Microalgal Hydrolysate Research Articles

Utilizing microalgal hydrolysate from dairy wastewater-grown Chlorella sorokiniana SU-1 as sustainable feedstock for polyhydroxybutyrate and β-carotene production by engineered Rhodotorula glutinis #100-29

The objective of this study was to explore the potential of utilizing Chlorella sorokiniana SU-1 biomass grown on dairy wastewater-amended medium as sustainable feedstock for the biosynthesis of β-carotene and polyhydroxybutyrate (PHB) by Rhodotorula glutinis #100-29. To break down the rigid cell wall, 100 g/L of microalgal biomass was treated with 3% sulfuric acid, followed by detoxification using 5% activated carbon to remove the hydroxymethylfurfural inhibitor. The detoxified microalgal hydrolysate (DMH) was used for flask-scale fermentation, which yielded a maximum biomass production of 9.22 g/L, with PHB and β-carotene concentration of 897 mg/L and 93.62 mg/L, respectively. Upon scaling up to a 5-L fermenter, the biomass concentration increased to 11.2 g/L, while the PHB and β-carotene concentrations rose to 1830 mg/L and 134.2 mg/L. These outcomes indicate that DMH holds promise as sustainable feedstock for the production of PHB and β-carotene by yeast.

Enzymatic hydrolysis of carbohydrate-rich Chlorella vulgaris for third-generation bioethanol production by cellulase-recombinant yeast

This study developed a multienzyme preparation for the hydrolysis of microalgal biomass. Possible synergism between enzymes was investigated using carbohydrate-rich Chlorella vulgaris biomass. Under optimum experimental conditions, α-amylase (Termamyl®) at 0.05 g−1 substrate and amyloglucosidase at 0.2 g−1 substrate achieved a maximum reducing sugars yield of 5.639 ± 0.32 g L−1 at a biomass loading of 10 g L−1. Saccharomyces cerevisiae JAYET cells modified with cellulase genes produced 8.015 g L−1 ethanol from unenriched whole microalgal hydrolysate. With the developed enzyme preparation, no pretreatment was necessary to convert the carbohydrate-rich fraction of microalgal biomass into third-generation bioethanol.

Improvement of methane production at alkaline and neutral pH from anaerobic co-digestion of microalgal biomass and cheese whey

Over the past decades, biofuels from microalgae have gained increasing attention. In the present work, methane production from mono-digestion of thermochemical microalgal hydrolysate and co-digestion with cheese whey was evaluated in an anaerobic sequencing batch reactor. The reactor was fed with 5 g total chemical oxygen demand (TCOD)/L and operated either at alkaline or neutral pH. The average methane content in the biogas was higher at alkaline pH (83 %) than at neutral pH (61 %), whereas the methane yield from co-digestion was similar at alkaline (233 N mL/g TCOD) and neutral pH (226 N mL/g TCOD), being 1.8-fold higher than the mono-digestion of the microalgal hydrolysate. After increasing stepwise the organic load up to 12 g TCOD/L, the methane production rate reached peaks around 1000 N mL/L-d, being between 200−800 mL/L-d in those stages with lower organic loads. The methane production from the particulate fraction of the microalgal hydrolysate and its co-digestion with cheese whey demonstrated that the particulate fraction was indeed used to produce methane, but co-digestion did not improve methane yield. The results showed that microalgal hydrolysate could be efficiently co-digested with a carbon-rich substrate enhancing methane production.

