Continuous Ethanol Production Research Articles

Development of a Highly Selective NAD+ -Dependent Glyceraldehyde Dehydrogenase and its Application in Minimal Cell-Free Enzyme Cascades.

Anthropogenic climate change has been caused by over-exploitation of fossil fuels and CO2 emissions. To counteract this, the chemical industry has shifted its focus to sustainable chemical production and the valorization of renewable resources. However, the biggest challenges in biomanufacturing are technical efficiency and profitability. In our minimal cell-free enzyme cascade generating pyruvate as the central intermediate, the NAD+ -dependent, selective oxidation of D-glyceraldehyde was identified as a key reaction step to improve the overall cascade flux. Successive genome mining identified one candidate enzyme with 24-fold enhanced activity and another whose stability is unaffected in 10 % (v/v) ethanol, the final product of our model cascade. Semi-rational engineering improved the substrate selectivity of the enzyme up to 21-fold, thus minimizing side reactions in the one-pot enzyme cascade. The final biotransformation of D-glucose showed a continuous linear production of ethanol (via pyruvate) to a final titer of 4.9 % (v/v) with a molar product yield of 98.7 %. Due to the central role of pyruvate in diverse biotransformations, the optimized production module has great potential for broad biomanufacturing applications.

Continuous ethanol production via ultrasound-enhanced yeast sedimentation

Continuous glucose fermentation produces bioethanol at higher volumetric rates than conventional batch or fed-batch systems. The retention of yeast cells via ultrasonic sedimentation in a lab-scale fermenter allowed for shearless, continuous cell upconcentration, and consequently process intensification. The cell separation efficiency of the ultrasonic system was predicted with a Response Surface Model (RSM) developed for yeast cells based on the linear, mixed, and quadratic effects of the operating variables and flow rates (3 levels, 5 variables). The experiments for the RSM calibration were designed via a central composite design. The efficiency model was validated and showed dependency to the Biomass concentration, Power input, and Harvest rate (R2calibration = 0.92, R2prediction = 0.83). A lab-scale fermenter fed with yeast growth medium was operated at varying dilution rates (0.1 – 0.6 h−1) based on the RSM to maximize the cell retention efficiency (23%−90%). Yeast cell concentration in the fermenter reached up to 31.5 ± 0.7 g/L while it remained at around 3 g/L in the harvest stream for all the dilution rates tested. The ethanol concentration ranged between 17 and 23 g/L and reached high volumetric productivity (8.8 g/L/h). A control run without ultrasonic sedimentation led to the washout of biomass at a dilution rate of 0.6 h−1. Ultrasonic yeast sedimentation is a promising technology for cell retention and enhanced productivity in continuous fermentation processes.

Continuous Production of Ethanol, 1-Butanol and 1-Hexanol from CO with a Synthetic Co-Culture of Clostridia Applying a Cascade of Stirred-Tank Bioreactors.

Syngas fermentation with clostridial co-cultures is promising for the conversion of CO to alcohols. A CO sensitivity study with Clostridium kluyveri monocultures in batch operated stirred-tank bioreactors revealed total growth inhibition of C. kluyveri already at 100 mbar CO, but stable biomass concentrations and ongoing chain elongation at 800 mbar CO. On/off-gassing with CO indicated a reversible inhibition of C. kluyveri. A continuous supply of sulfide led to increased autotrophic growth and ethanol formation by Clostridium carboxidivorans even at unfavorable low CO concentrations. Based on these results, a continuously operated cascade of two stirred-tank reactors was established with a synthetic co-culture of both Clostridia. An amount of 100 mbar CO and additional sulfide supply enabled growth and chain elongation in the first bioreactor, whereas 800 mbar CO resulted in an efficient reduction of organic acids and de-novo synthesis of C2-C6 alcohols in the second reactor. High alcohol/acid ratios of 4.5-9.1 (w/w) were achieved in the steady state of the cascade process, and the space-time yields of the alcohols produced were improved by factors of 1.9-5.3 compared to a batch process. Further improvement of continuous production of medium chain alcohols from CO may be possible by applying less CO-sensitive chain-elongating bacteria in co-cultures.

