 5.5 and thermophilic temperature (60 °C). This study suggests that chemical pre-treatment approaches manage to produce and improve the carbohydrate utilisation process further. Continuous fermentation in CSTR at the optimum conditions produce heightened 1.5-fold biohydrogen yield for 2.5% H3PO4 at 6 h HRT as compared to batch scale. Bacterial community via next-generation sequencing analysis at optimum HRT (6 h) revealed that Thermoanaerobacterium thermosaccharolyticum registered the highest relative frequency of 20.24%. At the class level, Clostridia, Bacilli, Bacteroidia, Thermoanaerobacteria, and Gammaproteobacteria were identified as the biohydrogen-producing bacteria in the continuous system. Insightful findings from this study suggest the substantial practical utility of dilute chemical pre-treatment in improving biohydrogen production.

D-Lactic acid production from agricultural residues by membrane integrated continuous fermentation coupled with B vitamin supplementation.

BACKGROUND: D-Lactic acid played an important role in the establishment of PLA as a substitute for petrochemical plastics. But, so far, the D-lactic acid production was limited in only pilot scale, which was definitely unable to meet the fast growing market demand. To achieve industrial scale D-lactic acid production, the cost-associated problems such as high-cost feedstock, expensive nutrient sources and fermentation technology need to be resolved to establish an economical fermentation process. RESULTS: In the present study, the combined effect of B vitamin supplementation and membrane integrated continuous fermentation on D-lactic acid production from agricultural lignocellulosic biomass by Lactobacillus delbrueckii was investigated. The results indicated the specific addition of vitamins B1, B2, B3 and B5 (VB1, VB2, VB3 and VB5) could reduce the yeast extract (YE) addition from 10 to 3 g/l without obvious influence on fermentation efficiency. By employing cell recycling system in 350 h continuous fermentation with B vitamin supplementation, YE addition was further reduced to 0.5 g/l, which resulted in nutrient source cost reduction of 86%. A maximum D-lactate productivity of 18.56 g/l/h and optical purity of 99.5% were achieved and higher than most recent reports. CONCLUSION: These findings suggested the novel fermentation strategy proposed could effectively reduce the production cost and improve fermentation efficiency, thus exhibiting great potential in promoting industrial scale D-lactic acid production from lignocellulosic biomass.

Continuous Culture of Auxenochlorella protothecoides on Biodiesel Derived Glycerol under Mixotrophic and Heterotrophic Conditions: Growth Parameters and Biochemical Composition.

As crude glycerol comprises a potential substrate for microalga fermentation and value added products' biosynthesis, Auxenochlorella protothecoides was grown on it under heterotrophic and mixotrophic conditions and its growth kinetics were evaluated in a continuous system under steady state conditions. Increasing initial glycerol concentration (from 30 to 50 g/L) in the heterotrophic culture led to reduced biomass yield (Yx/S) and productivity (Px), but favored lipid accumulation. Under heterotrophic conditions, the microalga was found to grow better (biomass up to 7.888 g/L) and faster (higher growth rates), the system functioned more effectively (higher Px) and crude glycerol was exploited more efficiently. Heterotrophy also favored proteins synthesis (up to 53%), lipids (up to 9.8%), and carbohydrates (up to 44.6%) accumulation. However, different trophic modes had no significant impact on the consistency of proteins and lipids. Oleic acid was the most abundant fatty acid detected (55-61.2% of the total lipids). The algal biomass contained many essential and non-essential amino acids, especially arginine, glutamic acid, lysine, aspartic acid, leucine, and alanine. In all the experimental trials, the protein contents in the microalgal biomass increased with the increasing dilution rate (D), with a concomitant decrease in the lipids and carbohydrates fractions.

Simultaneous Biological Pretreatment and Saccharification of Rice Straw by Ligninolytic Enzymes from Panus neostrigosus I9 and Commercial Cellulase.

