Fed-batch Experiments Research Articles

Mathematical model of the MenD-catalyzed 1,4-addition (Stetter reaction) of α-ketoglutaric acid to acrylonitrile

The Stetter reaction, a conjugate umpolung reaction, is well known for cyanide-catalyzed transformations of mostly aromatic aldehydes. Enzymatic Stetter reactions, however, have been largely unexplored, especially with respect to preparative transformations. We have investigated the kinetics of the MenD-catalyzed 1,4-addition of α-ketoglutaric acid to acrylonitrile which has shown that acrylonitrile, while an interesting candidate, is a poor substrate for MenD due to low affinity of the enzyme for this substrate. The kinetic model of the reaction was simplified to double substrate Michaelis–Menten kinetics where the reaction rate linearly depends on acrylonitrile concentration. Experiments at different initial concentrations of acrylonitrile under batch, repetitive batch, and fed-batch reactor conditions were carried out to validate the developed mathematical model. Thiamine diphosphate dependent MenD proved to be quite a robust enzyme; nevertheless, enzyme operational stability decay occurs in the reactor. The spontaneous reactivity of acrylonitrile towards polymerization was also taken into account during mathematical modeling. Almost quantitative conversion of acrylonitrile was achieved in all batch reactor experiments, while the yield of the desired product was dependent on initial acrylonitrile concentration (i.e., the concentration of the stabilizer additive). Using the optimized reactor parameters, it was possible to synthesize the product, 6-cyano-4-oxohexanoic acid, in a concentration of 250 mM. The highest concentration of product was achieved in a repetitive batch reactor experiment. A fed-batch reactor experiment also delivered promising results, especially regarding the short reaction time needed to achieve a 200 mM concentration of product. Hence, the enzymatic Stetter reaction with a highly reactive acceptor substrate can be performed on a preparative scale, which should enable similar transformations with acrylate, methacrylate, and methyl vinyl ketone.

Development of a recursive time series model for fed-batch mammalian cell culture

Recursive time series models are developed in this work for a fed-batch mammalian cell culture producing monoclonal antibodies, with key culture variables measured at different sampling frequencies. Glucose and glutamine feed rates are considered as inputs. A composite of an autoregressive moving average with exogenous input model and a dual rate-autoregressive with exogenous input model is used. Appropriate parameter constraints are imposed in parameter estimation algorithms and stability of these is examined and ensured. The data required for parameter estimation are generated from simulated fed-batch experiments using a well-tested first principles model. The predictions for glucose, glutamine, and viable cell concentrations track very well the data for these, with the errors for the high prediction horizons considered being limited to 10% or less. The prediction accuracy can be increased further if data from prior experiments with dynamic similarities are available. The models can be used reliably for model predictive control.

