Optimization of NaOH-catalyzed steam pretreatment of empty fruit bunch

Background

Empty fruit bunch (EFB) has many advantages, including its abundance, the fact that it does not require collection, and its year-round availability as a feedstock for bioethanol production. But before the significant costs incurred in ethanol production from lignocellulosic biomass can be reduced, an efficient sugar fractionation technology has to be developed. To that end, in the present study, an NaOH-catalyzed steam pretreatment process was applied in order to produce ethanol from EFB more efficiently.

Results

The EFB pretreatment conditions were optimized by application of certain pretreatment variables such as, the NaOH concentrations in the soaking step and, in the steam step, the temperature and time. The optimal conditions were determined by response surface methodology (RSM) to be 3% NaOH for soaking and 160°C, 11 min 20 sec for steam pretreatment. Under these conditions, the overall glucan recovery and enzymatic digestibility were both high: the glucan and xylan yields were 93% and 78%, respectively, and the enzymatic digestibility was 88.8% for 72 h using 40 FPU/g glucan. After simultaneous saccharification and fermentation (SSF), the maximum ethanol yield and concentration were 0.88 and 29.4 g/l respectively.

Conclusions

Delignification (>85%) of EFB was an important factor in enzymatic hydrolysis using CTec2. NaOH-catalyzed steam pretreatment, which can remove lignin efficiently and requires only a short reaction time, was proven to be an effective pretreatment technology for EFB. The ethanol yield obtained by SSF, the key parameter determining the economics of ethanol, was 18% (w/w), equivalent to 88% of the theoretical maximum yield, which is a better result than have been reported in the relevant previous studies.

     

Bioethanol production from brown seaweed, Undaria pinnatifida, using NaCl acclimated yeast

Strain improvement of Pichia angophorae KCTC 17574 was successfully carried out for bioethanol fermentation of seaweed slurry with high salt concentration. P. angophorae KCTC 17574 was cultured under increasing salinity from five practical salinity unit (psu, ‰) to as high as 100 psu for 723 h. The seaweed, Undaria pinnatifida (sea mustard, Miyuk), was fermented to produce bioethanol using high-salt acclimated yeast. The pretreatment of U. pinnatifida was optimized using thermal acid hydrolysis to obtain a high monosaccharide yield. Optimal pretreatment conditions of 75 mM H₂SO₄ and 13 % (w/v) slurry at 121 °C for 60 min were determined using response surface methodology. A maximum monosaccharide content of 28.65 g/L and the viscosity of 33.19 cP were obtained. The yeasts cultured under various salinity concentrations were collected and inoculated to the pretreated seaweed slurry after the neutralization using 5 N NaOH. The pretreated slurry was fermented with the inoculation of 0.1 g dcw/L of P. angophorae KCTC 17574 strain obtained at 90 psu. The maximum ethanol concentration of 9.42 g/L with 27 % yield of theoretical case of ethanol production from total carbohydrate of U. pinnatifida was obtained.     

Immobilized anaerobic fermentation for bio-fuel production by Clostridium co-culture

Clostridium thermocellum/Clostridium thermolacticum co-culture fermentation has been shown to be a promising way of producing ethanol from several carbohydrates. In this research, immobilization techniques using sodium alginate and alkali pretreatment were successfully applied on this co-culture to improve the bio-ethanol fermentation performance during consolidated bio-processing (CBP). The ethanol yield obtained increased by over 60 % (as a percentage of the theoretical maximum) as compared to free cell fermentation. For cellobiose under optimized conditions, the ethanol yields were approaching about 85 % of the theoretical efficiency. To examine the feasibility of this immobilization co-culture on lignocellulosic biomass conversion, untreated and pretreated aspen biomasses were also used for fermentation experiments. The immobilized co-culture shows clear benefits in bio-ethanol production in the CBP process using pretreated aspen. With a 3-h, 9 % NaOH pretreatment, the aspen powder fermentation yields approached 78 % of the maximum theoretical efficiency, which is almost twice the yield of the untreated aspen fermentation.     