Bioethanol production of cheese whey in continuous culture

The cheese industry produces a large amount of waste, the equivalent of 9L of cheese whey for every 10L of milk (Murari et al., 2018; Pendon et al., 2021; Sar et al., 2021). Whey is composed by lactose and proteins, mainly (Murari et al., 2018; Murari et al., 2019; Tesfaw et al., 2021). In most cases, cheese whey had not added value or specific use, being discarded and causing very important environmental implications (Maruri et al., 2018; Tesfaw et al., 2021; Sar et al., 2021). In the present study, the production of bioethanol from cheese whey were analyzed, using Kluyveromyces marxianus as inoculum, under continuous culture conditions (28°C, agitation at 100 rpm, aeration at 1 vvm, feed at 8.63 mL/min), where it was established that is possible to obtain yields of 0.62 g of ethanol per g of lactose in the first 18 h, this makes it feasible for the continuous production of ethanol with cheese whey with minimal pretreatment.

Continuous Ethanol Production from Molasses via Immobilized Saccharomyces cerevisiae on Different Carriers on Pilot Scale

IMMOBILIZATION of yeast cells for continuous ethanol production has been extensively studied worldwide during the past few years because it showed significant advantages compared to the production by free cell system. Using renewable substrates such as Egyptian cane and beet molasses becomes necessary to reduce food crops for bioethanol production. This study investigates the immobilization of commercial strain of Saccharomyces cerevisiae on different carriers (sugarcane bagasse, rice straw, wheat straw, and Na-alginate) for continuous ethanol production on a pilot scale. The results demonstrated that sugarcane bagasse was the best carrier for yeast cell immobilization. This substrate produced 66.30g/ L ethanol, 2.76g/ L/ hr as ethanol productivity, and 81% fermentation efficiency from the theoretical value, using a mixture of Egyptian cane and beet molasses with 16% initial sugar at 30°C and pH 4.5. Also, the production process is retained until 18 days continuously. From this study, it was clear that lignocellulosic materials provide suitable, cheap, and renewable carriers for immobilization of yeast cells used in the continuous ethanol production process.

Heat Integration and Batch Scheduling of Optimal Bioethanol Production

This paper examines bioethanol production from oil palm empty fruit bunches, 47,208 kgs of which could potentially produce 13,950 liters of ethanol per day, using the Aspen Plus Suite commercial software. Continuous ethanol production with efficiency, optimum plant design, process dynamics, and control were studied in a continuous process to minimize the loss of plant utility and prevent possible ethanol production failure, which can sometimes occur in real life. A Gantt chart was used for continuous production to obtain ethanol every 24.5 hours. The Gantt chart also increases equipment efficiency by up to 73.90%. Pinch analysis was conducted for minimal energy consumption at the plant. The proposed production without/with the heat exchanger network shows that the total costs per year are 1,343,861$/year and 886,253$/year, respectively. The implementation of a heat exchanger network can reduce costs by around 34.05% per year. The purification process is guaranteed by the controller of the column system due to its dynamic failure sensitivity. Details of the controller and the results in the dynamic mode are presented.

Feasibility Study on Long-Term Continuous Ethanol Production from Cassava Supernatant by Immobilized Yeast Cells in Packed Bed Reactor.

In this study, yeast cell immobilization was carried out in a packed bed reactor (PBR) to investigate the effects of the volumetric capacity of carriers as well as the different fermentation modes on fuel ethanol production. An optimal volumetric capacity of 10 g/l was found to obtain a high cell concentration. The productivity of immobilized cell fermentation was 16% higher than that of suspended-cell fermentation in batch and it reached a higher value of 4.28 g/l/h in repeated batches. Additionally, using this method, the ethanol yield (95.88%) was found to be higher than that of other tested methods due to low concentrations of residual sugars and free cells. Continuous ethanol production using four bioreactors showed a higher productivity (9.57 g/l/h) and yield (96.96%) with an ethanol concentration of 104.65 g/l obtained from 219.42 g/l of initial total sugar at a dilution rate of 0.092 h-1. Furthermore, we reversed the substrate-feed flow directions in the in-series bioreactors to keep the cells at their highest activity and to extend the length of continuous fermentation. Our study demonstrates an effective method of ethanol production with a new immobilized approach, and that by switching the flow directions, traditional continuous fermentation can be greatly improved, which could have practical and broad implications in industrial applications.

Use of electrospun threads in immobilized cell reactors for continuous ethanol production

Saccharomyces cerevisiae immobilized in electrospun Pluronic F127 dimethacrylate (FDMA) was successfully employed for the production of ethanol in an immobilized cell reactor. Yeast cells were immobilized into fibers formed through the process of electrospinning and cross-linking. The threads had an average diameter of 0.88 μm and were used in continuous-flow immobilized cell reactors. The immobilized cell reactors were able to maintain a high ethanol yield of >90% from day 4 through to the end of the time course at day 14. The reactor was able to achieve a maximum ethanol yield of 94.3%. This study shows that the use of electrospinning is a promising method for continuous ethanol production through immobilized cell-based continuous flow reactors.

