Use of spent brewer's yeast in L‐(+) lactic acid fermentation

The application of by‐products from the brewing industry in lactic acid (LA) production was investigated in order to replace expensive nitrogen sources (such as yeast extract) with cheaper and renewable nitrogenous materials such as brewer's yeast (BY). In this study, brewer's spent grain (BSG) hydrolysate was used for L‐(+)‐LA fermentation by Lactobacillus rhamnosus ATCC 7469. The effect of pH control during the fermentation and the addition of various BY contents (5–50 g/L) in BSG hydrolysate on fermentation parameters was evaluated. BY addition significantly increased free amino nitrogen (FAN) concentration (by 25.2% at 5 g/L to 616% at 50 g/L). A strong positive correlation between FAN concentration in the hydrolysate and concentration of L‐(+)‐LA produced was observed (correlation coefficient of 0.913). A high cell viability of L. rhamnosus ATCC 7469 (1.95–3.32 × 10⁹ CFU/mL at the end of fermentation) was achieved in all fermentations with the addition of brewer's yeast. The addition of BY increased L‐(+)‐lactic acid yield and volumetric productivity up to 8.4% (5 g/L) and 48.3% (50 g/L). The highest L‐(+)‐LA yield (89%) and volumetric productivity (0.89 g/L h^?1) were achieved in fermentation of BSG hydrolysate with 50 g/L of BY. © 2019 The Institute of Brewing & Distilling     

Fed‐batch l‐(+)‐lactic acid fermentation of brewer's spent grain hydrolysate

Brewer's spent grain (BSG) hydrolysates were used for l ‐(+)‐lactic acid (LA) fermentation by Lactobacillus rhamnosus ATCC 7469. The aim of this study was to evaluate fed‐batch LA fermentation of BSG hydrolysate with the addition of glucose, glucose and yeast extract, and wort during LA fermentation and its effect on fermentation parameters such as LA concentration, its volumetric productivity and yield, and L. rhamnosus cell viability. The highest LA yield, volumetric productivity and concentration of 93.3%, 2.0 g/L/h, and 116.1 g/L, respectively, were achieved with glucose and yeast extract addition during fermentation. In fed‐batch fermentation with glucose and yeast extract addition significantly higher LA concentration, yield and volumetric productivity (by 194.8; 2.2, and 20.7%, respectively) were achieved compared with batch fermentation. The results indicated that fed‐batch fermentation could be used to increase LA fermentation efficiency. Copyright © 2017 The Institute of Brewing & Distilling     

Lactic acid fermentation of brewer's spent grain hydrolysate by Lactobacillus rhamnosus with yeast extract addition and pH control

Lactic acid (LA) is a versatile chemical with a wide range of applications in food, pharmaceutical, cosmetic, textile and polymer industries. Brewer's spent grain (BSG) is the most abundant brewing by‐product. In this study BSG hydrolysates were used for LA fermentation by Lactobacillus rhamnosus ATCC 7469. The aim of this study was to evaluate the effects of pH control during fermentation, reducing sugar content and yeast extract content in BSG hydrolysate on LA fermentation parameters. The pH control greatly increased reducing sugar utilization, l ‐(+)‐LA content, yield and volumetric productivity. The highest l ‐(+)‐LA yield and volumetric productivity were achieved with the reducing sugar content of 54 g/L. Yeast extract addition significantly increased reducing sugar utilization, l ‐(+)‐LA content, L. rhamnosus cell viability, l ‐(+)‐LA yield and volumetric productivity. The highest l ‐(+)‐LA content (39.38 g/L), L. rhamnosus cell viability (9.67 log CFU/mL), l ‐(+)‐LA yield (91.29%) and volumetric productivity (1.69 g/L/h) were achieved with the reducing sugar content of 54 g/L and yeast extract content of 50 g/L. Copyright © 2017 The Institute of Brewing & Distilling     

The influence of calcium-carbonate and yeast extract addition on lactic acid fermentation of brewer's spent grain hydrolysate

Brewer's spent grain (BSG) is the major by-product of the brewing industry, representing around 85% of the total by-products generated. In this study BSG hydrolysate was produced using optimal conditions. Hydrolysates were used for lactic acid (LA) fermentation by Lactobacillus fermentum (PL-1) and Lactobacillus rhamnosus (ATCC 7469). The aim of this study was to evaluate possibilities of the BSG hydrolysate utilization as a substrate for LA fermentation. The effect of calcium-carbonate (2%) and yeast extract (0.5 to 5%) addition in hydrolysate on LA fermentation were investigated. The LA production by L. fermentum and L. rhamnosus in BSG hydrolysate was influenced by calcium-carbonate and yeast extract supplementation. L. fermentum produced a racemic mixture of L-(+)- and D-(−)-LA while L. rhamnosus produced mostly L-(+)-LA (95–98%) in all fermentations. Calcium-carbonate addition increased total LA yield by 13% in L. fermentum fermentations and by 17% in L. rhamnosus fermentations. Yeast extract addition increased total LA yield by 4–26% in L. fermentum fermentations and by 6–8% in L. rhamnosus fermentations.     

