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.     

活性污泥产酸发酵研究进展

有机物的厌氧生物处理一般经过三个阶段:水解阶段、产酸发酵阶段和产甲烷阶段;研究证明,产酸相不同发酵类型的形成对产甲烷相乃至整个工艺的稳定运行具有至关重要的作用,此外,污泥厌氧消化过程所产生的大量的挥发性脂肪酸(VFAs),如乙酸、丙酸、丁酸及戊酸等,还可作为化工原料用于发酵工业生产各种高附加值产品.近年来,产酸发酵受到越来越多的关注,该文主要对污泥产酸阶段的产酸发酵类型、产酸发酵细菌的生态学、产酸过程的影响因素和生态因子以及产酸发酵的液相末端产物VFAs的测定方法进行了论述.     

Characterization of anaerobic microbial culture with high acidogenic activity

The mixed cultures which were used were isolated from municipal sludge digesters, and the production of organic acids (acetic, propionic, butyric, etc.) from carbohydrates was tested. The behavior of the reference population (culture R) obtained directly from the sewage treatment plant, is compared to that obtained after three months in a plug-flow reactor (Gradostat fermentor) without pH control (culture A) and after six months with pH control (culture B). For culture B, the specific rate of acid production is related to the cell growth rate by (1/X)r_p= 17 µ + 1.6 with a maximal acid concentration of 40 g/liter. The batch culture yields are improved from 0.36g/g for the initial culture (R) to 0.72 g/g for culture B after six months in continuous culture, and 0.8 g/g in plug-flow continuous culture. The productivity of organic acids reaches 1.7 g/liter·hr. It is suggested that the acidogenic fermentation, the first step of methanogenesis, is a potential process to produce acetic, propionic, and butyric acids.     

A novel recycling process using the treated citric acid wastewater as ingredients water for citric acid production

In this study, an integrated process coupling citric acid and methane fermentations was proposed to solve severe wastewater pollution problem in cassava-based citric acid production. The accumulation patterns of the potential and major inhibitors in this process, including organic compounds, volatile fatty acids (VFAs), total ions and pigments were investigated. Both simulation and experimental results indicated that these inhibitors could reach their equilibrium levels after 3–7 fermentation runs when reutilizing the treated citric acid wastewater. As a result, the proposed citric acid fermentation process by recycling the wastewater treated in methane fermentation could be stably operated for more than 15 runs, which could save a large amount of fresh water and relieve the severe wastewater pollution in citric acid production potentially.     

Increased digestibility of bagasses by pretreatment with alkalis and steam explosion

Alkali treatment and steam explosion of bagasse were investigated in order to develop economical and effective methods of increasing the digestibility of bagasse. The treated bagasse was to be used as a substrate for the production of volatile fatty acids by anaerobic acidogenic bacteria. The alkalis examined were NaOH, NH(3) (aqueous), NaOH + NH(3), Ca(OH)(2), and Ca(OH)(2) + Na(2)CO(3), at ambient temperature and in combination with steam explosion at 200 degrees C, 6.9 MPa, and 5 min cooking times. Digestibilities of up to 733 g organic matter (OM)/kg bagasse dry matter (DM) were obtained for bagasse treated with NaOH and Ca(OH)(2) + Na(2)CO(3); less than 430 g OM was obtained for bagasse treated with aqueous NH(3); and up to 724 g OM was obtained for bagasse treated with Ca(OH)(2). This digestibility was only achieved by using high concentrations of Ca(OH)(2), i.e., 180-300 g/kg bagasse. Steam explosion increased the digestibility of bagasse up to 740 g OM in the presence of alkali but only to 610 g OM in the absence of alkali. The digestibility of bagasse without pretreatment was 190 g OM/kg bagasse DM. More than one-half the hemicellulose present was solubilized by pretreatment. The composition of the liquid fraction of steam-exploded material was examined and contained mainly xylose monomers and oligomers (112 g/kg original bagasse DM) and acetic acid (33 g/kg original DM). The relative costs of the alkalis used were obtained for the United States, Australia, and Europe. Lime [Ca(OH)(2)] was the least expensive alkali per unit of additional digestible OM obtained. Ammonia was the most expensive alkali to use, except in the United States where the difference in its cost relative to other alkalis was smaller. However, ammonia provides organic nitrogen for microbial growth, and could be recycled. With acidogenic fermentations, alkali is able to double as a neutralizing agent during fermentation. Thus, concentrations of alkali equal to that required for neutralization may be used in pretreatment. Concentrations of Ca(OH)(2) as high as 300 g/kg bagasse were needed for neutralization and should, therefore, be considered for pretreatment. Steam explosion of bagasse resulted in digestible, sterilized substrates of small particle size with readily separable liquid and pulp streams.     

