Optimal production of 4-deoxy-l-erythro-5-hexoseulose uronic acid from alginate for brown macro algae saccharification by combining endo- and exo-type alginate lyases

Algae are considered as third-generation biomass, and alginate is the main component of brown macroalgae. Alginate can be enzymatically depolymerized by alginate lyases into uronate monomers, such as mannuronic acid and guluronic acid, which are further nonenzymatically converted to 4-deoxy-l-erythro-5-hexoseulose uronic acid (DEH). We have optimized an enzymatic saccharification process using two recombinant alginate lyases, endo-type Alg7D and exo-type Alg17C, for the efficient production of DEH from alginate. When comparing the sequential and simultaneous additions of Alg7D and Alg17C, it was found that the final yield of DEH was significantly higher when the enzymes were added sequentially. The progress of saccharification reactions and production of DEH were verified by thin layer chromatography and gas chromatography–mass spectrometry, respectively. Our results showed that the two recombinant enzymes could be exploited for the efficient production of DEH that is the key substrate for producing biofuels from brown macro algal biomass.     

Production of bio-fuel ethanol from distilled grain waste eluted from Chinese spirit making process

Distilled grain waste eluted from Chinese spirit making is rich in carbohydrates, and could potentially serve as feedstock for the production of bio-fuel ethanol. Our study evaluated two types of saccharification methods that convert distilled grain waste to monosaccharides: enzymatic saccharification and concentrated H₂SO₄ saccharification. Results showed that enzymatic saccharification performed unsatisfactorily because of inefficient removal of lignin during pretreatment. Concentrated H₂SO₄ saccharification led to a total sugar recovery efficiency of 79.0 %, and to considerably higher sugar concentrations than enzymatic saccharification. The process of ethanol production from distilled grain waste based on concentrated H₂SO₄ saccharification was then studied. The process mainly consisted of concentrated H₂SO₄ saccharification, solid–liquid separation, decoloration, sugar–acid separation, oligosaccharide hydrolysis, and continuous ethanol fermentation. An improved simulated moving bed system was employed to separate sugars from acid after concentrated H₂SO₄ saccharification, by which 95.8 % of glucose and 85.8 % of xylose went into the sugar-rich fraction, while 83.3 % of H₂SO₄ went into the acid-rich fraction. A flocculating yeast strain, Saccharomyces cerevisiae KF-7, was used for continuous ethanol fermentation, which produced an ethanol yield of 91.9–98.9 %, based on glucose concentration.     

Whole slurry fermentation of maleic acid-pretreated oil palm empty fruit bunches for ethanol production not necessitating a detoxification process

The yield of ethanol from oil palm empty fruit bunches (EFB) was increased on exploiting maleic acid pretreatment combined with fermentation of the pretreated whole slurry. The optimized conditions for pretreatment were to expose EFB to a high temperature (190 °C) with 1 % (w/v) maleic acid for a short time duration (3 min ramping to the set temperature with no holding) in a microwave digester. An enzymatic digestibility of 60.9 % (based on theoretical glucose yield) was exhibited using pretreated and washed EFB after 48 h of hydrolysis. Simultaneous saccharification and fermentation (SSF) of the whole slurry of pretreated EFB for 48 h resulted in 61.3 % theoretical yield of ethanol based on the initial amount of glucan in untreated EFB. These results indicate that maleic acid is a suitable catalyst not requiring detoxification steps for whole slurry fermentation of EFB for ethanol production, thus improving the process economics. Also, the whole slurry fermentation can significantly increase the biomass utilization by converting sugar from both solid and liquid phases of the pretreated slurry.     

Comparisons of SPORL and dilute acid pretreatments for sugar and ethanol productions from aspen

This study reports comparative evaluations of sugar and ethanol production from a native aspen (Populus tremuloides) between sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) and dilute acid (DA) pretreatments. All aqueous pretreatments were carried out in a laboratory wood pulping digester using wood chips at 170°C with a liquid to oven dry (od) wood ratio (L/W) of 3:1 at two levels of acid charge on wood of 0.56 and 1.11%. Sodium bisulfite charge on od wood was 0 for DA and 1.5 or 3.0% for SPORL. All substrates produced by both pretreatments (except DA with pretreatment duration of 0) had good enzymatic digestibility of over 80%. However, SPORL produced higher enzymatic digestibility than its corresponding DA pretreatment for all the experiments conducted. As a result, SPORL produced higher ethanol yield from simultaneous saccharification and fermentation of cellulosic substrate than its corresponding DA pretreatment. SPORL was more effective than its corresponding DA pretreatment in reducing energy consumption for postpretreatment wood chip size-reduction. SPORL, with lower energy input and higher sugar and ethanol yield, produced higher sugar and ethanol production energy efficiencies than the corresponding DA pretreatment.     