稀碱预处理棕榈残渣制备纤维乙醇

以棕榈残渣(Empty fruit bunch,EFB)为原料,通过预处理、酶解、发酵等过程制备纤维乙醇.首先对比了碱、碱/过氧化氢等预处理条件对棕榈残渣组成及酶解的影响,结果表明稀碱预处理效果较好.适宜的稀碱预处理条件为:NaOH浓度为1％,固液比为1∶10,在40℃浸泡24 h后于121℃下保温30 min,在该条件下,EFB的固体回收率为74.09％,纤维素、半纤维素和木质素的含量分别为44.08％、25.74％和13.89％.对该条件下预处理后的固体样品,以底物浓度5％、酶载量30 FPU/g底物酶解72 h,纤维素和半纤维素的酶解率分别达到84.44％和89.28％.进一步考察了酶载量和底物浓度对酶解的影响以及乙醇批式同步糖化发酵,当酶载量为30 FPU/g底物,底物浓度由5％增加至25％时,利用酿酒酵母Saccharomyces cerevisiae(接种量为5％,VIV)发酵72 h后乙醇的浓度分别为9.76 g/L和35.25 g/L,可分别达到理论得率的79.09％和56.96％.     

Aqueous ammonia pretreatment of oil palm empty fruit bunches for ethanol production

Oil palm empty fruit bunches (EFB) were pretreated by aqueous ammonia soaking for ethanol production. Pretreated EFB, which were pretreated at the optimal conditions of 60 °C, 12 h, and 21% (w/w) aqueous ammonia, showed 19.5% and 41.4% glucose yields during an enzymatic digestibility test for 96 h when using 15 and 60 FPU of cellulase, respectively. Using the pretreated EFB, simultaneous saccharification and fermentation for 168 h with 5% (w/v) glucan loading and 60 FPU of cellulase and 30 CBU of β-glucosidase per gram glucan resulted in ethanol production of 18.6 g/L titer, 65.6% of theoretical maximum yield, and 0.11 g/L/h of productivity.     

Characterization of ethanol fermentation waste and its application to lactic acid production by Lactobacillus paracasei

In this study, an ethanol fermentation waste (EFW) was characterized for use as an alternative to yeast extract for bulk fermentation processes. EFW generated from a commercial plant in which ethanol is produced from cassava/rice/wheat/barley starch mixtures using Saccharomyces cerevisiae was used for lactic acid production by Lactobacillus paracasei. The effects of temperature, pH, and duration on the autolysis of an ethanol fermentation broth (EFB) were also investigated. The distilled EFW (DEFW) contained significant amounts of soluble proteins (2.91 g/l), nitrogen (0.47 g/l), and amino acids (24.1 mg/l). The autolysis of the EFB under optimum conditions released twice as much amino acids than in the DEFW. Batch fermentation in the DEFW increased the final lactic acid concentration, overall lactic acid productivity, and lactic acid yield on glucose by 17, 41, and 14 %, respectively, in comparison with those from comparable fermentation in a lactobacillus growth medium (LGM) that contained 2 g/l yeast extract. Furthermore, the overall lactic acid productivity in the autolyzed then distilled EFW (ADEFW) was 80 and 27 % higher than in the LGM and DEFW, respectively.     

Comparisons of five Saccharomyces cerevisiae strains for ethanol production from SPORL‐pretreated lodgepole pine

The performances of five yeast strains under three levels of toxicity were evaluated using hydrolysates from lodgepole pine pretreated by Sulfite Pretreatment to Overcome the Recalcitrance of Lignocelluloses (SPORL). The highest level of toxicity was represented by the whole pretreated biomass slurry, while intermediate toxicity was represented by the hydrolysate with partial loading of pretreatment spent liquor. The zero toxicity was represented using the enzymatic hydrolysate produced from thoroughly washed SPORL lodgepole pine solids. The results indicate that strains D5A and YRH400 can tolerate the whole pretreated biomass slurry to produce 90.1 and 73.5% theoretical ethanol yield. Strains Y1528, YRH403, and FPL450 did not grow in whole hydrolysate cultures and were observed to have lower ethanol productivities than D5A and YRH400 on the hydrolysate with intermediate toxicity. Both YRH400 and YRH403 were genetically engineered for xylose fermentation but were not able to consume xylose efficiently in hydrolysate. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1076–1083, 2014     

Sequential Thermochemical Hydrolysis of Corncobs and Enzymatic Saccharification of the Whole Slurry Followed by Fermentation of Solubilized Sugars to Ethanol with the Ethanologenic Strain <Emphasis Type="Italic">Escherichia coli</Emphasis> MS04