Production of bioethanol fuel using immobilized Sacharomyces cerevisiae

The use of Saccharomyces cerevisiae immobilized in alginate beads coated with chitosan show stable and continuous ethanol production along a 168 h fermentation utilizing 0,5 L bioreactor. Also, the beads stability has show excellent resistance.

Continuous production of bioethanol using microalgal sugars extracted from Nannochloropsis gaditana

We developed a continuous production process of bioethanol from sugars extracted from Nannochloropsis gaditana. To improve algal sugar production, the reaction conditions of acid-thermal hydrolysis were investigated based on five different types of acid and their concentrations (1-4%), and the loading ratio of solid/liquid (S/L). As a result, the maximum hydrolysis efficiency (92.82%) was achieved under 2% hydrochloric acid with 100 g/L biomass loading at 121 oC for 15 min. The hydrolysates obtained from N. gaditana were applied to the main medium of Bretthanomyces custersii H1-603 for bioethanol production. The maximum bioethanol production and yield by the microalgal hydrolysate were found to be 4.84 g/L and 0.37 g/g, respectively. In addition, the cell immobilization of B. custersii was carried out using sodium alginate, and the effect of the volume ratio of cell/sodium alginate on bioethanol productivity was investigated in a batch system. The optimal ratio was determined as 2 (v/v), and the immobilized cell beads were applied in the continuous stirred tank reactor (CSTR). Continuous ethanol production was performed using both free cells and immobilized cells at 1 L CSTR. In both groups, the maximum bioethanol production and yield were achieved at dilution rate of 0.04 h-1 (3.93 g/L and 0.3 g/g by free cell, and 3.68 g/L and 0.28 g/g by immobilized cell, respectively).

Fermentative energy conversion: Renewable carbon source to biofuels (ethanol) using Saccharomyces cerevisiae and downstream purification through solar driven membrane distillation and nanofiltration

Experimental studies on fermentative production of ethanol from renewable sugarcane juice as carbon source were carried out in a bench scale fermenter integrated with microfiltration, nanofiltration and membrane distillation units (MF-NF-MD) for downstream purification and concentration enrichment of ethanol. This new design is flexible, compact, eco-friendly, and modular and, operated in a cross flow module that ensures almost fouling-free membrane separation while overcoming the product inhibition of traditional batch fermenters. The system is blessed with steady and continuous ethanol production from sugarcane juice with comparatively high productivity 9.2gL−1h−1, yield 0.47gg−1 and concentration 54.3gL−1 using Saccharomyces cerevisiae (NCIM 3205) vis-à-vis the existing technologies. The new system enables continuous product withdrawal, separation and recycle of the biomass as well as residual carbon source back to the fermenter under high cell density without product inhibition. Membrane distillation in the final stage, increases product purity and results in significant concentration enrichment. The study culminates in development of a green and sustainable process for a biofuel production.

Performance investigation of a pilot scale integrated aerobic–anaerobic baffle reactor for continuous ethanol production process under various sugar-feeding rates

Ethanol as the promising alternative fuel in Indonesia fights against cost competitiveness with fossil fuel due to low productivity using a conventional batch process. Therefore, the production of ethanol employing continuous fermentation process in which it acquires higher productivity was investigated. The investigation was done using uncontrolled continuous process in the integrated aerobic–anaerobic baffled reactor for 168–340 h at 30℃ and medium pH of 4–5. The substrate of sugarcane molasses–water was varied in three concentrations, i.e. 20, 30, and 45% w/w. The productivity was indicated by ethanol concentration in the effluent stream and sugar to ethanol conversion (fermentation efficiency). The result showed that average ethanol concentration of 20, 30, and 45% molasses was 50.43, 62.70, and 57.17 g/l, respectively. Further, the fermentation efficiencies were 87.2, 80.3, and 46.7% for each of the molasses concentrations. The productivity can be monitored as follows: 1.75 g/l h (20% molasses), 2.31...