Production of L‐Lactic Acid from Spent Grain,a By‐Product of Beer Production

L‐lactic acid production from spent grain with immobilized lactic acid bacteria was investigated. Spent grains were liquefied by a steam explosion treatment to obtain liquefied sugar. When 1 kg of wet spent grain was treated under the 30 kg/cm²pressure for 1 min using a 5‐L steam explosion reactor, 60 g of total sugar was obtained from the liquefied spent grain. Furthermore, 1.3% (w/v) of glucose, 0.4% (w/v) of xylose, and 0.1% (w/v) of arabinose were produced when the liquefied spent grain was treated with glucoamylase, cellulase, and hemicellulase enzymes. When batch L‐lactic acid production was carried out by Lactobacillus rhamnosus NBRC14710, 19.0 g/L L‐lactic acid was produced from the Tween 80 liquefied spent grain after 5 days. Furthermore, during repeated batch production with immobilized Lactobacillus rhamnosus NBRC14710 from Tween 80 liquefied spent grain at 37°C, the productivity of L‐lactic acid was maintained at a 10 time higher level over a period of 40 days.     

Direct starch conversion into L‐(+)‐lactic acid by a novel amylolytic strain of Lactobacillus paracasei B41

A new amylolytic strain of Lactobacillus paracasei able to convert starch directly into L ‐(+)‐lactic acid (LA) was isolated. The identification of the by 16S rDNA sequencing proved that this strain, B41, is the first amylolytic representative of Lactobacillus casei group. The amylase activity assay revealed that L. paracasei B41 produced extracellular amylolytic enzyme, reaching 62 U/mL in the cell‐free supernatants. The optimal conditions for its action were pH 5.0 and temperature 45°C. The gene amy1 (1779 bp) encoding the putative B41 amylopullulanase was cloned, sequenced, and analyzed. The deduced protein contained a leader peptide of 28 amino acids and a mature peptide of 564 amino acids. Compared to the amylases of closely related species, B41 enzyme had several amino acid substitutions. An inducible control at amy1 expression was demonstrated. The starch fermentation abilities of L. paracasei B41 were studied in batch processes performed with and without pH control. The highest amount of LA from starch was obtained during 48 h fermentation from 40 g/L substrate at pH maintained at 5.0–37.3 g/L. In addition, 93.3% starch conversion into LA and the highest reported productivity for 24 h were achieved – 1.30 g/L/h.     

Impact of buffering capacity on the acidification of wort by brewing‐relevant lactic acid bacteria

Acidified wort produced biologically using lactic acid bacteria (LAB) has application during sour beer production and in breweries adhering to the German purity law (Reinheitsgebot ). LAB cultures, however, suffer from end product inhibition and low pH, leading to inefficient lactic acid (LA) yields. Three brewing‐relevant LAB (Pediococcus acidilactici AB39, Lactobacillus amylovorus FST2.11 and Lactobacillus plantarum FST1.7) were examined during batch fermentation of wort possessing increasing buffering capacities (BC). Bacterial growth was progressively impaired when exposed to higher LA concentrations, ceasing in the pH range of 2.9–3.4. The proteolytic rest (50°C) during mashing was found to be a major factor improving the BC of wort. Both a longer mashing profile and the addition of an external protease increased the BC (1.21 and 1.24, respectively) compared with a control wort (1.18), and a positive, linear correlation (R ² = 0.957) between free amino nitrogen and BC was established. Higher levels of BC led to significant greater LA concentration (up to +24%) after 48 h of fermentation, reaching a maximal value of 11.3 g/L. Even higher LA (maximum 12.8 g/L) could be obtained when external buffers were added to wort, while depletion of micronutrient(s) (monosaccharides, amino acids and/or other unidentified compounds) was suggested as the cause of LAB growth cessation. Overall, a significant improvement in LA production during batch fermentation of wort is possible when BC is improved through mashing and/or inclusion of additives (protease and/or external buffers), with further potential for optimization when strain‐dependent nutritional requirements, e.g. sugar and amino acids, are considered. Copyright © 2017 The Institute of Brewing & Distilling     

