Recent advances in ABE fermentation: hyper-butanol producing Clostridium beijerinckii BA101

This is an overview of the mutant strain Clostridium beijerinckii BA101 which produces solvents (acetone–butanol–ethanol, ABE) at elevated levels. This organism expresses high levels of amylases when grown on starch. C. beijerinckii BA101 hydrolyzes starch effectively and produces solvent in the concentration range of 27–29 g l⁻¹. C. beijerinckii BA101 has been characterized for both substrate and butanol inhibition. Supplementing the fermentation medium (MP2) with sodium acetate enhances solvent production to 33 g l⁻¹. The results of studies utilizing commercial fermentation medium and pilot plant-scale reactors are consistent with the results using small-scale reactors. Pervaporation, a technique to recover solvents, has been applied to fed-batch reactors containing C. beijerinckii BA101, and solvent production as high as 165 g l⁻¹ has been achieved. Immobilization of C. beijerinckii BA101 by adsorption and use in a continuous reactor resulted in reactor productivity of 15.8 g l⁻¹ h⁻¹. Recent economic studies employing C. beijerinckii BA101 suggested that butanol can be produced at US$0.20–0.25 lb⁻¹ by employing batch fermentation and distillative recovery. Application of new technologies such as pervaporation, fed-batch culture, and immobilized cell reactors is expected to further reduce these prices. Journal of Industrial Microbiology & Biotechnology (2001) 27, 287–291. Received 12 September 2000/ Accepted in revised form 27 January 2001     

Production of butanol from starch-based waste packing peanuts and agricultural waste

We examined the fermentation of starch-based packing peanuts and agricultural wastes as a source of fermentable carbohydrates using Clostridium beijerinckii BA101. Using semidefined P2 medium containing packing peanuts and agricultural wastes, instead of glucose as a carbohydrate source, we measured characteristics of the fermentation including solvent production, productivity, and yield. With starch as substrate (control), the culture produced 24.7 g l⁻¹ acetone–butanol–ethanol (ABE), while with packing peanuts it produced 21.7 g l⁻¹ total ABE with a productivity of 0.20 g l⁻¹ h⁻¹ and a solvent (ABE) yield of 0.37. Cell growth in starch, packing peanuts, and agricultural wastes medium was different, possibly due to the different nature of these substrates. Using model agricultural waste, 20.3g l⁻¹ ABE was produced; when using actual waste, 14.8 g l⁻¹ ABE was produced. The use of inexpensive substrates will increase the economic viability of the conversion of biomass to butanol, and can provide new markets for these waste streams. Journal of Industrial Microbiology & Biotechnology (2002) 29, 117–123 doi: 10.1038/sj.jim.7000285 Received 14 November 2001/ Accepted in revised form 07 June 2002     

Production of acetone,butanol and ethanol by Clostridium beijerinckii BA101 and in situ recovery by gas stripping

We examined the effect of gas-stripping on the in situ removal of acetone, butanol, and ethanol (ABE) from batch reactor fermentation broth. The mutant strain (Clostridium beijerinckii BA101) was not affected adversely by gas stripping. The presence of cells in the fermentation broth affected the selectivities of ABE. A considerable improvement in the productivity and yield was recorded in this work in comparison with the non-integrated process. In an integrated process of ABE fermentation-recovery using C. beijerinckii BA101, ABE productivities and yield were improved up to 200 and 118%, respectively, as compared to control batch fermentation data. In a batch reactor C. beijerinckii BA101 utilized 45.4 g glucose l^–1 and produced 17.7 g total ABE l^–1, while in the integrated process it utilized 161.7 g glucose l^–1 and produced total ABE of 75.9 g l^–1. In the integrated process, acids were completely converted to solvents when compared to the non-integrated process (batch fermentation) which contained residual acids at the end of fermentation. In situ removal of ABE by gas stripping has been reported to be one of the most important techniques of solvent removal. During these studies we were able to maintain the ABE concentration in the fermentation broth below toxic levels.     