The Performance of Start-Up Process in Continuous Ethanol Production Using Integrated Aerobic–Anaerobic Baffled Reactor (IAABR)

The Performance of Start-Up Process in Continuous Ethanol Production Using Integrated Aerobic–Anaerobic Baffled Reactor (IAABR)

Re-utilization of waste glycerol for continuous production of bioethanol by immobilized Enterobacter aerogenes

Waste glycerol, a by-product of biodiesel production, has been continuously increasing in abundance throughout the world since biodiesel was first commercially produced. Therefore, re-utilization of this waste substance is an important area of research at the present time. This study aims to utilize waste glycerol as a media source for bioethanol production. In addition, Enterobacter aerogenes ATCC 29007, one of the greatest ethanol producers, was immobilized using alginate and a continuous stirred tank reactor (CSTR) newly designed for continuous production with immobilized cells. For efficient bioethanol production, four major media components were statistically optimized by response surface methodology (RSM). The optimal concentrations of each factor were determined: about 6.5 g/L peptone, 3.8 g/L ammonium sulfate, 2.1 g/L citrate dehydrate and 12 g/L waste glycerol. Under the optimal conditions, the ethanol production and yield were about 5.38 g/L and 0.96 mol-ethanol/mol-glycerol with pure glycerol, whereas the ethanol production and yield were approximately 5.29 g/L and 0.91 mol-ethanol/mol-glycerol with the waste glycerol during continuous fermentation. Therefore, the suggested model for media optimization was determined to be reliable. Moreover, continuous ethanol production was successfully performed with waste glycerol using the CSTR system.

Continuous Ethanol Production from Synthesis Gas by Clostridium ragsdalei in a Trickle-Bed Reactor

A trickle-bed reactor (TBR) when operated in a trickle flow regime reduces liquid resistance to mass transfer because a very thin liquid film is in contact with the gas phase and results in improved gas–liquid mass transfer compared to continuous stirred tank reactors (CSTRs). In the present study, continuous syngas fermentation was performed in a 1-L TBR for ethanol production by Clostridium ragsdalei. The effects of dilution and gas flow rates on product formation, productivity, gas uptakes and conversion efficiencies were examined. Results showed that CO and H2 conversion efficiencies reached over 90% when the gas flow rate was maintained between 1.5 and 2.8 standard cubic centimeters per minute (sccm) at a dilution rate of 0.009 h−1. A 4:1 molar ratio of ethanol to acetic acid was achieved in co-current continuous mode with both gas and liquid entered the TBR at the top and exited from the bottom at dilution rates of 0.009 and 0.012 h−1, and gas flow rates from 10.1 to 12.2 sccm and 15.9 to 18.9 sccm, respectively.

Enhanced butanol production by solvent tolerance Clostridium acetobutylicum SE25 from cassava flour in a fibrous bed bioreactor

To enhance the butanol productivity and reduce the material cost, acetone, butanol, and ethanol fermentation by Clostridium acetobutylicum SE25 was investigated using batch, repeated-batch and continuous cultures in a fibrous bed bioreactor, where cassava flour was used as the substrate. With periodical nutrient supplementation, stable butanol production was maintained for about 360h in a 6-cycle repeated-batch fermentation with an average butanol productivity of 0.28g/L/h and butanol yield of 0.32g/g-starch. In addition, the highest butanol productivity of 0.63g/L/h and butanol yield of 0.36g/g-starch were achieved when the dilution rate were investigated in continuous production of acetone, butanol, and ethanol using a fibrous bed bioreactor, which were 231.6% and 28.6% higher than those of the free-cell fermentation. On the other hand, this study also successfully comfirmed that the biofilm can provide an effective protection for the microbial cells which are growing in stressful environment.

Butanol production by Clostridium acetobutylicum in a series of packed bed biofilm reactors

The continuous production of Acetone, n-Butanol and Ethanol (ABE) by immobilized cells of Clostridium acetobutylicum DSM 792 using glucose and lactose as carbon source is presented in this paper. The conversion process was successfully carried out for more than three months in 4 packed bed biofilm reactors (PBBRs) connected in series. The first PBBR of the series (fed with fresh medium) was kept under acidogenesis conditions and the three other PBBRs were kept under solventogenesis conditions.Each PBBR was a glass tube (4cm ID, 8cm high) with a 4 cm-bed of 3 mm-Tygon rings as carriers. The PBBR system was fed with 100g/L of lactose medium. The fermentation process was characterized in terms of metabolite production (butyric and acetic acids, acetone, butanol, and ethanol), sugar conversion and mass of biofilm. The overall dilution rate (DTOT) was varied between 0.15h−1 and 0.9h−1 to assess the PBBR system performance as a function of DTOT. The best PBBR system performance under optimized conditions was: butanol productivity 9.2g/Lh, butanol concentration 10.8g/L, acetone concentration 2.4g/L, ethanol concentration 1.8g/L, selectivity of butanol with respect to all solvents 72%w. To the authors’ knowledge, these butanol productivity and concentration values are the highest in the literature on lactose/(cheese whey) fermentation.An interpretation of the biofilm structure in the PBBR was put forward.