Interest in the use of corncobs as feedstock for bioethanol production is growing. This study assesses the feasibility of sequential thermochemical diluted sulfuric acid pretreatment of corncobs at moderate temperature to hydrolyze the hemicellulosic fraction, followed by enzymatic hydrolysis of the whole slurry, and fermentation of the obtained syrup. The total sugar concentration after enzymatic hydrolysis was 85.21 g/l, i.e., 86 % of the sugars were liberated from the polymeric fractions, together with a low amount of furfural (0.26 g/l) and 4.01 g/l of acetic acid. The syrups, which contained 36.3, 40.9, 4.47, and 1.84 g/l of xylose, glucose, arabinose, and mannose, respectively, were fermented (pH 7, 37 °C, 150 rpm) to ethanol with the metabolically engineered acetate-tolerant Escherichia coli strain MS04 under non-aerated conditions, producing 35 g/l of ethanol in 18 h (1.94 g_EtOH/l/h), i.e., a conversion yield greater than 80 % of the theoretical value based on total sugars was obtained. Hence, using the procedures developed in this study, 288 l of ethanol can be produced per metric ton of dry corncobs. Strain MS04 can ferment sugars in the presence of acetate, and the amount of furans generated during the sequential thermochemical and enzymatic hydrolysis was low; hence, the detoxification step was avoided. The residual salts, acetic acid, and solubilized lignin present in the syrup did not interfere with the production of ethanol by E. coli MS04 and the results show that this strain can metabolize mixtures of glucose and xylose simultaneously.     

Removal of enzymatic and fermentation inhibitory compounds from biomass slurries for enhanced biorefinery process efficiencies

Within the biorefinery paradigm, many non-monomeric sugar compounds have been shown to be inhibitory to enzymes and microbial organisms that are used for hydrolysis and fermentation. Here, two novel separation technologies, polyelectrolyte polymer adsorption and resin-wafer electrodeionization (RW-EDI), have been evaluated to detoxify a dilute acid pretreated biomass slurry. Results showed that detoxification of a dilute acid pretreated ponderosa pine slurry by sequential polyelectrolyte and RW-EDI treatments was very promising, with significant removal of acetic acid, 5-hydroxymethyl furfural, and furfural (up to 77%, 60%, and 74% removed, respectively) along with >97% removal of sulfuric acid. Removal of these compounds increased the cellulose conversion to 94% and elevated the hydrolysis rate to 0.69 g glucose/L/h. When using Saccharomyces cerevisiae D₅A for fermentation of detoxified slurry, the process achieved 99% of the maximum theoretical ethanol yield and an ethanol production rate nearly five-times faster than untreated slurry.     

Conversion of acid hydrolysate of oil palm empty fruit bunch to L-lactic acid by newly isolated Bacillus coagulans JI12

Cost-effective conversion of lignocellulose hydrolysate to optically pure lactic acid is commercially attractive but very challenging. Bacillus coagulans JI12 was isolated from natural environment and used to produce L-lactic acid (optical purity?>?99.5 %) from lignocellulose sugars and acid hydrolysate of oil palm empty fruit bunch (EFB) at 50 °C and pH 6.0 without sterilization of the medium. In fed-batch fermentation with 85 g/L initial xylose and 55 g/L xylose added after 7.5 h, 137.5 g/L lactic acid was produced with a yield of 98 % and a productivity of 4.4 g/L?h. In batch fermentation of a sugar mixture containing 8.5 % xylose, 1 % glucose, and 1 % L-arabinose, the lactic acid yield and productivity reached 98 % and 4.8 g/L?h, respectively. When EFB hydrolysate was used, 59.2 g/L of lactic acid was produced within 9.5 h at a yield of 97 % and a productivity of 6.2 g/L?h, which are the highest among those ever reported from lignocellulose hydrolysates. These results indicate that B. coagulans JI12 is a promising strain for industrial production of L-lactic acid from lignocellulose hydrolysate.     

Alkaline-sulfite pretreatment and use of surfactants during enzymatic hydrolysis to enhance ethanol production from sugarcane bagasse