Production of acetone butanol (AB) from liquefied corn starch,a commercial substrate,using <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis> coupled with product recovery by gas stripping

A potential industrial substrate (liquefied corn starch; LCS) has been employed for successful acetone butanol ethanol (ABE) production. Fermentation of LCS (60 g l⁻¹) in a batch process resulted in the production of 18.4 g l⁻¹ ABE, comparable to glucose: yeast extract based medium (control experiment, 18.6 g l⁻¹ ABE). A batch fermentation of LCS integrated with product recovery resulted in 92% utilization of sugars present in the feed. When ABE was recovered by gas stripping (to relieve inhibition) from the fed-batch reactor fed with saccharified liquefied cornstarch (SLCS), 81.3 g l⁻¹ ABE was produced compared to 18.6 g l⁻¹ (control). In this integrated system, 225.8 g l⁻¹ SLCS sugar (487 % of control) was consumed. In the absence of product removal, it is not possible for C. beijerinckii BA101 to utilize more than 46 g l⁻¹ glucose. A combination of fermentation of this novel substrate (LCS) to butanol together with product recovery by gas stripping may economically benefit this fermentation. Mention of trade names of commercial products in this article/publication is solely for the purpose of providing scientific information and does not imply recommendation or endorsement by the United States Department of Agriculture.     

<Emphasis Type="Italic">Clostridium beijerinckii</Emphasis> mutant with high inhibitor tolerance obtained by low-energy ion implantation

Clostridium beijerinckii mutant strain IB4, which has a high level of inhibitor tolerance, was screened by low-energy ion implantation and used for butanol fermentation from a non-detoxified hemicellulosic hydrolysate of corn fiber treated with dilute sulfuric acid (SAHHC). Evaluation of toxicity showed C. beijerinckii IB4 had a higher level of tolerance than parent strain C. beijerinckii NCIMB 8052 for five out of six phenolic compounds tested (the exception was vanillin). Using glucose as carbon source, C. beijerinckii IB4 produced 9.1 g l⁻¹ of butanol with an acetone/butanol/ethanol (ABE) yield of 0.41 g g⁻¹. When non-detoxified SAHHC was used as carbon source, C. beijerinckii NCIMB 8052 grew well but ABE production was inhibited. By contrast, C. beijerinckii IB4 produced 9.5 g l⁻¹ of ABE with a yield of 0.34 g g⁻¹, including 2.2 g l⁻¹ acetone, 6.8 g l⁻¹ butanol, and 0.5 g l⁻¹ ethanol. The remarkable fermentation and inhibitor tolerance of C. beijerinckii IB4 appears promising for ABE production from lignocellulosic materials.     

ABE production from corn: a recent economic evaluation

This article details an economic assessment of butanol production from corn using the newly developed hyper-butanol-producing strain of Clostridium beijerinckii BA101. Butanol is produced in batch reactors and recovered by distillation. For a plant with 153,000 metric tons of acetone, butanol, and ethanol (ABE) production capacity, the production equipment cost and total working capital cost is US$33.47×10⁶ and US$110.46×10⁶, respectively. Based on a corn price (C _p) of US$79.23 ton⁻¹ (US$2.01 bushel⁻¹), an ABE yield of 0.42 (g ABE/g glucose) butanol price is projected to be US$0.34 kg⁻¹. An improved yield of 0.50 will reduce this price to US$0.29 kg⁻¹. Assumptions, such as by-product credit for gases and complete conversion of corn steep liquor (CSL) to fermentation by-products, have been taken into consideration. An increased price of corn to US$197.10 ton⁻¹ would result in a butanol price of US$0.47 kg⁻¹. A grass-rooted plant would result in a butanol price of US$0.73 kg⁻¹ (C _p US$79.23 ton⁻¹). In a worst case scenario, the price of butanol would increase to US$1.07 kg⁻¹ (C _p 197.10 ton⁻¹ for a grass-rooted plant and assuming no credit for gases). This is based on the assumption that corn price would not increase to more than US$197.10 ton⁻¹. Journal of Industrial Microbiology & Biotechnology (2001) 27, 292–297. Received 12 September 2000/ Accepted in revised form 12 January 2001     