Comparative efficiency of different pretreatment methods on enzymatic digestibility of Parthenium sp.

The potential of Parthenium sp. as a feedstock for enzymatic saccharification was investigated by using chemical and biological pretreatment methods. Mainly chemical pretreatments (acid and alkali) were compared with biological pretreatment with lignolytic fungi Marasmiellus palmivorus PK-27. Structural and chemical changes as well as crystallinity of cellulose were examined through scanning electron microscopy, fourier transform infra red and X-ray diffraction analysis, respectively after pretreatment. Total reducing sugar released during enzymatic saccharification of pretreated substrates was also evaluated. Among the pretreatment methods, alkali (1 % NaOH) treated substrate showed high recovery of acid perceptible polymerised lignin (7.53 ± 0.5 mg/g) and significantly higher amount of reducing sugar (513.1 ± 41.0 mg/gds) compared to uninoculated Parthenium (163.4 ± 21.2) after 48 h of hydrolysis. This is the first report of lignolytic enzyme production from M. palmivorus, prevalent in oil palm plantations in Malaysia and its application in biological delignification of Parthenium sp. Alkali (1 % NaOH) treatment proves to be the suitable method of pretreatment for lignin recovery and enhanced yield of reducing sugar which may be used for bioethanol production from Parthenium sp.     

Optimization of Dilute Acid Pretreatment and Enzymatic Hydrolysis of <Emphasis Type="Italic">Phalaris aquatica</Emphasis> L. Lignocellulosic Biomass in Batch and Fed-Batch Processes

Phalaris aquatica L., a rich in holocellulose (69.80 %) and deficient in lignin (6.70 %) herbaceous, perennial grass species, was utilized in a two-step (biomass pretreatment-enzymatic hydrolysis) saccharification process for sugars recovery. The Taguchi methodology was employed to determine the dilute acid pretreatment and enzymatic hydrolysis conditions that optimized hemicellulose conversion (75.04 %), minimized the production of inhibitory compounds (1.41 g/L), and maximized the cellulose to glucose yield (69.69 %) of mixed particulate biomass (particles <1000 μm) under batch conditions. The effect of biomass particle size on saccharification process efficiency was also investigated. It was found that small-size biomass particles (53–106 μm) resulted in maximum hemicellulose conversion (81.12 %) and cellulose to glucose yield (93.24 %). The determined optimal conditions were then applied to a combined batch pretreatment process followed by a fed-batch enzymatic hydrolysis process that maximized glucose concentration (62.24 g/L) and yield (92.48 %). The overall efficiency of the saccharification process was 88.13 %.     

Comparing the Recalcitrance of Eucalyptus, Pine, and Switchgrass Using Ionic Liquid and Dilute Acid Pretreatments

Pine, eucalyptus, and switchgrass were evaluated for the production of fermentable sugars via ionic liquid and dilute acid pretreatments and subsequent enzymatic hydrolysis. The results show that among the three feedstocks, switchgrass has the highest sugar yields and faster hydrolysis rates for both pretreatment technologies by achieving 48 % (dilute acid) and 96 % (ionic liquid) sugar yields after 24 h. Of the two wood species, eucalyptus has a higher and faster sugar recovery after ionic liquid pretreatment than pine (93 vs. 62 % in 24 h) under 160 °C for 3 h with [C₂mim][OAc]. Pretreatment of pine and eucalyptus is observed to be ineffective under 1.2 % dilute acid condition and 160 °C for 15 min, indicating that further enhancement of reaction temperature or acid concentration is necessary to increase the digestibility of pretreated materials. Raman spectroscopy data show that the extent of lignin depolymerization that occurs during pretreatment also varies for the three different feedstocks. Under similar hemicellulose removal conditions, lignin removal in ionic liquid pretreatment can help improve cellulose conversion. This finding may help explain the observed variation in the saccharification yields and kinetics. These results indicate that ionic liquid pretreatment not only improved saccharification over dilute acid for all three feedstocks but also better dealt with the differences among them, suggesting better tolerance to feedstock variability.     