Sugarcane bagasse is a by-product from the sugar and ethanol industry which contains approximately 70 % of its dry mass composed by polysaccharides. To convert these polysaccharides into fuel ethanol it is necessary a pretreatment step to increase the enzymatic digestibility of the recalcitrant raw material. In this work, sugarcane bagasse was pretreated by an alkaline-sulfite chemithermomechanical process for increasing its enzymatic digestibility. Na₂SO₃ and NaOH ratios were fixed at 2:1, and three increasing chemical loads, varying from 4 to 8 % m/m Na₂SO₃, were used to prepare the pretreated materials. The increase in the alkaline-sulfite load decreased the lignin content in the pretreated material up to 35.5 % at the highest chemical load. The pretreated samples presented enhanced glucose yields during enzymatic hydrolysis as a function of the pretreatment severity. The maximum glucose yield (64 %) was observed for the samples pretreated with the highest chemical load. The use of 2.5 g l^?1 Tween 20 in the hydrolysis step further increased the glucose yield to 75 %. Semi-simultaneous hydrolysis and fermentation of the pretreated materials indicated that the ethanol yield was also enhanced as a function of the pretreatment severity. The maximum ethanol yield was 56 ± 2 % for the sample pretreated with the highest chemical load. For the sample pretreated with the lowest chemical load (2 % m/m NaOH and 4 % m/m Na₂SO₃), adding Tween 20 during the hydrolysis process increased the ethanol yield from 25 ± 3 to 39.5 ± 1 %.     

The effects of phosphoric acid pretreatment on conversion of cellulose to ethanol at 45°C using the thermotolerant yeast Kluyveromyces marxianus IMB3

Summary Here we report on the effects of phosphoric acid pretreated cellulose as a substrate for ethanol production by K. marxianus IMB3 using simultaneous saccharification and fermentation systems at 45°C. With untreated, milled filter paper as substrate the maximum amount of ethanol produced was 25% of the maximum theoretical yield. After pre-treatment with 100% phosphoric acid, the yield increased to 42% of the maximum theoretical yield. When untreated microcrystalline cellulose was used as the fermentation substrate, yields of ethanol as 45°C amounted to 16% of the maximum theoretical yield whereas pretreatment of the substrate with phosphoric acid resulted in an increase in ethanol production to 69% of the maximum theoretical yield. This suggests that pretreatment of substrate with phosphoric acid would contribute to a reduction in the amount of exogenous enzyme needed.     

A comparison between simultaneous saccharification and fermentation and separate hydrolysis and fermentation using steam-pretreated corn stover

Two different process configurations, simultaneous saccharification and fermentation (SSF) and separate hydrolysis and fermentation (SHF), were compared, at 8% water-insoluble solids (WIS), regarding ethanol production from steam-pretreated corn stover. The enzymatic loading in these experiments was 10 FPU/g WIS and the yeast concentration in SSF was 1 g/L (dry weight) of a Saccharomyces cerevisiae strain. When the whole slurry from the pretreatment stage was used as it was, diluted to 8% WIS with water and pH adjusted, SSF gave a 13% higher overall ethanol yield than SHF (72.4% versus 59.1% of the theoretical). The impact of the inhibitory compounds in the liquid fraction of the pretreated slurry was shown to affect SSF and SHF in different ways. The overall ethanol yield (based on the untreated raw material) decreased when SSF was run in absence on inhibitors compared to SSF with inhibitors present. On the contrary, the presence of inhibitors decreased the overall ethanol yield in the case of SHF. However, the SHF yield achieves in the absence of inhibitors was still lower than the SSF yield achieves with inhibitors present.     

Integrated process of starch ethanol and cellulosic lactic acid for ethanol and lactic acid production

The sequential production of bioethanol and lactic acid from starch materials and lignocellulosic materials was investigated as ethanol fermentation broth (EFB) can provide nutrients for lactic acid bacteria. A complete process was developed, and all major operations are discussed, including ethanol fermentation, broth treatment, lactic acid fermentation, and product separation. The effect of process parameters, including ethanol fermentation conditions, treatment methods, and the amount of EFB used in simultaneous saccharification and fermentation (SSF), is investigated. Under the selected process conditions, the integrated process without additional chemical consumption provides a 1.08 acid/alcohol ratio (the broth containing 22.4 g/L ethanol and 47.6 g/L lactic acid), which corresponds to a polysaccharide utilization ratio of 86.9 %. Starch ethanol can thus promote cellulosic lactic acid by providing important nutrients for lactic acid bacteria, and in turn, cellulosic lactic acid could promote starch ethanol by improving the profit of the ethanol production process. Two process alternatives for the integration of starch ethanol and cellulosic lactic acid are compared, and some suggestions are given regarding the reuse of yeast following the cellulosic SSF step for lactic acid production.     