Cellulase hyper-production by Trichoderma reesei mutant SEU-7 on lactose

BackgroundThe induction of cellulase production by insoluble carbon source cellulose was a common and efficient strategy, but has some drawbacks, such as difficult fermentation operation, substantial cellulase loss, long fermentation time, and high energy-consumption, resulting in high cost of cellulase production in industry. These drawbacks can be overcome if soluble carbon sources are utilized as the inducers for cellulase production. However, until now the induction efficiency of most soluble carbon sources, especially lactose and glucose, is still inferior to cellulose despite extensive efforts have been made by either optimizing the fermentation process or constructing the recombinant strains. Therefore, strain improvement by metabolic engineering for high induction efficiency of soluble carbon sources is of great interest.ResultsTrichoderma reesei mutant SEU-7 was constructed from T. reesei RUT-C30 with the overexpression of endogenous gene β-glucosidase (BGL1) by insertional mutagenesis via Agrobacterium tumefaciens-mediated transformation (AMT). Compared to RUT-C30, SEU-7 displays substantially enhanced activities of both cellulase and hemicellulase when grown on either lactose or cellulose. The induction efficiency with lactose was found to be higher than cellulose in strain SEU-7. To the best of our knowledge, we achieved the highest FPase activity in SEU-7 in both batch culture (13.0 IU/mL) and fed-batch culture (47.0 IU/mL) on lactose. Moreover, SEU-7 displayed unrivaled pNPGase activity on lactose in both batch culture (81.0 IU/mL) and fed-batch culture (144.0 IU/mL) as compared to the other reported T. reesei strains in the literature grown in batch or fed-batch experiments on cellulose or lactose. This superiority of SEU-7 over RUT-C30 improves markedly the saccharification ability of SEU-7 on pretreated corn stover. The overexpression of gene BGL1 was found either at the mRNA or at the protein level in the mutant strains with increased cellulase production in comparison with RUT-C30, but only SEU-7 displayed much higher expression of gene BGL1 on lactose than on cellulose. Two copies of gene BGL1 were inserted into the chromosome of T. reesei SEU-7 between KI911141.1:347357 and KI911141.1:347979, replacing the original 623-bp fragment that is not within any genes’ coding region. The qRT-PCR analysis revealed that the mRNA levels of both cellulase and hemicellulase were upregulated significantly in SEU-7, together with the MFS transporter CRT1 and the XYR1 nuclear importer KAP8.ConclusionsRecombinant T. reesei SEU-7 displays hyper-production of both cellulase and hemicellulase on lactose with the highest FPase activity and pNPGase activity for T. reesei, enabling highly efficient saccharification of pretreated biomass. For the first time, the induction efficiency for cellulase production by lactose in T. reesei was reported to be higher than that by cellulose. This outperformance of T. reesei SEU-7, which is strain-specific, is attributed to both the overexpression of gene BGL and the collateral mutation. Moreover, the increased transcription levels of cellulase genes, the related transcription factors, and the MFS transporter CRT1 contribute to the outstanding cellulase production of SEU-7. Our research advances strain improvement to enhance the induction efficiency of soluble carbon sources to produce cost-effective cellulase and hemicellulase in industry.

Production of High Levels of Chirally Pure d-2,3-Butanediol with a Newly Isolated Bacillus Strain

2,3-Butanediol (2,3-BD), especially when it exists in the chirally pure form, is a valuable chemical feedstock with numerous applications in various industries. However, in most cases, the 2,3-BD naturally produced by the microorganism is a mixture of two of the three isomers (d-2,3-BD, l-2,3-BD, and meso-2,3-BD), which has restricted applications and thus a much lower value than the chirally pure one. In this study, we report a new isolate Bacillus spp. FJ-4 strain, which can produce highly chirally pure d-2,3-BD (>99.9%) to high levels, with very high productivity and yield. The composition of the fermentation medium for the strain was sequentially optimized using statistical experimental methodologies. During a fed-batch experiment, 100.0 g/L of d-2,3-BD was produced from 226.8 g/L glucose, with a yield of 0.44 g/g (88.2% of the theoretical maximum). Acetoin was the primary byproduct, and no other byproduct such as lactate, acetate, or ethanol was detected, which would be advantageous for the downstrea...

Nutrient Recovery From Cyanobacteria Biomasses Using Purple Nonsulfur Bacterium Rhodopseudomonas palustris

Occurrences of harmful cyanobacterial blooms are a worldwide environmental problem in most eutrophic lake ecosystems. But what should be noticed is that cyanobacteria can be used as a useful resource due to the wide range of metabolites they produce. Nutrient partitioning using purple nonsulfur bacteria (PNSB) has the potential to biologically concentrate nutrients. The present study evaluated the kinetics of nutrients released from decomposed field blue green algae (BGA) biomasses. The potential of nutrient acquisition from decomposed BGA biomasses for culturing Rhodopseudomonas palustris (R. palustris) was investigated via fed-batch experiments. Results indicated that R. palustris stimulated in algae substrates with algae biomasses ranging from 3.33 to 10 g/L. Removal efficiencies of N and P in the stationary phase of growth were at least 40% and 95%, respectively, of all the nitrogen (N) and phosphorus (P) released. Additionally, the cellular contents like total lipid and poly-beta-hydroxybutyrate (PHB), as well as the fatty acids produced by R. palustris, were consistent. Hence, practice based on the bacterial production for the nutrient recovery from BGA biomasses provides a new insight in field algae disposal. It will lower the chances of secondary pollution due to algae decay and produce giant cells of R. palustris and surely will prosper the industries applying R. palustris.