Candida shehatae and Saccharomyces cerevisiae work synergistically to improve ethanol fermentation from sugarcane bagasse and rice straw hydrolysate in immobilized cell bioreactor

Sugarcane bagasse (SCB) and rice straw (RS), abundant lignocellulosic agro‐industrial residues in South‐East Asia, are potent feedstocks for bioethanol production as they contain significant amount of glucose and xylose monomers after fractionation and subsequent enzymatic hydrolysis. To simultaneously convert glucose and xylose to ethanol, it requires co‐cultivation of Saccharomyces cerevisiae and Candida shehatae which are hexose and pentose‐fermenting yeasts, respectively. Xylose‐fermenting strain grows slower than glucose‐fermenting one, therefore low efficiency of xylose‐to‐ethanol conversion was found. To enhance the efficiency of ethanol fermentation, the present work proposed to improve xylose assimilation by using co‐immobilization of two strains in a packed bed bioreactor and to increase oxygenation of the medium by applying a recycled batch system when the recycle stream was intervened by a mixing system in a naturally aerated vessel. Initially, conversion of glucose and xylose to ethanol using pure culture was investigated. Subsequently, influence of different immobilization techniques was investigated. Cells entrapment in Ca‐alginate beads provided considerably high ethanol yield over cells immobilized on delignified cellulose, and thus it was selected to use as inoculum in an immobilized cell bioreactor (ICB). The results showed that continuous ethanol production yielded 0.38 and 0.40 g/g corresponding to 74.5% and 78.4% theoretical yields from SCB and RS hydrolysate, respectively. However, recycled batch system produced significantly improved ethanol yield to 0.49 g/g and 0.50 g/g corresponding to 96.1% and 98.0% theoretical yields for SCB and RS hydrolysate, respectively. In this study, higher ethanol concentration and less unfermented sugar concentration was successfully achieved in the ICB with recycled batch system when using SCB and RS hydrolysate as the substrate.

Lignocellulosic ethanol production employing immobilized Saccharomyces cerevisiae in packed bed reactor

Most of ethanol production processes are limited by lower ethanol production rate and recyclability problem of ethanologenic organism. In the present study, immobilized co-fermenting Saccharomyces cerevisiae GSE1618 was employed for ethanol fermentation using rice straw enzymatic hydrolysate in a packed bed reactor (PBR). The immobilization of S. cerevisiae was performed by entrapment in Ca-alginate for optimization of ethanol production by varying alginic acid concentration, bead size, glucose concentration, temperature and hardening time. Remarkably, extra hardened beads (EHB) immobilized with S. cerevisiae could be used up to repeated 40 fermentation batches. In continuous PBR, maximum 81.82 g L−1 ethanol was obtained with 29.95 g L−1 h−1 productivity with initial glucose concentration of 180 g L−1 in feed at dilution rate of 0.37 h−1. However, maximum ethanol concentration of 40.33 g L−1 (99% yield) with 24.61 g L−1 h−1 productivity was attained at 0.61 h−1 dilution rate in fermentation of un-detoxified rice straw enzymatic hydrolysate (REH). At commercial scale, EHB has great potential for continuous ethanol production with high productivity using lignocellulosic hydrolysate in PBR.

Improved operating strategy for continuous fermentation of carbon monoxide to fuel-ethanol by clostridia

Ethanol production from C1 compounds, such as carbon monoxide, using acetogenic bacteria is an attractive alternative to produce renewable fuels. However, the process is generally associated with acetic acid accumulation, which is often produced in larger quantities than ethanol itself. This study shows the continuous production of ethanol and complete conversion of produced acetic acid to ethanol, which is possible through pH shifts from high pH to low pH using an optimized medium and one single gas-fed bioreactor. A first study with a single pH shift resulted in the production of 2408mg/L of ethanol without any accumulation of acetic acid. In a next study a final ethanol concentration of 4260mg/L was reached with just one pH shift and one partial medium renewal. These results indicate that maintaining an optimal growth pH at the beginning of the process in order to obtain a high cell mass, and later shifting the pH to a low value will improve ethanol productivity and avoid any acetic acid accumulation.