Aqueous ammonia pretreatment, saccharification, and fermentation evaluation of oil palm fronds for ethanol production

Oil palm fronds are the most abundant lignocellulosic biomass in Malaysia. In this study, fronds were tested as the potential renewable biomass for ethanol production. The soaking in aqueous ammonia pretreatment was applied, and the fermentability of pretreated fronds was evaluated using simultaneous saccharification and fermentation. The optimal pretreatment conditions were 7?% (w/w) ammonia, 80?°C, 20?h of pretreatment, and 1:12 S/L ratio, where the enzymatic digestibility was 41.4?% with cellulase of 60?FPU/g-glucan. When increasing the cellulase loading in the hydrolysis of pretreated fronds, the enzymatic digestibility increased until the enzyme loading reached 60?FPU/g-glucan. With 3?% glucan loading in the SSF of pretreated fronds, the ethanol concentration and yield based on the theoretical maximum after 12 and 48?h of the SSF were 7.5 and 9.7?g/L and 43.8 and 56.8?%, respectively. The ethanol productivities found at 12 and 24?h from pretreated fronds were 0.62 and 0.36?g/L/h, respectively.     

Optimization of organic acid pretreatment of wheat straw

The objective of this study was to determine the effectiveness of different organic acids (maleic, succinic, and oxalic acid) on enzymatic hydrolysis and fermentation yields of wheat straw. It was also aimed to optimize the process conditions (temperature, acid concentration, and pretreatment time) by using response surface methodology (RSM). In line with this objective, the wheat straw samples were pretreated at three different temperatures (170, 190, and 210°C), acid concentrations (1%, 3%, and 5%) and pretreatment time (10, 20, and 30 min). The findings show that at extreme pretreatment conditions, xylose was solubilized in liquid phase, causing an increase in cellulose and lignin content of biomass. Enzymatic hydrolysis experiments revealed that maleic and oxalic acids were quite effective at achieving high sugar yields (>90%) from wheat straw. In contrast, the highest sugar yields were 50–60%, when the samples were pretreated with succinic acid, indicating that succinic acid was not as effective. The optimum process conditions for maleic acid were, 210°C, 1.08% acid concentration, and 19.8 min; for succinic acid 210°C, 5% acid concentration, and 30 min; for oxalic acid 210°C, 3.6% acid concentration, and 16.3 min. The ethanol yields obtained at optimum conditions were 80, 79, and 59% for maleic, oxalic and succinic acid, respectively. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1487–1493, 2016     

Impact of recycling stillage on conversion of dilute sulfuric acid pretreated corn stover to ethanol

Both the current corn starch to ethanol industry and the emerging lignocellulosic biofuels industry view recycling of spent fermentation broth or stillage as a method to reduce fresh water use. The objective of this study was to understand the impact of recycling stillage on conversion of corn stover to ethanol. Sugars in a dilute‐acid pretreated corn stover hydrolysate were fermented to ethanol by the glucose–xylose fermenting bacteria Zymomonas mobilis 8b. Three serial fermentations were performed at two different initial sugar concentrations using either 10% or 25% of the stillage as makeup water for the next fermentation in the series. Serial fermentations were performed to achieve near steady state concentration of inhibitors and other compounds in the corn stover hydrolysate. Little impact on ethanol yields was seen at sugar concentrations equivalent to pretreated corn stover slurry at 15% (w/w) with 10% recycle of the stillage. However, ethanol yields became progressively poorer as the sugar concentration increased and fraction of the stillage recycled increased. At an equivalent corn stover slurry concentration of 20% with 25% recycled stillage the ethanol yield was only 5%. For this microorganism with dilute‐acid pretreated corn stover, recycling a large fraction of the stillage had a significant negative impact on fermentation performance. Although this finding is of concern for biochemical‐based lignocellulose conversion processes, other microorganism/pretreatment technology combinations will likely perform differently. Biotechnol. Bioeng. 2010;105: 992–996. © 2009 Wiley Periodicals, Inc.     