The physiology of trace elements in biological methane production

Trace element (TE) requirements of Methanothermobacter okinawensis and Methanothermobacter marburgensis were examined in silico, and using closed batch and fed-batch cultivation experiments. In silico analysis revealed genomic differences among the transport systems and enzymes related to the archaeal Wood-Ljungdahl pathway of these two methanogens. M. okinawensis responded to rising concentrations of TE by increasing specific growth rate (µ) and volumetric productivity (MER) during closed batch cultivation, and can grow and produce methane (CH4) during fed-batch cultivation. M. marburgensis showed higher µ and MER during fed-batch cultivation and was therefore prioritized for subsequent optimization of CO2-based biological CH4 production. Multiple-parameter cultivation dependency on growth and productivity of M. marburgensis was finally examined using exponential fed-batch cultivation at different medium-, TE- and sulphide dilution rates, and different gas inflow rates. MER of 476mmolL−1h−1 and µ of 0.69h−1 were eventually obtained during exponential fed-batch cultivations employing M. marburgensis.

Pilot Scale System of Two Horizontal Rotating Bioreactors for Bioethanol Production from Household Food Waste at High Solid Concentrations

Household food wastes (HFW) a complex biomass containing soluble sugars, lipids, proteins, cellulose, starch was used for bioethanol production in a newly designed pilot scale system consisting of two horizontal rotating bioreactors (HRRs) operating at high solids content under non-isothermal simultaneous saccharification and fermentation (NSSF) in fed-batch mode. Operational conditions were determined in lab-scale experiments. More specifically, enzymes including cellulases, α-amylase and glucoamylase at different loadings were tested one at the time for the pre-hydrolysis and subsequent fermentation by Saccharomyces cerevisiae of the pretreated HFW. The highest ethanol production (42.74 g/L, corresponding to 72.33% of the maximum theoretical) was obtained when cellulases (at 60 FPU/g cellulose) and glucoamylase (60 FPU/g starch) were used. Fed-batch experiments were conducted in a 20 L bioreactor. Increasing batch additions resulted in a higher ethanol titer. Ethanol production of 60.69 g/L (corresponding to 69.77% of the maximum theoretical) was achieved when three additions were made. Implementation of the NSSF process operating in fed-batch mode in the pilot scale facility resulted in ethanol production of 53.90 g/L (corresponding to 73.26% of maximum theoretical yield). The pilot scale facility of the present study can produce 188 L of ethanol from one ton of dry HFW.

Wastewater recycling technology for fermentation in polyunsaturated fatty acid production

To reduce fermentation-associated wastewater discharge and the cost of wastewater treatment, which further reduces the total cost of DHA and ARA production, this study first analyzed the composition of wastewater from Aurantiochytrium (DHA) and Mortierella alpina (ARA) fermentation, after which wastewater recycling technology for these fermentation processes was developed. No negative effects of DHA and ARA production were observed when the two fermentation wastewater methods were cross-recycled. DHA and ARA yields were significantly inhibited when the wastewater from the fermentation process was directly reused. In 5-L fed-batch fermentation experiments, using this cross-recycle technology, the DHA and ARA yields were 30.4 and 5.13gL−1, respectively, with no significant changes (P>0.05) compared to the control group, and the water consumption was reduced by half compared to the traditional process. Therefore, this technology has great potential in industrial fermentation for polyunsaturated fatty acid production.