A coupled two-stage continuous fermentation for solvent production by Clostridium acetobutylicum

The effects of nitrogen and phosphate in batch and continuous AEB fermentations were tested. Both nitrogen- and phosphate-limited fermentations favored acid formation but not solvent production. A coupled two-stage continuous fermentation was performed for 30 days with a nitrogen-limited first stage fermentation for enhanced acid production. The bacteria from the acidogenic phase (first stage) fermentation were continuously pumped into a 14-l second stage fermentor with supplemental glucose and nitrogen for solvent production. The second stage fermentor had a maximum butanol productivity of 0.4 g l⁻¹ h⁻¹ (total solvent production was 0.6 g l⁻¹ h⁻¹) at a dilution rate of 0.06 h⁻¹.     

Anaerobic digestion: microbial and biochemical aspects of volatile acid production

Summary The production of organic acids has been tested with bacterial flora selected from a municipal sludge digestor. In order to elucidate the basic mechanisms by which glucose is converted to volatile fatty acids, the examination of non-methanogenic bacteria was attempted. Both lactate-producers and lactate-utilizers were found among these bacteria. When mixed isolates were used as the inoculum, the accumulation of lactic acid and its further conversion to propionic and butyric acids was demonstrated at a carbon conversion rate of about 0.75. It is therefore suggested that this metabolic sequence may occur as a normal process in acidogenic fermentation, which is the first step in anaerobic digestion.     

Production of polyhydroxyalkanoates by activated sludge treating a paper mill wastewater

Production of polyhydroxyalkanoates (PHAs) in activated sludge treating wastewater represents an economical and environmental promising alternative to pure culture fermentations. A process for production of PHA from a paper mill wastewater was examined at laboratory scale. The three stage process examined consisted of acidogenic fermentation to convert wastewater organic matter to volatile fatty acids (VFAs), an activated sludge system operating under feast/famine conditions to enrich for PHA producing organisms and accumulation of PHA in batch experiments. After fermentation of the wastewater, 74% of the soluble COD was present as VFA (acetate, propionate, butyrate and valerate) and the resulting PHA after batch accumulation consisted of 31-47 mol% hydroxybutyrate and 53-69 mol% hydroxyvalerate. The maximum PHA content achieved was 48% of the sludge dry weight and the three stage process exhibited a potential to produce 0.11 kg of PHA per kg of influent COD treated.     

Conversion of cereal residues into biogas in a rumen-derived process

A recently developed high-rate, two-phase process, which employs rumen microorganisms for efficient acidogenesis, was tested for anaerobic degradation of barley straw, rye straw, and maize stover. Under conditions similar to those of the rumen and loading rates varying between 9.8 and 26.0 g of organic matter/I/day in the first phase (acidogenic reactor), total fibre degradation efficiencies ranged between 42% and 57%, irrespective of the loading rate applied. Average specific production of volatile fatty acids and biogas/g volatile solid digested in the acidogenic reactor varied between 6.9 and 11.2 mmol and 0.10 and 0.25 l, respectively.The effect of varying solid retention times on the extent of degradation of barley straw was examined. Changing of retention times in the range of 60 to 156 h had no effect on degradation efficiency, but a decrease in efficiency was observed at retention times below 60 h.By connecting the acidogenic reactor in series to an Upflow Anaerobic Sludge Blanket (UASB) methanogenic reactor the volatile fatty acids were converted into biogas. Average methane contents of the gases produced in the acidogenic reactor and in the UASB reactor were 30±3% and 78±3%, respectively.     

A high-throughput transient gene expression system for switchgrass (Panicum virgatum L.) seedlings

Background

Fermentations using Escherichia coli KO11, Saccharomyces cerevisiae 424A(LNH-ST), and Zymomonas mobilis AX101 are compared side-by-side on corn steep liquor (CSL) media and the water extract and enzymatic hydrolysate from ammonia fiber expansion (AFEX)-pretreated corn stover.