Effect of post-treatment process of microalgal hydrolysate on bioethanol production

Microalgae accumulate abundant lipids and are a promising source for biodiesel. However, carbohydrates account for 40% of microalgal biomass, an important consideration when using them for the economically feasible production of biodiesel. In this study, different acid hydrolysis and post-treatment processing of Chlorella sp. ABC-001 was performed, and the effect of these different hydrolysates on bioethanol yield by Saccharomyces cerevisiae KL17 was evaluated. For hydrolysis using H2SO4, the neutralization using Ca(OH)2 led to a higher yield (0.43 g ethanol/g sugars) than NaOH (0.27 g ethanol/g sugars). Application of electrodialysis to the H2SO4 + NaOH hydrolysate increased the yield to 0.35 g ethanol/g sugars, and K+ supplementation further enhanced the yield to 0.41 g ethanol/g sugars. Hydrolysis using HNO3 led to the generation of reactive species. Neutralization using only NaOH yielded 0.02 g ethanol/g sugars, and electrodialysis provided only a slight enhancement (0.06 g ethanol/g sugars). However, lowering the levels of reactive species further increased the yield to 0.25 g ethanol/g sugars, and K+ supplementation increased the yield to 0.35 g ethanol/g sugars. Overall, hydrolysis using H2SO4 + Ca(OH)2 provided the highest ethanol yield, and the yield was almost same as from conventional medium. This research emphasizes the importance of post-treatment processing that is modified for the species or strains used for bioethanol fermentation.

Biobutanol production from lignocellulosic biomass using immobilized Clostridium acetobutylicum

Biobutanol produced by acetone-biobutanol-ethanol (ABE) fermentation process has been revisited in the light of its use as “drop in” liquid biofuel to be blended with gasoline. In this study, renewable feedstock like rice straw, sugarcane bagasse and microalgal hydrolysate were used in ABE fermentation via separate hydrolysis and fermentation. Clostridium acetobutylicum ATCC 824 was used as the fermenting organism. Alkali pretreatment followed by enzymatic hydrolysis was used for rice straw and sugarcane bagasse. In batch fermentation, a biobutanol titer and yield of 9.10 g/L and 0.42 mol/mol glucose (0.17 g biobutanol/g glucose), respectively was obtained from rice straw, while sugarcane bagasse achieved a biobutanol titer and yield of 8.40 g/L and 0.40 mol/mol glucose (0.16 g biobutanol/g glucose), respectively. Higher microalgal biomass loading with 3% acid pretreatment severely inhibited fermentation performance. Unhydrolyzed microalgal biomass at a loading of 180 g/L in ABE fermentation resulted in 4.32 g/L biobutanol and 0.09 g biobutanol/g microalgae as yield. C. acetobutylicum was immobilized in polyvinyl alcohol (PVA) for improving the cell loading in fermentation and protect the cells from biobutanol toxicity. With rice straw hydrolysate as a feedstock and in the absence of yeast extract, a biobutanol titer, yield and productivity of 13.80 g/L, 0.90 g/L/h, and 0.58 mol biobutanol/mol glucose (0.23 g biobutanol/g glucose), respectively were obtained. Hence, rice straw is a potential feedstock for biobutanol production for fuel use.

Bioethanol production from acid pretreated microalgal hydrolysate using microwave-assisted heating wet torrefaction

This study focused on the bioethanol production from the co-production of solid biochar and liquid hydrolysate under microwave-assisted heating wet torrefaction towards a sustainable green technology. The two indigenous microalgal biomass undergone dilute acid pretreatment using wet torrefaction to produce microalgal hydrolysates and biochar at operating conditions of 160–170 °C with holding times of 5–10 min. The hydrolysates were utilized for fermentation with the yeast Saccharomyces cerevisiae at 29 °C in a dark condition at a non-agitation state for 120 h. The concentrations of total reducing sugar, reducing sugar by-product, and bioethanol in the hydrolysates were determined. The carbohydrate-rich microalga C. vulgaris ESP-31 showed a good performance in bioethanol production. Microalgal hydrolysate obtained after the pretreatment consisted of a total reducing sugar with the highest concentration of 98.11 g/L. The formation of by-product 5-hydroxymethyl-2-furaldehyde (5-HMF), which might act as the fermentation inhibitor that led to the low ethanol yield, was also analyzed. The highest ethanol yield achieved was 7.61% with a maximum experimental conversion probability of 95.22%. This study has demonstrated the feasible bioethanol production from microalgal hydrolysate through microwave-assisted heating wet torrefaction using dilute acids and the optimization of bioethanol production can be carried out for better performance in the future study.