Hybrid modeling of microbial exopolysaccharide (EPS) production: The case of Enterobacter A47

Enterobacter A47 is a bacterium that produces high amounts of a fucose-rich exopolysaccharide (EPS) from glycerol residue of the biodiesel industry. The fed-batch process is characterized by complex non-linear dynamics with highly viscous pseudo-plastic rheology due to the accumulation of EPS in the culture medium. In this paper, we study hybrid modeling as a methodology to increase the predictive power of models for EPS production optimization. We compare six hybrid structures that explore different levels of knowledge-based and machine-learning model components. Knowledge-based components consist of macroscopic material balances, Monod type kinetics, cardinal temperature and pH (CTP) dependency and power-law viscosity models. Unknown dependencies are set to be identified by a feedforward artificial neural network (ANN). A semiparametric identification schema is applied resorting to a data set of 13 independent fed-batch experiments. A parsimonious hybrid model was identified that describes the dynamics of the 13 experiments with the same parameterization. The final model is specific to Enterobacter A47 but can be easily extended to other microbial EPS processes.

Effects of glycerol supply and specific growth rate on methanol-free production of CALB by P. pastoris: functional characterisation of a novel promoter

As Pichia pastoris (syn. Komagataella sp.) yeast can secrete pure recombinant proteins at high rates, it is a desirable production system. The function of a novel synthetic variant of the AOX1 promoter was characterised comprehensively using a strain secreting Candida antarctica lipase B (CALB) as a model. A new time-saving approach was introduced to determine, in only one experiment, the hitherto unknown relationship between specific product formation rate (qp) and specific growth rate (μ). Tight control of recombinant protein formation was possible in the absence of methanol, while using glycerol as a sole carbon/energy source. CALB was not synthesised during batch cultivation in excess glycerol (>10 g l−1) and at a growth rate close to μmax (0.15 h−1). Between 0.017 and 0.115 h−1 in glycerol-limited fedbatch cultures, basal levels of qp > 0.4 mg g−1 h−1 CALB were reached, independent of the μ at which the culture grew. At μ > 0.04 h−1, an elevated qp occurred temporarily during the first 20 h after changing to fedbatch mode and decreased thereafter to basal. In order to accelerate the determination of the qp(μ) relationship (kinetics of product formation), the entire μ range was covered in a single fedbatch experiment. By linearly increasing and decreasing glycerol addition rates, μ values were repeatedly shifted from 0.004 to 0.074 h−1 and vice versa. Changes in qp were related to changes in μ. A rough estimation of μ range suitable for production was possible in a single fedbatch, thus significantly reducing the experimental input over previous approaches comprising several experiments.

Production of docosahexaenoic acid by Aurantiochytrium sp. ATCC PRA-276.

The high costs and environmental concerns associated with using marine resources as sources of oils rich in polyunsaturated fatty acids have prompted searches for alternative sources of such oils. Some microorganisms, among them members of the genus Aurantiochytrium, can synthesize large amounts of these biocompounds. However, various parameters that affect the polyunsaturated fatty acids production of these organisms, such as the carbon and nitrogen sources supplied during their cultivation, require further elucidation. The objective of this investigation was to study the effect of different concentrations of carbon and total nitrogen on the production of polyunsaturated fatty acids, particularly docosahexaenoic acid, by Aurantiochytrium sp. ATCC PRA-276. We performed batch system experiments using an initial glucose concentration of 30 g/L and three different concentrations of total nitrogen, including 3.0, 0.44, and 0.22 g/L, and fed-batch system experiments in which 0.14 g/L of glucose and 0.0014 g/L of total nitrogen were supplied hourly. To assess the effects of these different treatments, we determined the biomass, glucose, total nitrogen and polyunsaturated fatty acids concentration. The maximum cell concentration (23.9 g/L) was obtained after 96 h of cultivation in the batch system using initial concentrations of 0.22 g/L total nitrogen and 30 g/L glucose. Under these conditions, we observed the highest level of polyunsaturated fatty acids production (3.6 g/L), with docosahexaenoic acid and docosapentaenoic acid ω6 concentrations reaching 2.54 and 0.80 g/L, respectively.