Results

The three ethanologens are able produce ethanol from a CSL-supplemented co-fermentation at a metabolic yield, final concentration and rate greater than 0.42 g/g consumed sugars, 40 g/L and 0.7 g/L/h (0-48 h), respectively. Xylose-only fermentation of the tested ethanologenic bacteria are five to eight times faster than 424A(LNH-ST) in the CSL fermentation. All tested strains grow and co-ferment sugars at 15% w/v solids loading equivalent of ammonia fiber explosion (AFEX)-pretreated corn stover water extract. However, both KO11 and 424A(LNH-ST) exhibit higher growth robustness than AX101. In 18% w/w solids loading lignocellulosic hydrolysate from AFEX pretreatment, complete glucose fermentations can be achieved at a rate greater than 0.77 g/L/h. In contrast to results from fermentation in CSL, S. cerevisiae 424A(LNH-ST) consumed xylose at the greatest extent and rate in the hydrolysate compared to the bacteria tested.

Conclusions

Our results confirm that glucose fermentations among the tested strains are effective even at high solids loading (18% by weight). However, xylose consumption in the lignocellulosic hydrolysate is the major bottleneck affecting overall yield, titer or rate of the process. In comparison, Saccharomyces cerevisiae 424A(LNH-ST) is the most relevant strains for industrial production for its ability to ferment both glucose and xylose from undetoxified and unsupplemented hydrolysate from AFEX-pretreated corn stover at high yield.     

Determination of Steam-Volatile Organic Acids in Fermentation Media by Gas-Liquid Chromatography

Five gas chromatographic liquid phases (25% Carbowax 20 M plus 4% H₃PO₄, 17.5% dioctyl sebacate plus 7.5% sebacic acid, 17.5% dioctyl sebacate plus 7.5% docosanoic acid, 5% Tween 80, and 20% LAC-296 [poly (diethylene glycol adipate)] plus 2% H₃PO₄) were studied with respect to their utility in the separation and quantitation of steam-volatile organic acids commonly produced in fermentation. Optimal operating conditions and column stability for routine analysis were established. An Aerograph Hy-Fi gas chromatograph was used for all work, except the studies with Tween 80 in which an Aerograph A-90-C was employed. Chromatographic traces are presented of volatile fatty acid analyses with each of the liquid phases. Complete separation of all isomers of the fatty acids from C₂ to C₅ was accomplished by the Carbowax 20 M plus H₃PO₄, dioctyl sebacate plus sebacic acid, and dioctyl sebacate plus docosanoic acid columns. The latter two liquid phases were extremely unstable and proved to be unsatisfactory for analysis of aqueous samples. A column of Carbowax 20 M + H₃PO₄ separated steam-volatile organic acids completely. The volatile fatty acid isomers were separated by 5% Tween 80 somewhat less completely, and the peak shapes were not as sharp and symmetrical as that desired for good quantitative work. LAC-296 (20%) plus 2% H₃PO₄ proved to be the most satisfactory of the liquid phases for routine analysis of deproteinated in vitro rumen fermentation media. The column has been used for routine analysis of rumen fermentation fluid and in vitro rumen incubation fluid. All the organic acids from C₂ to C₅, except isobutyric, could be quantitated with this column. Stability of the column with the aqueous solutions was extremely good. The standard deviation of the analysis of each volatile acid component in a fermentation fluid was less than 0.5 molar per cent. The short-chain organic acids (C₂ to C₅) were shown to be extremely stable in aqueous solution for as long as 6 months after preparation for gas chromatographic analysis by protein precipitation with metaphosphoric acid-H₂SO₄ and refrigeration at 4 C in stoppered tubes.     

Production of volatile fatty acids from bagasse by rumen bacteria

Summary Conditions are described for converting bagasse lignocellulose to volatile fatty acids (VFA) by anaerobic fermentation. Although yields of VFA were as high as 74% by weight of digestible organic matter (or 54% of dry bagasse), limitations were imposed by both fermenter design and fibre digestibility. All fermentations were substrate-limited up to the maximum initial concentration examined of 50 g bagasse · l^-1 and no product inhibition was evident (up to 260 mM VFA produced). Maximum VFA productivities of 0.25 to 0.65 g · l^-1 · h^-1 were obtained in batch fermentations and this is greater than those previously reported using lignocellulosic substrates. Batch fermentations neared completion after 66 h.     