Development of a cost-effective glucose-corn steep medium for production of butanol by Clostridium beijerinckii

Corn steep water (CSW) medium (1.6% solids plus 6% glucose) was evaluated for growth and butanol production by Clostridium beijerinckii NCIMB 8052 wild-type and hyper-amylolytic, hyper-butanol-producing mutant strain BA101. CSW alone was not a suitable substrate, whereas addition of glucose supported growth and butanol production by both strains. In a batch-scale fermentation using an optimized 6% glucose-1.6% solids CSW medium, C. beijerinckii NCIMB 8052 and strain BA101 produced 10.7 g L⁻¹ and 14.5 g L⁻¹ of butanol, respectively. The total solvents (acetone, butanol, and ethanol) produced by C. beijerinckii NCIMB 8052 and strain BA101 were 14 g L⁻¹ and 20 g L⁻¹, respectively. Initial fermentation in small-scale flasks containing 6% maltodextrin-1.6% solids concentration CSW medium resulted in 6 g L⁻¹ and 12.6 g L⁻¹ of butanol production by C. beijerinckii NCIMB 8052 and strain BA101, respectively. CSW can serve as an economic source of nitrogen, vitamins, amino acids, minerals, and other nutrients. Thus, it is feasible to use 6% glucose-1.6% solids CSW medium in place of semi-defined P2 medium. Received 9 February 1998/ Accepted in revised form 1 September 1998     

Butanol production by Clostridium beijerinckii BA101 using cassava flour as fermentation substrate: enzymatic versus chemical pretreatments

Cassava flour (CF), a cost-effective source of starch, was employed as a substrate for successful acetone-butanol-ethanol (ABE) production by batch-fermentation with Clostridium beijerinckii. The effect of temperature, initial concentration of CF and chemical/enzymatic hydrolysis were studied in a 2³ factorial design. Results revealed that temperature and initial concentration of substrate exert a significant effect on ABE production, as well as interactions of temperature with the other variables. Solvent production was maximized when working at 40°C, 60 g l⁻¹ CF and enzymatic pretreatment. An average of 31.38 g l⁻¹ ABE was produced after 96 h, with a productivity of 0.33 g l⁻¹ h⁻¹. A posterior randomized block design (3 × 2) showed that enzymatic hydrolysis (with saccharification periods of 6 h at 60°C) enhances both reducing sugar and solvent production if compared to chemical pretreatments. Average ABE production in this case was 27.28 g l⁻¹, with a productivity of 0.28 g l⁻¹ h⁻¹. Results suggest that CF may be a suitable substrate for industrial ABE production.     

Butanol production by Clostridium beijerinckii ATCC 55025 from wheat bran

Wheat bran, a by-product of the wheat milling industry, consists mainly of hemicellulose, starch and protein. In this study, the hydrolysate of wheat bran pretreated with dilute sulfuric acid was used as a substrate to produce ABE (acetone, butanol and ethanol) using Clostridium beijerinckii ATCC 55025. The wheat bran hydrolysate contained 53.1 g/l total reducing sugars, including 21.3 g/l of glucose, 17.4 g/l of xylose and 10.6 g/l of arabinose. C. beijerinckii ATCC 55025 can utilize hexose and pentose simultaneously in the hydrolysate to produce ABE. After 72 h of fermentation, the total ABE in the system was 11.8 g/l, of which acetone, butanol and ethanol were 2.2, 8.8 and 0.8 g/l, respectively. The fermentation resulted in an ABE yield of 0.32 and productivity of 0.16 g l⁻¹ h⁻¹. This study suggests that wheat bran can be a potential renewable resource for ABE fermentation.     