The utilization of rice straw for biofuel production is limited by its composition. The pretreatment process is required to improve the enzymatic accessibility of polysaccharides in the biomass prior to enzymatic saccharification. In this study, simultaneous biological pretreatment and saccharification (SPS) of rice straw starting from laccase production by Panus neostrigosus I9 was operated in a 2-L fermenter. It was found that fungal physiology was strongly influenced by the agitation, and that the highest laccase production was obtained at an agitation speed of 750 rpm (209.96 ± 0.34 U/L). The dilution rate of 0.05 h-1 was set in continuous fermentation which resulted in laccase activity of 678.49 ± 20.39 U/L, approximately three times higher than that in batch culture. Response surface methodology (RSM) was applied to achieve the condition for maximum percentage of delignification. The maximum percentage of delignification of 45.55% was accomplished after pretreatment of rice straw with laccase enzyme 39.40 U/g rice straw at 43.70 °C for 11.19 h. Reducing sugar of 3.85 ± 0.15 g/L was obtained from the digested rice straw in a SPS reactor, while non-pretreated rice straw gave only 1.13 ± 0.10 g/L within 12 h of incubation. The results indicated that simultaneous biological pretreatment and saccharification (SPS) of rice straw by laccase helped to improve the accessibility of cellulose by cellulolytic enzymes.

A quantitative metabolic analysis reveals Acetobacterium woodii as a flexible and robust host for formate-based bioproduction.

Cheap and renewable feedstocks such as the one-carbon substrate formate are emerging for sustainable production in a growing chemical industry. We investigated the acetogen Acetobacterium woodii as a potential host for bioproduction from formate alone and together with autotrophic and heterotrophic co-substrates by quantitatively analyzing physiology, transcriptome, and proteome in chemostat cultivations in combination with computational analyses. Continuous cultivations with a specific growth rate of 0.05 h-1 on formate showed high specific substrate uptake rates (47 mmol g-1 h-1). Co-utilization of formate with H2, CO, CO2 or fructose was achieved without catabolite repression and with acetate as the sole metabolic product. A transcriptomic comparison of all growth conditions revealed a distinct adaptation of A. woodii to growth on formate as 570 genes were changed in their transcript level. Transcriptome and proteome showed higher expression of the Wood-Ljungdahl pathway during growth on formate and gaseous substrates, underlining its function during utilization of one-carbon substrates. Flux balance analysis showed varying flux levels for the WLP (0.7-16.4 mmol g-1 h-1) and major differences in redox and energy metabolism. Growth on formate, H2/CO2, and formate + H2/CO2 resulted in low energy availability (0.20-0.22 ATP/acetate) which was increased during co-utilization with CO or fructose (0.31 ATP/acetate for formate + H2/CO/CO2, 0.75 ATP/acetate for formate + fructose). Unitrophic and mixotrophic conversion of all substrates was further characterized by high energetic efficiencies. In silico analysis of bioproduction of ethanol and lactate from formate and autotrophic and heterotrophic co-substrates showed promising energetic efficiencies (70-92%). Collectively, our findings reveal A. woodii as a promising host for flexible and simultaneous bioconversion of multiple substrates, underline the potential of substrate co-utilization to improve the energy availability of acetogens and encourage metabolic engineering of acetogenic bacteria for the efficient synthesis of bulk chemicals and fuels from sustainable one carbon substrates.

Reliable determination of the growth and hydrogen production parameters of the photosynthetic bacterium Rhodobacter capsulatus in fed batch culture using a combination of the Gompertz function and the Luedeking-Piret model.

In this study, experimental results of hydrogen producing process based on anaerobic photosynthesis using the purple non-sulfur bacterium Rhodobacter capsulatus are scrutinized. The bacterial culture was carried out in a photo-bioreactor operated in a quasi-continuous mode, using lactate as a carbon source. The method is based on the continuous stirred tank reactors (CSTR) technique to access kinetic parameters. The dynamic evolution of hydrogen production as a function of time was accurately simulated using Luedeking-Piret model and the growth of R. capsulatus was computed using Gompertz model. The combination of both models was successfully applied to determine the relevant parameters (λ, µmax, α and ß) for two R. capsulatus strains studied: the wild-type strain B10 and the H2 over-producing mutant IR3. The mathematical description indicates that the photofermentation is more promising than dark fermentation for the conversion of organic substrates into biogas.

Microbial biofilms in biorefinery - Towards a sustainable production of low-value bulk chemicals and fuels.