Fermentation of soybean hulls to ethanol while preserving protein value

Soybean hulls were evaluated as a resource for production of ethanol by the simultaneous saccharification and fermentation (SSF) process, and no pretreatment of the hulls was found to be needed to realize high ethanol yields with Saccharomyces cerevisiae D₅A. The impact of cellulase, β-glucosidase and pectinase dosages were determined at a 15% biomass loading, and ethanol concentrations of 25–30 g/L were routinely obtained, while under these conditions corn stover, wheat straw, and switchgrass produced 3–4 times lower ethanol yields. Removal of carbohydrates also concentrated the hull protein to over 25% w/w from the original roughly 10%. Analysis of the soybean hulls before and after fermentation showed similar amino acid profiles including an increase in the essential amino acids lysine and threonine in the residues. Thus, eliminating pretreatment should assure that the protein in the hulls is preserved, and conversion of the carbohydrates to ethanol with high yields produces a more concentrated and valuable co-product in addition to ethanol. The resulting upgraded feed product from soybean hulls would likely to be acceptable to monogastric as well as bovine livestock.     

Photo-fermentation of Rhodobacter sphaeroides for hydrogen production using lignocellulose-derived organic acids

In this study, the corn stover was pretreated, enzymatically hydrolyzed, and then fermented in the lipid production fermentation. The corn stover fermentation effluent was utilized for the photo-fermentation of a Rhodobacter sphaeroides ZX-5 for hydrogen production. The hydrogen production was more than twofolds greater than that in the synthetic medium under the similar organic acid concentration range. The synergism among the pure organic acids was found to facilitate cell growth and hydrogen production, although some organic acid was not utilized for hydrogen production directly. The synergism among the components in the corn stover fermentation effluent was also found. The initial pH value was found to be an important parameter for the photo-fermentation of R. sphaeroide ZX-5 using the corn stover fermentation effluent. The results provided a possible way to utilize lignocellulose-derived organic acids for hydrogen production, and to treat fermentation wastewater in biofuel production using lignocellulose.     

Xylose fermentation by genetically modified Saccharomyces cerevisiae 259ST in spent sulfite liquor

Spent sulfite pulping liquor (SSL) is a high-organic content byproduct of acid bisulfite pulp manufacture which is fermented to make industrial ethanol. SSL is typically concentrated to 240 g/l (22% w/w) total solids prior to fermentation, and contains up to 24 g/l xylose and 30 g/l hexose sugars, depending upon the wood species used. The xylose present in SSL is difficult to ferment using natural xylose-fermenting yeast strains due to the presence of inhibitory compounds, such as organic acids. Using sequential batch shake flask experiments, Saccharomyces cerevisiae 259ST, which had been genetically modified to ferment xylose, was compared with the parent strain, 259A, and an SSL adapted strain, T2, for ethanol production during SSL fermentation. With an initial SSL pH of 6, without nutrient addition or SSL pretreatment, the ethanol yield ranged from 0.32 to 0.42 g ethanol/g total sugar for 259ST, compared to 0.15-0.32 g ethanol/g total sugar for non-xylose fermenting strains. For most fermentations, minimal amounts of xylitol (<1 g/l) were produced, and glycerol yields were approximately 0.12 g glycerol/g sugar consumed. By using 259ST for SSL fermentation up to 130% more ethanol can be produced compared to fermentations using non-xylose fermenting yeast.     

Anaerobic thermophilic fermentation for carboxylic acid production from in-storage air-lime-treated sugarcane bagasse

Wet storage and in situ lime pretreatment (50 °C, 1-atm air, 56 days, excess lime loading of 0.3 g Ca(OH)₂/g dry biomass) of sugarcane bagasse (4,000 g dry weight) was performed in a bench-scale pile pretreatment system. Under thermophilic conditions (55 °C, NH₄HCO₃ buffer, methane inhibitors), air-lime-treated bagasse (80 wt.%) and chicken manure (20 wt.%) were anaerobically co-digested in 1-L rotary fermentors by a mixed culture of marine microorganisms (Galveston, TX). During four-stage countercurrent fermentation, the resulting carboxylic acids consisted of primarily acetate (average 87.7 wt.%) and butyrate (average 9.0 wt.%). The experimental fermentation trains had the highest yield (0.47 g total acids/g volatile solids (VS) fed) and highest selectivity (0.79 g total acids/g VS digested) at a total acid concentration of 28.3 g/L, which is equivalent to an ethanol yield of 105.2 gal/(tonne VS fed). Both high total acid concentrations (>44.7 g/L) and high substrate conversions (>77.5%) are predicted for countercurrent fermentations of bagasse at commercial scale, allowing for an efficient conversion of air-lime-treated biomass to liquid transportation fuels and chemicals via the carboxylate platform.     