Ethanol production from cotton-based waste textiles

Ethanol production from cotton linter and waste of blue jeans textiles was investigated. In the best case, alkali pretreatment followed by enzymatic hydrolysis resulted in almost complete conversion of the cotton and jeans to glucose, which was then fermented by Saccharomyces cerevisiae to ethanol. If no pretreatment applied, hydrolyses of the textiles by cellulase and beta-glucosidase for 24 h followed by simultaneous saccharification and fermentation (SSF) in 4 days, resulted in 0.140-0.145 g ethanol/g textiles, which was 25-26% of the corresponding theoretical yield. A pretreatment with concentrated phosphoric acid prior to the hydrolysis improved ethanol production from the textiles up to 66% of the theoretical yield. However, the best results obtained from alkali pretreatment of the materials by NaOH. The alkaline pretreatment of cotton fibers were carried out with 0-20% NaOH at 0 degrees C, 23 degrees C and 100 degrees C, followed by enzymatic hydrolysis up to 4 days. In general, higher concentration of NaOH resulted in a better yield of the hydrolysis, whereas temperature had a reverse effect and better results were obtained at lower temperature. The best conditions for the alkali pretreatment of the cotton were obtained in this study at 12% NaOH and 0 degrees C and 3 h. In this condition, the materials with 3% solid content were enzymatically hydrolyzed at 85.1% of the theoretical yield in 24 h and 99.1% in 4 days. The alkali pretreatment of the waste textiles at these conditions and subsequent SSF resulted in 0.48 g ethanol/g pretreated textiles used.     

Production of isoprene,one of the high-density fuel precursors,from peanut hull using the high-efficient lignin-removal pretreatment method

Background

Isoprene as the feedstock can be used to produce renewable energy fuels, providing an alternative to replace the rapidly depleting fossil fuels. However, traditional method for isoprene production could not meet the demands for low-energy consumption and environment-friendliness. Moreover, most of the previous studies focused on biofuel production out of lignocellulosic materials such as wood, rice straw, corn cob, while few studies concentrated on biofuel production using peanut hull (PH). As is known, China is the largest peanut producer in the globe with an extremely considerable amount of PH to be produced each year. Therefore, a novel, renewable, and environment-friendly pretreatment strategy to increase the enzymatic hydrolysis efficiency of cellulose and reduce the inhibitors generation was developed to convert PH into isoprene.

Results

The optimal pretreatment conditions were 100 °C, 60 min, 10% (w/v) solid loading with a 2:8 volume ratio of phosphoric acid and of hydrogen peroxide. In comparison with the raw PH, the hemicellulose and lignin were reduced to 85.0 and 98.0%, respectively. The cellulose–glucose conversion of pretreated PH reached up to 95.0% in contrast to that of the raw PH (19.1%). Only three kinds of inhibitors including formic acid, levulinic acid, and a little furfural were formed during the pretreatment process, whose concentrations were too low to inhibit the isoprene yield for Escherichia coli fermentation. Moreover, compared with the isoprene yield of pure glucose fermentation (298 ± 9 mg/L), 249 ± 6.7 and 294 ± 8.3 mg/L of isoprene were produced using the pretreated PH as the carbon source by the engineered strain via separate hydrolysis and fermentation and simultaneous saccharification and fermentation (SSF) methods, respectively. The isoprene production via SSF had a 9.8% glucose–isoprene conversion which was equivalent to 98.8% of isoprene production via the pure glucose fermentation.

Conclusions

The optimized phosphoric acid/hydrogen peroxide combination pretreatment approach was proved effective to remove lignin and hemicellulose from lignocellulosic materials. Meanwhile, the pretreated PH could be converted into isoprene efficiently in the engineered Escherichia coli. It is concluded that this novel strategy of isoprene production using lignocellulosic materials pretreated by phosphoric acid/hydrogen peroxide is a promising alternative to isoprene production using traditional way which can fully utilize non-renewable fossil sources.

     

Efficacy of acidic pretreatment for the saccharification and fermentation of alginate from brown macroalgae

This study was performed to evaluate the effectiveness of acidic pretreatment in increasing the enzymatic digestibility of alginate from brown macroalgae. Pretreatment with 1 % (w/v) sulfuric acid at 120 °C for 30 min produced oligosaccharides, mannuronic acid, and guluronic acid. Enzymatic saccharification of pretreated alginate by alginate lyases produced 52.2 % of the theoretical maximal sugar yield, which was only 7.5 % higher than the sugar yield obtained with unpretreated alginate. Mass spectrometric analyses of products of the two reactions revealed that acidic pretreatment and enzymatic saccharification produced saturated monomers (i.e., mannuronic and guluronic acid) with saturated oligosaccharides and unsaturated monomers (i.e., 4-deoxy-l-erythro-5-hexoseulose uronic acid; DEH), respectively. While DEH is further metabolized by microorganisms, mannuronic acid and guluronic acid are not metabolizable. Because of the poor efficacy in increasing enzymatic digestibility and owing to the formation of non-fermentable saturated monomers, acidic pretreatment cannot be recommended for enzymatic saccharification and fermentation of alginate.