Continuous solvent production by Clostridium beijerinckii BA101 immobilized by adsorption onto brick

The performance of a continuous bioreactor containing Clostridium beijerinckii BA101 adsorbed onto clay brick was examined for the fermentation of acetone, butanol, and ethanol (ABE). Dilution rates from 0.3 to 2.5 h^–1 were investigated with the highest solvent productivity of 15.8 g l^–1 h^–1 being obtained at 2.0 h^–1. The solvent yield at this dilution rate was found to be 0.38 g g^–1 and total solvent concentration was 7.9 g l^–1. The solvent yield was maximum at 0.45 at a dilution rate of 0.3 h^–1. The maximum solvent productivity obtained was found to be 2.5 times greater than most other immobilized continuous and cell recycle systems previously reported for ABE fermentation. A higher dilution rate (above 2.0 h^–1) resulted in acid production rather than solvent production. This reactor was found to be stable for over 550 h. Scanning electron micrographs (SEM) demonstrated that a large amount of C. beijerinckii cells were adsorbed onto the brick support.     

Butanol production from wheat straw hydrolysate using <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

In these studies, butanol (acetone butanol ethanol or ABE) was produced from wheat straw hydrolysate (WSH) in batch cultures using Clostridium beijerinckii P260. In control fermentation 48.9 g L⁻¹ glucose (initial sugar 62.0 g L⁻¹) was used to produce 20.1 g L⁻¹ ABE with a productivity and yield of 0.28 g L⁻¹ h⁻¹ and 0.41, respectively. In a similar experiment where WSH (60.2 g L⁻¹ total sugars obtained from hydrolysis of 86 g L⁻¹ wheat straw) was used, the culture produced 25.0 g L⁻¹ ABE with a productivity and yield of 0.60 g L⁻¹ h⁻¹ and 0.42, respectively. These results are superior to the control experiment and productivity was improved by 214%. When WSH was supplemented with 35 g L⁻¹ glucose, a reactor productivity was improved to 0.63 g L⁻¹ h⁻¹ with a yield of 0.42. In this case, ABE concentration in the broth was 28.2 g L⁻¹. When WSH was supplemented with 60 g L⁻¹ glucose, the resultant medium containing 128.3 g L⁻¹ sugars was successfully fermented (due to product removal) to produce 47.6 g L⁻¹ ABE, and the culture utilized all the sugars (glucose, xylose, arabinose, galactose, and mannose). These results demonstrate that C. beijerinckii P260 has excellent capacity to convert biomass derived sugars to solvents and can produce over 28 g L⁻¹ (in one case 41.7 g L⁻¹ from glucose) ABE from WSH. Medium containing 250 g L⁻¹ glucose resulted in no growth and no ABE production. Mixtures containing WSH + 140 g L⁻¹ glucose (total sugar approximately 200 g L⁻¹) showed poor growth and poor ABE production. Mention of trade names or commercial products in this article is solely for the purpose of providing scientific information and does not imply recommendation or endorsement by the United States Department of Agriculture.     

Pilot-scale production of butanol by Clostridium beijerinckii BA101 using a low-cost fermentation medium based on corn steep water