Effect of dilute acid pretreatment on the saccharification and fermentation of rye straw

This research shows the effect of dilute acid pretreatment with various sulfuric acid concentrations (0.5–2.0% [wt/vol]) on enzymatic saccharification and fermentation yield of rye straw. After pretreatment, solids of rye straw were suspended in Na citrate buffer or post-pretreatment liquids (prehydrolysates) containing sugars liberated after hemicellulose hydrolysis. Saccharification was conducted using enzymes dosage of 15 or 25 FPU/g cellulose. Cellulose saccharification rate after rye straw pretreatment was enhanced by performing enzymatic hydrolysis in sodium citrate buffer in comparison with hemicellulose prehydrolysate. The maximum cellulose saccharification rate (69%) was reached in sodium citrate buffer (biomass pretreated with 2.0% [wt/vol] H₂SO₄). Lignocellulosic complex of rye straw after pretreatment was subjected to separate hydrolysis and fermentation (SHF) or separate hydrolysis and co-fermentation (SHCF). The SHF processes conducted in the sodium citrate buffer using monoculture of Saccharomyces cerevisiae (Ethanol Red) were more efficient compared to hemicellulose prehydrolysate in respect with ethanol yields. Maximum fermentation efficiency of SHF processes obtained after rye straw pretreatment at 1.5% [wt/vol] H₂SO₄ and saccharification using enzymes dosage of 25 FPU/g in sodium citrate buffer, achieving 40.6% of theoretical yield. However, SHCF process using cocultures of pentose-fermenting yeast, after pretreatment of raw material at 1.5% [wt/vol] H₂SO₄ and hydrolysis using enzymes dosage of 25 FPU/g, resulted in the highest ethanol yield among studied methods, achieving 9.4 g/L of ethanol, corresponding to 55% of theoretical yield.     

Dilute sulfuric acid cycle spray flow-through pretreatment of corn stover for enhancement of sugar recovery

A cycle spray flow-through reactor was designed and used to pretreat corn stover in dilute sulfuric acid medium. The dilute sulfuric acid cycle spray flow-through (DCF) process enhanced xylose sugar yields and cellulose digestibility while increasing the removal of lignin. Within the DCF system, the xylose sugar yields of 90–93% could be achieved for corn stover pretreated with 2% (w/v) dilute sulfuric acid at 95 °C during the optimal reaction time (90 min). The remaining solid residue exhibited enzymatic digestibility of 90–95% with cellulase loading of 60 FPU/g glucan that was due to the effective lignin removal (70–75%) in this process. Compared with flow-through and compress-hot water pretreatment process, the DCF method produces a higher sugar concentration and higher xylose monomer yield. The novel DCF process provides a feasible approach for lignocellulosic material pretreatment.     

Lessons from the cow: What the ruminant animal can teach us about consolidated bioprocessing of cellulosic biomass

Consolidated bioprocessing (CBP) of cellulosic biomass is a promising source of ethanol. This process uses anaerobic bacteria, their own cellulolytic enzymes and fermentation pathways that convert the products of cellulose hydrolysis to ethanol in a single reactor. However, the engineering and economics of the process remain questionable. The ruminal fermentation is a very highly developed natural cellulose-degrading system. We propose that breakthroughs developed by cattle and other ruminant animals in cellulosic biomass conversion can guide future improvements in engineered CBP systems. These breakthroughs include, among others, an elegant and effective physical pretreatment; operation at high solids loading under non-aseptic conditions; minimal nutrient requirements beyond the plant biomass itself; efficient fermentation of nearly all plant components; efficient recovery of primary fermentation end-products; and production of useful co-products. Ruminal fermentation does not produce significant amounts of ethanol, but it produces volatile fatty acids and methane at a rapid rate. Because these alternative products have a high energy content, efforts should be made to recover these products and convert them to other organic compounds, particularly transportation fuels.