Production of 2-Hydroxyisobutyric Acid from Methanol by Methylobacterium extorquens AM1 Expressing (R)-3-Hydroxybutyryl Coenzyme A-Isomerizing Enzymes.

In this study, we genetically modified a methylotrophic bacterium in order to channel intermediates of its overflow metabolism to the C4 carboxylic acid 2-hydroxyisobutyric acid, a precursor of acrylic glass. This has implications for biotechnology, as it shows that reduced C1 substrates, such as methanol and formic acid, can be alternative feedstocks for producing today's commodities. We found that product titers and yields depend more on host physiology than on the activity of the introduced heterologous function modifying the overflow metabolism. In addition, we show that the fitness of recombinant strains substantially varies when they express orthologous genes from different origins. Further studies are needed to extend the overflow production phase in methylotrophic microorganisms for the implementation of biotechnological processes.

Production of human pro-relaxin H2 in the yeast Pichia pastoris

BackgroundInitially known as the reproductive hormone, relaxin was shown to possess other therapeutically useful properties that include extracellular matrix remodeling, anti-inflammatory, anti-ischemic and angiogenic effects. All these findings make relaxin a potential drug for diverse medical applications. Its precursor, pro-relaxin, is an 18 kDa protein, that shows activity in in vitro assays. Since extraction of relaxin from animal tissues raises several issues, prokaryotes and eukaryotes were both used as expression systems for recombinant relaxin production. Most productive results were obtained when using Escherichia coli as a host for human relaxin expression. However, in such host, relaxin precipitated in the form of inclusion bodies and, therefore, required several expensive recovery steps as cell lysis, refolding and reduction.ResultsTo overcome the issues related to prokaryotic expression here we report the production and purification of secreted human pro-relaxin H2 by using the methylotrophic yeast Pichia pastoris as expression host. The methanol inducible promoter AOX1 was used to drive expression of the native and histidine tagged forms of pro-relaxin H2 in dual phase fed-batch experiments on the 22 L scale. Both protein forms presented the correct structure, as determined by mass spectrometry and western blotting analyses, and demonstrated to be biologically active in immune enzymatic assays. The presence of the tag allowed to simplify pro-relaxin purification obtaining higher purity.ConclusionsThis work presents a strategy for microbial production of recombinant human pro-relaxin H2 in Pichia pastoris that allowed the obtainment of biologically active pro-hormone, with a final concentration in the fermentation broth ranging between 10 and 14 mg/L of product, as determined by densitometric analyses.

A discretized model for enzymatic hydrolysis of cellulose in a fed-batch process

In the enzymatic hydrolysis of cellulose, several phenomena have been proposed to cause a decrease in the reaction rate with increasing conversion. The importance of each phenomenon is difficult to distinguish from batch hydrolysis data. Thus, kinetic models for the enzymatic hydrolysis of cellulose often suffer from poor parameter identifiability. This work presents a model that is applicable to fed-batch hydrolysis by discretizing the substrate based on the feeding time. Different scenarios are tested to explain the observed decrease in reaction rate with increasing conversion, and comprehensive assessment of the parameter sensitivities is carried out. The proposed model performed well in the broad range of experimental conditions used in this study and when compared to literature data. Furthermore, the use of data from fed-batch experiments and discretization of the model substrate to populations was found to be very informative when assessing the importance of the rate-decreasing phenomena in the model.