To improve the economic competitiveness of the acetone/butanol/ethanol fermentation process, glucose/corn steep water (CSW) medium was used on a pilot scale for the production of solvents. The production of butanol by the Clostridium beijerinckii NCIMB 8052 parent strain and the solvent-hyperproducing BA101 mutant was compared. In a 20-l fermentation using 5% glucose/CSW medium,  C. beijerinckii 8052 produced 8.5 g butanol/l and 5 g acetone/l, while  C. beijerinckii BA101 produced 16 g butanol/l and 7.5 g acetone/l. Further studies were carried out on a larger scale using an optimized 6% glucose/CSW medium. In a 200-l pilot-scale fermentor,  C. beijerinckii 8052 produced 12.7 g butanol/l and 6 g acetone/l following 96 h of fermentation.  C. beijerinckii BA101 produced 17.8 g/l and 5.5 g/l butanol and acetone respectively, following 130 h of fermentation. These results represent a 40% increase in final butanol concentration by the C. beijerinckii BA101 mutant strain when compared to the 8052 parent strain. The total solvents (acetone, butanol, and ethanol) produced by C. beijerinckii NCIMB 8052 and BA101 in a 200-l fermentation were 19.2 g/l and 23.6 g/l respectively. This is the first report of pilot-scale butanol production by the solvent-hyperproducing C. beijerinckii BA101 mutant employing an inexpensive glucose/CSW medium. Received: 26 May 1998 / Received revision: 21 September 1998 / Accepted: 11 October 1998     

Effects of 3 days of carbohydrate supplementation on muscle glycogen content and utilisation during a 1-h cycling performance

This study compared the effects of supplementing the normal diets of six trained cyclists [maximal oxygen uptake O_2max) 4.5 (0.36)l · min⁻¹; values are mean (SD)] with additional carbohydrate (CHO) on muscle glycogen utilisation during a 1-h cycle time-trial (TT). Using a randomised crossover design, subjects consumed either their normal diet (NORM) for 3 days, which consisted of 426 (137) g · day⁻¹ CHO [5.9 (1.4) g · kg⁻¹ body mass (BM)], or additional CHO (SUPP) to increase their intake to 661 (76) g · day⁻¹ [9.3 (0.7) g · kg⁻¹ BM]. The SUPP diet elevated muscle glycogen content from 459 (83) to 565 (62) mmol · kg⁻¹ dry weight (d.w.) (P < 0.05). However, despite the increased pre-exercise muscle glycogen stores, there was no difference in the distance cycled during the TT [40.41 (1.44) vs 40.18 (1.76) km for NORM and SUPP, respectively]. With NORM, muscle glycogen declined from 459 (83) to 175 (64) mmol · kg⁻¹ d.w., whereas with SUPP the corresponding values were 565 (62) and 292 (113) mmol · kg⁻¹ d.w. Accordingly, both muscle glycogen utilisation [277 (64) vs 273 (114) mmol · kg⁻¹ d.w.] and total CHO oxidation [169 (20) vs 165 (30) g · h⁻¹ for NORM and SUPP, respectively] were similar. Neither were there any differences in plasma glucose or lactate concentrations during the two experimental trials. Plasma glucose concentration averaged 5.5 (0.5) and 5.6 (0.6) mmol · l⁻¹, while plasma lactate concentration averaged 4.4 (1.9) and 4.4 (2.3) mmol · l⁻¹ for NORM and SUPP, respectively. The results of this study show that when well-trained subjects increase the CHO content of their diet for 3 days from 6 to 9 g · kg⁻¹ BM there is only a modest increase in muscle glycogen content. Since supplementary CHO did not improve TT performance, we conclude that additional CHO provides no benefit to performance for athletes who compete in intense, continuous events lasting 1 h. Furthermore, the substantial muscle CHO reserves observed at the termination of exercise indicate that whole-muscle glycogen depletion does not determine fatigue at this exercise intensity and duration. Accepted: 25 November 1996     

Acetate enhances solvent production and prevents degeneration in Clostridium beijerinckii BA101

Addition of sodium acetate to chemically defined MP2 medium was found to increase and stabilize solvent production by Clostridium beijerinckii BA101, a solvent-hyperproducing mutant derived from C. beijerinckii NCIMB 8052. C. beijerinckii BA101 demonstrated a greater increase in solvent production than C. beijerinckii NCIMB 8052 when sodium acetate was added to MP2 medium. In 1-l batch fermentations, C. beijerinckii BA101 produced 32.6 g/l total solvents, with butanol at 20.9 g/l, when grown in MP2 medium containing 60 mM sodium acetate and 8% glucose. To our knowledge, these values represent the highest solvent and butanol concentrations produced by a solventogenic Clostridium strain when grown in batch culture. Received: 29 September 1998 / Received revision: 13 February 1999 / Accepted: 26 February 1999     