Structural Studies of Alginic acid,using a bacterial poly-α-L-guluronate lyase

An extracellular poly-α-L-guluronate lyase from Klebsiella aerogenes degrades those blocks from alginate which contain both mannuronic and guluronic acid residues (poly-MG blocks) to a mixture of oligosaccharides. From an analysis of these products, it is concluded that poly-MG blocks do not have a strictly alternating sequence of the two uronic acid residues. Enzymic degradation of various samples of algal alginate to leave the poly-M blocks intact has shown that these blocks have a uniform chain-length, estimated at 24 residues.     

Isolation of Marine Bacteria Capable of Producing Specific Lyases for Alginate Degradation

Alginate lyases (EC 4.2.2.3) were isolated from cultures of several marine bacterial isolates. The lyases were induced by native alginate and had activity toward both the mannuronic acid and the guluronic acid blocks of the alginate polymer. The guluronic acid-specific lyase was recovered from the medium, whereas the mannuronic acid-specific lyase was retained with the bacteria.     

Variations in the chemical composition of Costaria costata during harvest

In this study, variations in the chemical composition of Costaria costata collected during 3 months of the harvest period were analyzed. Moisture (4.94–10.50 %), ash (29.25–38.19 %), protein (9.77–18.15 %), lipid (0.60–2.21 %), crude fiber (4.45–5.68 %), alginate (22.49–29.13 %), fucoxanthin (0.07–0.32 mg g^?1), polyphenol (1.579–4.796 mg g^?1) were analyzed from dried alga. Six mineral elements were analyzed and the most abundant were calcium (6.64–11.56 mg g^?1) and magnesium (7.02–7.92 mg g^?1). Analysis of fatty acid composition indicated that the polyunsaturated fatty acids palmitoleic acid and linoleic acid were abundant in May and June, whereas the saturated fatty acid palmitic acid was abundant in July. Amino acid composition was also analyzed and the most abundant amino acids were aspartic acid, glutamic acid, glycine, and alanine. The ratio of mannuronic acid to guluronic acid of alginate was 2.57, 2.17, and 1.66 in May, June, and July, respectively. The gel strength of alginate was 1,449.0, 1,935.0, and 980.5 g cm^?1 in May, June, and July, respectively. The results of this study indicate that C. costata is an excellent resource that provides extensively applications in the industrial areas of chemicals, food, cosmetics, and pharmacy.     

Comparative study on chemical pretreatment methods for improving enzymatic digestibility of crofton weed stem

In order to utilize and control the invasive weed, crofton weed (Eupatorium adenophorum Spreng), a potential pathway was proposed by using it as a feedstock for production of fermentable sugars. Three chemical pretreatment methods were used for improving enzymatic saccharification of the weed stem. Mild H2SO4 pretreatment could obtain a relatively high yield of sugars in the pretreatment (32.89%, based on initial holocellulose), however, it led to only a slight enhancement of enzymatic digestibility. NaOH pretreatment could obtain a higher enzymatic conversion ratio of cellulose compared with H2SO4 pretreatment. Peracetic acid (PAA) pretreatment seemed to be the most effective for improving enzymatic saccharification of the weed stem in the three chemical pretreatment methods under the same conditions. The conversion ratio of cellulose in the sample pretreated by PAA under the "optimal" condition was increased to 50% by cellulase loading of 80 FPU/g cellulose for 72 h incubation. A number of empirical quadratic models were successfully developed according to the experimental data to predict the yield of sugar and degree of delignification.     

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.

     

Synergistic effects of mixing hybrid poplar and wheat straw biomass for bioconversion processes

Background

Low cost of raw materials and good process yields are necessary for future lignocellulosic biomass biorefineries to be sustainable and profitable. A low cost feedstock will be diverse, changing as a function of seasonality and price and will most likely be available from multiple sources to the biorefinery. The efficacy of the bioconversion process using mixed biomass, however, has not been thoroughly investigated. Considering the seasonal availability of wheat straw and the year round availability of hybrid poplar in the Pacific Northwest, this study aims to determine the impact of mixing wheat straw and hybrid poplar biomass on the overall sugar production via steam pretreatment and enzymatic saccharification.