Robust at-line quantification of poly(3-hydroxyalkanoate) biosynthesis by flow cytometry using a BODIPY 493/503-SYTO 62 double-staining

Poly(3-hydroxyalkanoates) (PHAs) are bio-based and biodegradable polyesters which have been considered as a promising alternative to petrol-based plastics. Their bacterial production is a dynamic process in which intracellular polymerization and depolymerization are closely linked and depend on the availability of carbon substrates and other nutrients. These dynamics require a fast and quantitative method to determine the optimal harvest-time of PHA containing cells or to adjust carbon supply. In principle, flow cytometry (FCM) is an ideal tool that suits these requirements and, in addition, provides data on the PHA content of different cell populations. However, FCM-based PHA quantification methods have often relied on laborious sample preparation including washing steps and long incubation times. Here, we introduce a fast method based on double-staining using BODIPY 493/503 for PHA staining and SYTO 62 for DNA that allows acquiring reliable fluorescence and cell count data in <10min. Finally, fed-batch experiments with Pseudomonas putida KT2440 and Rhodospirillum rubrum S1 revealed that the method was robust and independent of the strain and type of PHA (medium-chain-length [mcl-] and short-chain-length [scl-] PHA, respectively). Interestingly, the specific PHA fluorescence was in case of mcl-PHA larger than for scl-PHA, probably reflecting the different material properties (e.g., specific density, hydrophilicity and crystallinity).

Biogas Production from Anaerobic Co-Digestion of Food Waste Mixed with Domestic Wastewater

This research was to investigate the potential of biogas production from the co- digestion of canteen wastewater and food waste. Batch experiments were carried out under various substrate ratios1 : 1 : 0, 1 : 1 : 1, 1 : 1 : 2, 1 : 2 : 1, 2 : 1 : 1, 2 : 2 : 1, 3 : 2 : 1, 4 : 2 : 1, 6 : 5 : 1, 8 : 5 : 1, 10 : 3 : 1, 10 : 4 : 1 and 10 : 5 : 1 (canteen wastewater : swine manure : food residue waste) at room temperature. The biogas production was carried for a retention period of 7 days to investigate suitable mixing ratio. The suitable ratio was tested in a plastic container 200 liter with bath and fed-batch experiment for a retention period of 45 days. The results revealed that fermentation slurry mixing ratio of 1 : 2 : 1 was found to be optimum, which gave the methane production with composition 47.34-61 %CH4. The biogas yield and thermal energy were 88.86 L/day and 69 kcal respectively.These primary results indicated the significance of co-digestion of canteen wastewater with food waste for biodegradation and biogas production.

Novel homologous lactate transporter improves l-lactic acid production from glycerol in recombinant strains of Pichia pastoris

BackgroundCrude glycerol is the main byproduct of the biodiesel industry. Although it can have different applications, its purification is costly. Therefore, in this study a biotechnological route has been proposed for further utilization of crude glycerol in the fermentative production of lactic acid. This acid is largely utilized in food, pharmaceutical, textile, and chemical industries, making it the hydroxycarboxylic acid with the highest market potential worldwide. Currently, industrial production of lactic acid is done mainly using sugar as the substrate. Thus here, for the first time, Pichia pastoris has been engineered for heterologous l-lactic acid production using glycerol as a single carbon source. For that, the Bos taurus lactate dehydrogenase gene was introduced into P. pastoris. Moreover, a heterologous and a novel homologous lactate transporter have been evaluated for l-lactic acid production.ResultsBatch fermentation of the P. pastoris X-33 strain producing LDHb allowed for lactic acid production in this yeast. Although P. pastoris is known for its respiratory metabolism, batch fermentations were performed with different oxygenation levels, indicating that lower oxygen availability increased lactic acid production by 20 %, pushing the yeast towards a fermentative metabolism. Furthermore, a newly putative lactate transporter from P. pastoris named PAS has been identified by search similarity with the lactate transporter from Saccharomyces cerevisiae Jen1p. Both heterologous and homologous transporters, Jen1p and PAS, were evaluated in one strain already containing LDH activity. Fed-batch experiments of P. pastoris strains carrying the lactate transporter were performed with the batch phase at aerobic conditions followed by an aerobic oxygen-limited phase where production of lactic acid was favored. The results showed that the strain containing PAS presented the highest lactic acid titer, reaching a yield of approximately 0.7 g/g.ConclusionsWe showed that P. pastoris has a great potential as a fermentative organism for producing l-lactic acid using glycerol as the carbon source at limited oxygenation conditions (below 0.05 % DO in the bioreactor). The best strain had both the LDHb and the homologous lactate transporter encoding genes expressed, and reached a titer 1.5 times higher than the strain with the S. cerevisiae transporter. Finally, it was also shown that increased lactic acid production was concomitant to reduction of acetic acid formation by half.