Acetone butanol ethanol (ABE) production from concentrated substrate: reduction in substrate inhibition by fed-batch technique and product inhibition by gas stripping

Acetone butanol ethanol (ABE) was produced in an integrated fed-batch fermentation-gas stripping product-recovery system using Clostridium beijerinckii BA101, with H₂ and CO₂ as the carrier gases. This technique was applied in order to eliminate the substrate and product inhibition that normally restricts ABE production and sugar utilization to less than 20 g l^–1 and 60 g l^–1, respectively. In the integrated fed-batch fermentation and product recovery system, solvent productivities were improved to 400% of the control batch fermentation productivities. In a control batch reactor, the culture used 45.4 g glucose l^–1 and produced 17.6 g total solvents l^–1 (yield 0.39 g g^–1, productivity 0.29 g l^–1 h^–1). Using the integrated fermentation-gas stripping product-recovery system with CO₂ and H₂ as carrier gases, we carried out fed-batch fermentation experiments and measured various characteristics of the fermentation, including ABE production, selectivity, yield and productivity. The fed-batch reactor was operated for 201 h. At the end of the fermentation, an unusually high concentration of total acids (8.5 g l^–1) was observed. A total of 500 g glucose was used to produce 232.8 g solvents (77.7 g acetone, 151.7 g butanol, 3.4 g ethanol) in 1 l culture broth. The average solvent yield and productivity were 0.47 g g^–1 and 1.16 g l^–1 h^–1, respectively.     

Effects of acetic and formic acid on ABE production by <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> and <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

The effect of acetic acid and formic acid on acetone-butanol-ethanol (ABE) production by solventogenic Clostridia was investigated. The ABE concentration in Clostridium acetobutylicum was found to have increased slightly on addition of 3.7 ∼ 9.7 g/L acetic acid, but was found to have drastically reduced in the presence of 11.7 g/L acetic acid. However, the solvent production of C. beijerinckii was not affected by addition of acetic acid in the range of 3.7 ∼ 11.7 g/L. C. acetobutylicum was more vulnerable to formic acid than C. beijerinckii. In C. acetobutylicum, the total ABE production decreased to 77% on addition of 0.4 g/L formic acid and 25% with 1.0 g/L formic acid. The total ABE production by C. acetobutylicum was also noted to have decreased from 15.1 to 8.6 g/L when 8.7 g/L acetic acid and 0.4 g/L formic acid co-existed. The solvent production by C. beijerinckii was not affected at all under the tested concentration range of formic acid (0.0 ∼ 1.0 g/L) and co-presence of acetic acid and formic acid. Therefore, C. beijerinckii is more favorable than C. acetobutylicum when the ABE is produced using lignocellulosic hydrolysate containing acetic and formic acid.     

β-Galactosidase from Talaromyces thermophilus immobilized on to Eupergit C for production of galacto-oligosaccharides during lactose hydrolysis in batch and packed-bed reactor

The β-galactosidase from Talaromyces thermophilus CBS 236.58 immobilized onto Eupergit C produced galacto-oligosaccharides (GalOS) in batchwise and continuous packed-bed mode of operation. A maximum yield of GalOS of 12, 39 and 80 g l⁻¹ was obtained for initial lactose concentrations of 50, 100 and 200 g l⁻¹, respectively, for batch conversion experiments. The immobilized enzyme could be re-used for several cycles for lactose hydrolysis and transformation. The maximum GalOS concentration of approximately 50 g l⁻¹ was obtained with the dilution rate of 0.375 h⁻¹ in a packed-bed reactor, when using an initial lactose concentration of 200 g l⁻¹. Continuous conversion of lactose in the packed-bed reactor resulted in the formation of relatively more trisaccharides than when employing the immobilized enzyme in discontinuous mode of operation.     