Results

Steam pretreatment proved to be effective for processing different mixtures of hybrid poplar and wheat straw. Following SO₂-catalyzed steam explosion pretreatment, on average 22 % more sugar monomers were recovered using mixed feedstock than either single biomass. Improved sugar recovery with mixtures of poplar and wheat straw continued through enzymatic hydrolysis. After steam pretreatment and saccharification, the mixtures showed 20 % higher sugar yields than that produced from hybrid poplar and wheat straw alone.

Conclusions

Blending hybrid poplar and wheat straw resulted in more monomeric sugar recovery and less sugar degradation. This synergistic effect is attributable to interaction of hybrid poplar’s high acetic acid content and the presence of ash supplied by wheat straw. As a consequence on average 20 % more sugar was yielded by using the different biomass mixtures. Combining hybrid poplar and wheat straw enables sourcing of the lowest cost biomass, reduces seasonal dependency, and results in increasing biofuels and chemicals productivity in a cellulosic biorefinery.

     

Comparison of Dilute Acid and Sulfite Pretreatment for Enzymatic Saccharification of Earlywood and Latewood of Douglas fir

This study applied dilute acid (DA) and sulfite pretreatment to overcome the recalcitrance of lignocelluloses (SPORL) to deconstruct earlywood and latewood cell walls of Douglas fir for fermentable sugars production through subsequent enzymatic hydrolysis. DA pretreatment removed almost all the hemicelluloses, while SPORL at initial pH?=?4.5 (SP-B) removed significant amount of lignin between 20 and 25 %. But both are not sufficient for effective enzymatic saccharification. SPORL at low initial pH?=?2 (SP-AB) combines the advantage of both DA and SPORL-B to achieve approximately 90 % hemicellulose removal and delignification of 10–20 %. As a result, SP-AB effectively removed recalcitrance and thereby significantly improved enzymatic saccharification compared with DA and SP-B. Results also showed that earlywood with significantly lower density produced less saccharification after DA pretreatment, suggesting that wood density does not contribute to recalcitrance. The thick cell wall of latewood did not limit chemical penetration in pretreatments. The high lignin content of earlywood limited the effectiveness of DA pretreatment for enzymatic saccharification, while hemicellulose limits the effectiveness of high pH pretreatment of SP-B. The higher hemicellulose content in the earlywood and latewood of heartwood reduced saccharification relative to the corresponding earlywood and latewood in the sapwood using DA and SP-AB.     

Polysaccharide production and the possible occurrence of GDP-d-mannose dehydrogenase inAzotobacter vinelandii

During the growth ofAzotobacter vinelandii in batch culture in Burk's 2% glucose medium supplemented with 50mg EDTA per litre, water-insoluble capsular polysaccharide material accumulated in cultures prior to the appearance of water-soluble polysaccharide in the culture medium. On isolation, hydrolysis and chromatography, both these polysaccharides were observed to be composed of carbohydrate monomers having the same chromatographic mobilities as glucose, rhamnose, guluronic acid and mannuronic acid. The activity of GDP-d-mannose dehydrogenase recorded in crude cell-free extracts fromAzotobacter vinelandii, when these polysaccharides were produced, may indicate a close similarity between the biosynthetic pathway of alginate synthesis in marine Phaeophyceae and this soil microorganism.     

Comparative evaluation of acid and alkaline sulfite pretreatments for enzymatic saccharification of bagasses from three different sugarcane hybrids