A simple method to determine IgG light chain to heavy chain polypeptide ratios expressed by CHO cells

To establish a high-throughput method for determination of antibodies intra- and extracellular light chain (LC) to heavy chain (HC) polypeptide ratio as screening parameter during cell line development. Chinese Hamster Ovary (CHO) TurboCell pools containing different designed vectors supposed to result in different LC:HC polypeptide ratios were generated by targeted integration. Cell culture supernatants and cell lysates of a fed batch experiment were purified by combined Protein A and anti-kappa affinity batch purification in 96-well format. Capture of all antibodies and their fragments allowed the determination of the intra- and extracellular LC:HC peptide ratios by reduced SDS capillary electrophoresis. Results demonstrate that the method is suitable to show the significant impact of the vector design on the intra- and extracellular LC:HC polypeptide ratios. Determination of LC:HC polypeptide ratios can give important information in vector design optimization leading to CHO cell lines with optimized antibody assembly and preferred product quality.

Simultaneous Saccharification and Fermentation of Low Strength Sodium Hydroxide Pre-treated Rice Straw for Higher Ethanol Yields: Batch and Fed Batch Hybrid Approach

Low strength sodium hydroxide pre-treated rice straw was hydrolysed at two different solid loadings i.e. 10 and 15 % (w/v) with in-house cellulases (IC) produced by Aspergillus terreus along with commercial cellulases (CC) by using batch and fed batch hybrid simultaneous saccharification and fermentation process. Optimization of process parameters enhanced the crude cellulase activities i.e. filter paper, β-glucosidase and endoglucanase by 1.6, 2.7 and 2.2 fold in 6 days as compared to activities obtained on a single substrate i.e. rice straw. Out of four fed batch approaches, approach III (A-III) at 10 % solid loading yielded higher ethanol concentrations of 30.55 and 28.66 g L−1 by using thermo tolerant in-house yeast strain Kluyveromyces marxianus at 42 °C. Combination of 9 FPU g−1 substrate and CC plus 30 CBU of commercial β-glucosidase (Cβ) yielded slightly higher theoretical ethanol yields of 92.24 % as compared to 86.54 % obtained from IC+Cβ. Dunnett’s Post Hoc Annova test also proved that CC+Cβ were found to be significant in batch as well fed batch experiments as compared to other combinations tried. Thus, fed batch A-III seems to be an optimistic one to overcome the problems associated with batch mode to achieve higher ethanol yields.

High-purity propionate production from glycerol in mixed culture fermentation

High-purity propionate production from glycerol in mixed culture fermentation (MCF) induced by high ammonium concentration was investigated. Fed-batch experiments revealed that higher ammonium concentration (>2.9g/L) had simultaneous negative effects on acetate and propionate degradation. Propionate production and yield was up to 22.6g/L and 0.45g COD/g COD glycerol, respectively, with a purity of 96%. Sequential batch experiments demonstrated that the yields of propionate were 0.3±0.05, 0.32±0.01, and 0.34±0.03g COD/g COD at a glycerol concentration of 2.78, 4.38, and 5.56g/L, respectively, and the purity of propionate was 91–100%. Microbial community analysis showed that the phylum Firmicutes dominated the bacterial community at different glycerol concentrations. However, the Methanosaeta population decreased from 46% to 6% when glycerol concentration increased from 2.78 to 5.56g/L, resulting in lower acetate degradation rate. Thus, the present study might provide an alternative option for the production of propionate from glycerol via MCF.