Evaluation of Arthrospira sp. enzyme hydrolysate as dietary additive in gilthead seabream (Sparus aurata) juveniles

The aim of this work was to evaluate the effects of the dietary inclusion of Arthrospira sp. enzyme hydrolysate on gilthead seabream (Sparus aurata) juveniles in a 128-day feeding trial. Algal hydrolysate was tested at low inclusion level, namely, 2 and 4%, against a control diet without Arthrospira sp. supplementation. At the end of the feeding trial, fish body weight was recorded for growth evaluation. No significant differences were found among the experimental groups regarding growth performance or nutrient utilization, despite the fact that those animals fed with diets enriched with Arthrospira displayed higher trypsin, chymotrypsin, and leucine aminopeptidase enzyme activities, compared to fish fed with control diet. The ultrastructural study of the intestinal mucosa also revealed increased microvilli length and absorptive capacity in fish fed with Arthrospira sp. diets, especially at 4% inclusion level. Arthrospira supplementation was also responsible for lower lipid oxidation in muscle tissue, as well as for remarkable colour differences in skin, compared to control animals. These results suggest, overall, that low dietary supplementation with this microalgal hydrolysate might improve not only the intestinal ultrastructure and functionality but also muscle pigmentation and antioxidant capacity of juvenile gilthead seabream.

Exploring fermentation strategies for enhanced lactic acid production with polyvinyl alcohol-immobilized Lactobacillus plantarum 23 using microalgae as feedstock

Lactic acid (LA) fermentation was conducted with suspended and immobilized cells of an isolated Lactobacillus plantarum 23 strain using various fermentation strategies. Glucose and an alternative, relatively inexpensive carbon source - the hydrolysate of microalga Chlorella vulgaris ESP-31, were used as the carbon source. Batch fermentation using immobilized cells of L. plantarum 23 could enhance LA titer and yield by 43% and 39%, respectively, when compared with the suspended culture. Fed-batch culture integrated with in situ LA removal via ion exchange raised LA productivity by 72% by overcoming product inhibition. The highest LA productivity from glucose with PVA immobilized cells was 14.22 g/L/h, achieved under continuous operation at 50% w/v loading of immobilized beads and hydraulic retention time (HRT) of 2 h. PVA immobilized L. plantarum 23 could also use microalgal hydrolysate as the renewable carbon source, and the highest LA productivity was 9.93 g/L/h under continuous fermentation at 4 h HRT.

Improvement in energy recovery from Chlorella sp. biomass by integrated dark-photo biohydrogen production and dark fermentation-anaerobic digestion processes

Chlorella sp. biomass was used as the sole substrate for the production of hydrogen and methane through integrated dark fermentation (DF) and photo-fermentation (PF), and DF and anaerobic digestion (AD) processes. Prior to use in fermentations, the biomass was pretreated by acid-hydrothermal method, which yielded a maximum reducing sugar yield of 162.9 ± 4.2 mg g-biomass−1. The use of the microalgal hydrolysate to produce hydrogen by DF gave a hydrogen yield (HY) of 47.2 ± 1.1 mL g-volatile-solids−1 (VS). The subsequent use of the hydrogenic effluent in PF gave a HY of 125.0 ± 1.5 mL g-VS−1, while AD of the hydrogenic effluent gave a methane yield of 152.8 ± 1.3 mL g-VS−1. The total energy yield attained by the use of DF alone, the integrated DF-PF, and DF-AD processes were 0.51, 1.86 and 5.98 kJ g-VS−1, respectively. These results indicate that the integrated DF-AD process was effective in recovering energy from Chlorella sp. biomass. However, an energy balance analysis indicated that the process was not energetically feasible due to the high energy demand for the acid-hydrothermal pretreatment.

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).