Residual phosphate concentration under nitrogen-limiting conditions regulates curdlan production in Agrobacterium species

We investigated the influence of inorganic phosphate concentration on the production of curdlan by Agrobacterium species. A two-step culture method was employed where cells were first cultured, followed by curdlan production under nitrogen-limiting conditions. In the curdlan production step, cells did not grow but metabolized sugar into curdlan. Shake-flask experiments showed that the optimal phosphate concentration for curdlan production was in the range of 0.1–0.3 g l⁻¹. As the cell concentration increased from 0.42 to 1.68 g l⁻¹ in shake-flask cultures, curdlan production increased from 0.44 to 2.80 g l⁻¹. However, the optimal phosphate concentration range was not dependent upon cell concentration. The specific production rate was about 70 mg curdlan g-cell⁻¹ h⁻¹ irrespective of cell concentration. When the phosphate concentration was maintained at 0.5 g l⁻¹ under nitrogen-limiting conditions, as high as 65 g l⁻¹ of curdlan was obtained in 120 h. Journal of Industrial Microbiology & Biotechnology (2000) 25, 180–183. Received 25 October 1999/ Accepted in revised form 21 July 2000     

Production of acetone–butanol–ethanol (ABE) in a continuous flow bioreactor using degermed corn and Clostridium beijerinckii

An examination of the sustainability of the long-term cultivation of C. beijerinckii BA101 in degermed corn/saccharified degermed corn based P2 medium has been described in this work. It was found that long-term continuous cultivation of C. beijerinckii BA101 in a degermed corn based medium was not possible due to the instability of the gelatinized degermed corn starch during storage often called “retrogradation”. Using this substrate, continuous ABE fermentation was run for 228 h, before the fermentation turned acidogenic. However continuous fermentations of saccharified degermed corn with normal and half P2 medium nutrients were successful. In saccharified degermed corn continuous fermentation, ABE concentration up to 14.28 g/L was achieved at a dilution rate of 0.03 h⁻¹. This work demonstrated that byproduct (germ/oil, corn fiber) credit can be obtained by fermenting saccharified degermed corn in continuous flow bioreactors. Additionally significant savings can be achieved by supplementing with half of normal P2 medium nutrients.     

Cold hardiness in Entomobrya nivalis (Collembola, Entomobryidae): annual cycle of polyols and antifreeze proteins, and antifreeze triggering by temperature and photoperiod

In the Swiss Prealps Entomobrya nivalis hibernates in an inactive state, hidden under bark flakes on spruce. For freeze avoidance it relies on thermal hysteresis proteins (THPs) and polyols (mainly ribitol, with small amounts arabitol and threitol). Polyols are present only during the inactive state, THPs additionally protect during the transition phase in spring and autumn, when animals are still active but frosts may occur. Peak values were recorded in February/March for THPs (3.5 °C hysteresis between melting and freezing point) and for polyols (26 μg mg⁻¹ FW; hemolymph osmolality 680 mosmol l⁻¹). E. nivalis is able to control its hemolymph osmolality independently of body water content. Mean osmolality in summer was 350– 440 mosmol l⁻¹, in winter it was elevated to 650 mosmol l⁻¹, due to a synthesis mainly of ribitol. Body water content varied between 1.8 and 3.3 mg H₂O mg⁻¹ DW, depending on humidity conditions. Experiments on triggering of antifreeze synthesis showed the action of temperature and photoperiod as cues, but there was also evidence for an endogenous rhythm. No clear correlation between antifreeze concentration and supercooling ability could be established, suggesting that gut content or other parameters also play an inportant role. Accepted: 18 November 1995