Sugarcane bagasses from three experimental sugarcane hybrids and a mill‐reference sample were used to compare the efficiency and mode of action of acid and alkaline sulfite pretreatment processes. Varied chemical loads and reaction temperatures were used to prepare samples with distinguished characteristics regarding xylan and lignin removals, as well as sulfonation levels of residual lignins. The pretreatment with low sulfite loads (5%) under acidic conditions (pH 2) provided maximum glucose yield of 70% during enzymatic hydrolysis with cellulases (10 FPU/g) and β‐glucosidases (20 UI/g bagasse). In this case, glucan enzymatic conversion from pretreated materials was mostly associated with extensive xylan removal (70–100%) and partial delignification occurred during the pretreatment. The use of low sulfite loads under acidic conditions required pretreatment temperatures of 160°C. In contrast, at a lower pretreatment temperature (120°C), alkaline sulfite process achieved similar glucan digestibility, but required a higher sulfite load (7.5%). Residual xylans from acid pretreated materials were almost completely hydrolysed by commercial enzymes, contrasting with relatively lower xylan to xylose conversions observed in alkaline pretreated samples. Efficient xylan removal during acid sulfite pretreatment and during enzymatic digestion can be useful to enhance glucan accessibility and digestibility by cellulases. Alkaline sulfite process also provided substrates with high glucan digestibility, mainly associated with delignification and sulfonation of residual lignins. The results demonstrate that temperature, pH, and sulfite can be combined for reducing lignocellulose recalcitrance and achieve similar glucan conversion rates in the alkaline and acid sulfite pretreated bagasses. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:944–951, 2018     

Affinity precipitation of Aspergillus niger pectinase by microwave-treated alginate

Affinity precipitation is a simple, single plate separation process in which the complex of a smart macroaffinity ligand with the target protein (from a crude broth) can be selectively precipitated by application of a suitable stimulus. Alginate is a copolymer of guluronic acid and mannuronic acid residues and precipitates with Ca(2+) ions. It was found to bind to pectinase present in a commercial preparation of Aspergillus niger, Pectinex Ultra-SPL. Microwave pretreatment of alginate at 75 degrees C was found to enhance the selectivity of the affinity precipitation. Using microwave-treated alginate, 83% of the enzyme activity with 20-fold purification could be recovered. SDS-PAGE upon silver staining confirmed the enhanced selectivity of affinity precipitation when microwave-treated alginate was used.     

Catalytic properties and specificity of a recombinant, overexpressed D-mannuronate lyase

Lysis of alginates and of their saturated and unsaturated fragments was monitored by 1H NMR spectroscopy. AlxM(B) alginate lyase performs beta-elimination on the mannuronic acid (M) residues. It does not cleave the guluronic acid (G) sequences, nor the M-G or the G-M diads. In consequence, it is a true mannuronate lyase. The end product of the reaction is O-(4-deoxy-alpha-L-ery-thro-hex-4-enopyranosyl-uronic acid)-(1->(4)-O-(beta-D-mannopyranosyluronic acid)-(1->4)-O-beta-D-mannpyranuronic acid. Viscosity measurements made during degradation of a polymannuronate alginate showed that AlxM(B) behaves as an endo-enzyme. HPLC analysis of the degradation products of oligomannuronates and oligoalginates suggested that the beta-elimination requires the interaction of the enzyme with at least three sequential mannuronic acid residues. The catalytic site may possess 5 sub-sites and accommodate pentamers with different M/G ratio. Kinetic measurements showed that the specificity constant Vm/Km increased with the number of mannuronic acid residues. AlxM(B) may be reversibly inhibited by heteropolymeric blocks in a competitive manner.     

Formation of effective channels in alginate gel for immobilization of anchorage-dependent animal cells

Summary The conditions for formation of effective channels in alginate gels for growth of anchorage-dependent animal cells were examined. Many channels were formed in the gels by adding a low concentration solution of a high molecular weight polymer of alginate to a high concentration solution of divalent cations. It is recommended that an alginate with a high molecular weight and a low mannuronic acid/guluronic acid ratio be gelled by contact with strontium ions for the cultivation of immobilized anchorage-dependent cells because the gels produced have many channels and are mechanically strong.     

Consequences of the association of calcium with alginate during batch culture of Azotobacter vinelandii

Summary Azotobacter vinelandii strain E was grown in batch culture in the presence of radioactive Ca⁴⁵. The partitioning of Ca⁴⁵ in solution and associated with extracellular polysaccharide (alginate) during the growth was studied. The amount of alginate produced was estimated and its composition was determined by ¹H-n.m.r. Alginate produced early in the growth cycle was characterised by high guluronic acid block mole fraction (0.45) whilst later, alginate with a preponderance of mannuronic acid blocks was observed. Mixed block synthesis occurred throughout the incubation. The level of free Ca²⁺ fell rapidly (from 97% to 22%) during the first 33 h of incubation.