Application of the enzymatic fuel cell system using waste microalgal hydrolysate

To optimize sorbitol production through solid state fermentation (SSF) process using response surface methodology (RSM), the central composite design (CCD) was employed in this study in order to minimize the number of experiments needed for the determination of the best combination of parameters for the process optimization. The raw material used in this study was Meranti wood sawdust (MWS) obtained from Gambang Sawmill (M) Sdn Bhd, Gambang Kuantan, Pahang. The type of bacteria used for the study was Lactobacillus plantarum (BAA 793; NCIMB 8826), purchased from the America Type Culture Collection (ATCC). The physical and chemical pretreatment methods were used in the study to recover cellulose from the MSW, followed by the enzymatic hydrolysis process to produce glucose. The last phase of this work was the fermentation process using SSF to convert the produced glucose to sorbitol; this is the section of the study where the parameters were optimized using the RSM. The result of the RSM studies showed that the interactions between moisture content and temperature had a very significant effect with an F value of <0.0001. The highest yield of sorbitol (29.0625 g/L) was obtained when using 50% of moisture content, at a temperature of 35 °C for 10 h of fermentation time. The RSM helped in the optimization of the effective parameters as well as to analyze the interaction between the parameters.

The potential of waste microalgal hydrolysate for power generation in enzymatic fuel cell

Abstract The purpose of this study is to generate power by using the waste hydrolysate from microalgal extracts (AH), substrates of enzymatic fuel cell (EFC), and analyze the effects of microalgal hydrolysate containing a neutralizing agent on EFC. The effect of different dilute acids on extraction of microalgae was also investigated. The maximum concentration (∼5 g/L) of extraction was achieved for Chlorella pyrenoiudosa under the optimum conditions (2% hydrochloric acid and 100 g/L biomass loading). In order to investigate the effects of various neutralization agents used for AH on the EFC system, CV and EIS were investigated. Higher current and lower resistance values appeared during the neutralization using KOH. Maximum power density of 3637 μW/cm2 was obtained for the EFC system, 6.59 folds higher than the control. The result implies that AH used as a substrate has a significant influence on the power generation in the EFC system.

A systematic study regarding hydrolysis and ethanol fermentation from microalgal biomass

Abstract In this study, acidic and enzymatic hydrolysis were carried out with Chlorella vulgaris and Scenedesmus obliquus, and a systematic evaluation of the yeast inoculum optimization (Saccharomyces cerevisiae and Pichia stipitis) during the hydrolysates fermentation was made. Acidic hydrolysis with 3% of sulphuric acid at 121 °C and 30 min of reaction time (50–100 g/L of biomass) was found as best condition, and more than 90% of sugars recovery in the liquid phase was achieved even though S. obliquus be more sensible to thermal degradation than C. vulgaris. On the other hand, during the enzymatic hydrolysis, ultrasonication showed to be an effective method of biomass pretreatment to provide higher enzyme accessibility what together with a combination between amylases, cellulase/hemicellulase mix and pectinases achieved more than 90% of sugars recovery in 8 h of hydrolysis time. Thus, both treatments could recover efficiently the sugars present in microalgae. After inoculum optimization (concentration and consortium between S. cerevisiae and P. stipitis) and saline influence on yeast performance were evaluated, the fermentation of microalgal hydrolysate exhibited very different profiles in comparison with the control conditions and ethanol biochemical yield changed between 45% and 61%, emphasizing the importance to optimize this last but not less important step of ethanol production.

Production of 2,3‐butanediol by Klebsiella oxytoca from various sugars in microalgal hydrolysate

A new fermentation process using a mixed sugar medium is proposed in this study for 2,3-butanediol (2,3-BDO) production. The medium contained seven different monosugars known to be present in Nannochloropsis oceanica hydrolysate. The performance of each sugar when existing alone or together with glucose was evaluated. All the sugars except fucose were successfully metabolized for 2,3-BDO production. A 2,3-BDO yield of 0.31g/g was achieved with the mixed sugar medium, which was very close to that with the glucose-only medium. However, the 2,3-BDO productivity (0.28 g L(-1) h(-1) ) was found to be about 30% lower than that with glucose, implying, as expected, the existence of glucose repression on the uptake of other sugars. Strain development is in need to remove such negative effect of glucose for improved process efficiency. Fucose with the lowest uptake rate and no contribution to 2,3-BDO production can be a high value-added byproduct, once recovered and purified.