Harnessing the potential of biocatalytic conversion of renewable biomass into value-added products is still hampered by unfavorable process economics. This has promoted the use of biofilms as an alternative to overcome the limitations of traditional planktonic systems. In this paper, the benefits and challenges of biofilm fermentations are reviewed with a focus on the production of low-value bulk chemicals and fuels from waste biomass. Our study demonstrates that biofilm fermentations can potentially improve productivities and product yields by increasing biomass retention and allowing for continuous operation at high dilution rates. Furthermore, we show that biofilms can tolerate hazardous environments, which improve the conversion of crude biomass under substrate and product inhibitory conditions. Additionally, we present examples for the improved conversion of pure and crude substrates into bulk chemicals by mixed microbial biofilms, which can benefit from microenvironments in biofilms for synergistic multi-species reactions, and improved resistance to contaminants. Finally, we suggest the use of mathematical models as useful tools to supplement experimental insights related to the effects of physico-chemical and biological phenomena on the process. Major challenges for biofilm fermentations arise from inconsistent fermentation performance, slow reactor start-up, biofilm carrier costs and carrier clogging, insufficient biofilm monitoring and process control, challenges in reactor sterilization and scale-up, and issues in recovering dilute products. The key to a successful commercialization of the technology is likely going to be an interdisciplinary approach. Crucial research areas might include genetic engineering combined with the development of specialized biofilm reactors, biofilm carrier development, in-situ biofilm monitoring, model-based process control, mixed microbial biofilm technology, development of suitable biofilm reactor scale-up criteria, and in-situ product recovery.

Anaerobic caproate production on carbon chain elongation: Effect of lactate/butyrate ratio, concentration and operation mode.

The objective of this study was to understand how lactate-to-butyrate ratio and substrates concentrations affect the caproate production and product structure. The results showed that a higher butyrate-to-lactate ratio is beneficial to caproate production at low initial lactate concentration. Low pH (5.0) and low substrate concentration (20 mM and 40 mM) effectively decreased propionate production via restrained acrylate pathway, resulting in higher electron efficiency of caproate. With the optimum mole ratio of lactate to butyrate (1:4) and 80 mM initial butyrate concentration, the electron efficiency of caproate reached the maximum (43.10%). Moreover, high butyrate concentration suppressed the production of odd-carbon-number carboxylates while promoting the production of caproate. Compared with the batch operation, the caproate production in semi-continuous operation was enhanced by 3.45 times to 30.91 ± 1.07 mM as the acrylate pathway was successfully inhibited in semi-continuous experiments due to low pH and low lactate concentration.

Continuous production of enzymes under carbon-limited conditions by Trichoderma harzianum P49P11.

Carbon-limited chemostat cultures were performed using different carbon sources (glucose, 10 and 20 g/L; sucrose, 10 g/L; fructose/glucose, 5.26/5.26 g/L; carboxymethyl cellulose, 10 g/L; and carboxymethyl cellulose/glucose, 5/5 g/L) to verify the capability of the wild type strain Trichoderma harzianum to produce extracellular enzymes. All chemostat cultures were carried out at a fixed dilution rate of 0.05 h-1. Experiments using glucose, fructose/glucose and sucrose were performed in duplicate. Glucose condition was found to induce the production of enzymes that can catalyse the hydrolysis of p-nitrophenyl-ß-d-glucopyranoside (PNPGase). A concentration of 20 g/L of glucose in the feed provided the highest productivity (1048 ± 16 U/mol h). Extracellular polysaccharides were considered the source of inducers. Based on the obtained results, a new PNPGase production process was developed using mainly glucose. This process raises interesting possibilities of synthesizing the inducer substrate and the induced enzymes in a single step using an easily assimilated carbon source under carbon-limited conditions.

Sustainable lipid and lutein production from Chlorella mixotrophic fermentation by food waste hydrolysate.

Bioconversion of food waste into value-added products is a promising way to tackle the global food waste management problem. In this study, a novel valorisation strategy for bioenergy and lutein production via microalgal fermentation was investigated. Significant amount of glucose was recovered from enzymatic hydrolysis of food waste. The resultant hydrolysate was then utilised as culture medium in mixotrophic cultivation of Chlorella sp. to obtain high levels of lipid and lutein, whose accumulation patterns were consistent with molecular analyses. The resultant algal lipid derived from microalgal biomass using food hydrolysate was at high quality in terms of biodiesel properties. Further, in semi-continuous fermentation, the average algal biomass was 6.1 g L-1 with 2.5 g L-1 lipid and 38.5 mg L-1 lutein using hydrolysate with an initial glucose concentration of 10 g L-1. Meanwhile, the resultant algal biomass was 6.9 g L-1 with 1.8 g L-1 lipid and 63.0 mg L-1 lutein using hydrolysate with an initial glucose concentration of 20 g L-1, which suggests food waste hydrolysate could trigger algal products preferences. The experimental results of this study suggested the potential of microalgae as a platform for bioconversion of food waste into high-value products, especially sustainable bioenergy.