Production of DagA and ethanol by sequential utilization of sugars in a mixed-sugar medium simulating microalgal hydrolysate

A novel two-step fermentation process using a mixed-sugar medium mimicking microalgal hydrolysate has been proposed to avoid glucose repression and thus to maximize substrate utilization efficiency. When DagA, a β-agarase was produced in one step in the mixed-sugar medium by using a recombinant Streptomyces lividans, glucose was found to have negative effects on the consumption of the other sugars and DagA biosynthesis causing low substrate utilization efficiency and low DagA productivity. To overcome such difficulties, a new strategy of sequential substrate utilization was developed. In the first step, glucose was consumed by Saccharomyces cerevisiae together with galactose and mannose producing ethanol, after which DagA was produced from the remaining sugars of xylose, rhamnose and ribose. Fucose was not consumed. By adopting this two-step process, the overall substrate utilization efficiency was increased approximately 3-fold with a nearly 2-fold improvement of DagA production, let alone the additional benefit of ethanol production.

Bioethanol production from the nutrient stress-induced microalga Chlorella vulgaris by enzymatic hydrolysis and immobilized yeast fermentation

The microalga Chlorella vulgaris is a potential feedstock for bioenergy due to its rapid growth, carbon dioxide fixation efficiency, and high accumulation of lipids and carbohydrates. In particular, the carbohydrates in microalgae make them a candidate for bioethanol feedstock. In this study, nutrient stress cultivation was employed to enhance the carbohydrate content of C. vulgaris. Nitrogen limitation increased the carbohydrate content to 22.4% from the normal content of 16.0% on dry weight basis. In addition, several pretreatment methods and enzymes were investigated to increase saccharification yields. Bead-beating pretreatment increased hydrolysis by 25% compared with the processes lacking pretreatment. In the enzymatic hydrolysis process, the pectinase enzyme group was superior for releasing fermentable sugars from carbohydrates in microalgae. In particular, pectinase from Aspergillus aculeatus displayed a 79% saccharification yield after 72h at 50°C. Using continuous immobilized yeast fermentation, microalgal hydrolysate was converted into ethanol at a yield of 89%.

Fermentative hydrogen production by Clostridium butyricum CGS5 using carbohydrate-rich microalgal biomass as feedstock

In this work, a carbohydrate-rich microalga, Chlorella vulgaris ESP6, was grown photoautotrophically to fix the CO2. The resulting microalgal biomass was hydrolyzed by acid or alkaline/enzymatic treatment and was then used for biohydrogen production with Clostridium butyricum CGS5. The C. vulgaris biomass could be effectively hydrolyzed by acid pretreatment while similar hydrolysis efficiency was achieved by combination of alkaline pretreatment and enzymatic hydrolysis. The biomass of C. vulgaris ESP6 containing a carbohydrate content of 57% (dry weight basis) was efficiently hydrolyzed by acid treatment with 1.5% HCl, giving a reducing sugars (RS) yield of nearly 100%. C. butyricum CGS5 could utilize RS from C. vulgaris ESP6 biomass to produce hydrogen without any additional organic carbon sources. The optimal conditions for hydrogen production were 37 °C and a microalgal hydrolysate loading of 9 g RS/L with pH-controlled at 5.5. Under the optimal conditions, the cumulative H2 production, H2 production rate, and H2 yield were 1476 ml/L, 246 ml/L/h, and 1.15 mol/mol RS, respectively. The results demonstrate that the C. vulgaris biomass has the potential to serve as effective feedstock for dark fermentative H2 production.