Integral utilisation of barley husk for the production of food additives

A new and effective chemical–biotechnological process for the global utilisation of barley husk (obtained from the spent grains in the brewing process) is reported. With the proposed process the three main components of the lignocellulosic residue (cellulose, hemicellulose and lignin) are utilised. A first treatment with sulfuric acid (pre‐hydrolysis) allowed the solubilisation of hemicelluloses to give xylose and glucose‐containing liquors (suitable to make fermentation media for the continuous lactic acid (LA) production with L. pentosus) and a solid phase containing cellulose and lignin. In this set of experiments, a maximum volumetric productivity (Q_P) = 2.077 g L^?1 h^?1 and product yield (Y_P/S) = 0.62 g g^?1 were obtained for a dilution rate of 0.01 h^?1. The solid residues from pre‐hydrolysis were treated with NaOH in order to increase their cellulase digestibility, and dissolve the lignin content. The cellulose residue was used as substrates for lactic acid production by simultaneous saccharification and fermentation (SSF) in media containing Trichoderma reesei cellulases and Lactobacillus rhamnosus cells using the complete MRS broth or a cheaper medium. In both cases similar LA concentrations and volumetric productivities were achieved (P = 73.4–71.0 g L^?1 and Q_P = 1.28–1.25 g L^?1 h^?1, respectively), where P is LA concentration. The lignin solution obtained after the alkaline treatment was extracted with ethyl acetate in order to obtain the phenolic components. The extract obtained at pH 3 showed three times more antioxidant activity than the one extracted at pH 12.8, with an EC₅₀ of 1.396 g L^?1 for pH 3 and 4.604 g L^?1 for pH 12.8. The best extracts showed twice antioxidant activity than BHT. Copyright © 2007 Society of Chemical Industry     

Improvement of L(+)‐lactic acid production from cassava wastewater by Lactobacillus rhamnosus B 103

BACKGROUND: L (+)‐Lactic acid is used in the pharmaceutical, textile and food industries as well as in the synthesis of biodegradable plastics. The aim of this study was to investigate the effects of different medium components added in cassava wastewater for the production of L (+)‐lactic acid by Lactobacillus rhamnosus B 103. RESULTS: The use of cassava wastewater (50 g L^?1 of reducing sugar) with Tween 80 and corn steep liquor, at concentrations (v/v) of 1.27 mL L^?1 and 65.4 mL L^?1 respectively led to a lactic acid concentration of 41.65 g L^?1 after 48 h of fermentation. The maximum lactic acid concentration produced in the reactor after 36 h of fermentation was 39.00 g L^?1 using the same medium, but the pH was controlled by addition of 10 mol L^?1 NaOH. CONCLUSION: The use of cassava wastewater for cultivation of L. rhamnosus is feasible, with a considerable production of lactic acid. Furthermore, it is an innovative proposal, as no references were found in the scientific literature on the use of this substrate for lactic acid production. Copyright © 2010 Society of Chemical Industry     

固定化热带假丝酵母发酵氨浸稻秸水解液生产木糖醇

采用海藻酸钙固定化热带假丝酵母细胞发酵氨水浸泡稻秸半纤维素水解液生产木糖醇。为了提高木糖醇的转化率,对发酵条件进行了研究。发酵在250 mL锥形瓶中进行。向水解液中补充适量氮源和营养盐等营养物质提高了木糖醇的生产速率,但木糖醇转化率没有因此而提高。适宜的初始pH和细胞干浓度分别为4-5和1.22 g/L。在这些条件下,进行了固定化细胞重复法较高浓缩度水解液的试验。结果发现,固定化细胞能在初始木糖浓度为104.2 g/L的水解液中重复批式发酵5次,木糖醇平均得率和生产速率分别为0.737 g/g和0.533 g/(L.h)。     

Bioconversion of agro‐industrial by‐products to lactic acid using Lactobacillus sakei and two Pediococcus spp. strains

The aim of this study was to evaluate the bioconversion efficiency of rich in cellulose agro‐industrial by‐products such as wheat bran (WB), spent distiller's grain with solids (DGS), brewer's spent grain (BSG) and lupin (Lupinus angustifolius L.) wholemeal fraction (LF) to lactic acid (LA) using acid tolerant lactic acid bacteria (LAB) strains Lactobacillus sakei KTU05‐06, Pediococcus acidilactici KTU05‐7 and P. pentosaceus KTU05‐9. Carbohydrase preparation Depol^? 692L was used for the hydrolysis of non‐starch polysaccharides. Analysed raw materials were suitable substrates for LAB propagation and L‐lactic acid production. The lowest pH (3.6) was found in LF medium after 48 h fermentation with P. acidilactici and P. pentosaceus strains. The lowest pH (3.86) was measured in WB fermented with L. sakei, and in DGS and BSG (pH 3.8 and 3.9 respectively) fermented with P. acidilactici. The highest endoxylanase activity was excreted by the P. acidilactici and P. pentosaceus (84 and 69 XU g^?1 respectively), and the highest α‐amylase activity was of L. sakei (255.6 AU g^?1) after 24 h incubation in WB medium. The L‐lactic acid concentration of 86.11 g kg^?1 was reached after the bioconversion of hydrolysed WB in combination with 48 h fermentation by P. pentosaceus KTU05‐9 strain. LA contents between 222 and 282 mg kg^?1 was produced from lupin processing residues via fermentation using P. acidilactici and P. pentosaceus KTU05‐9 strains. The major challenge within the presented study is the viability of tested LAB in cereal waste media and effective LA production at a low pH (3.6–3.8).     

Production of lactic acid from vine‐trimming wastes and viticulture lees using a simultaneous saccharification fermentation method

An effective process for the chemical–biotechnological utilization of trimming wastes of vineshoots, an agricultural waste with little use, is reported. Initial treatment with sulfuric acid (prehydrolysis) allowed the solubilization of hemicelluloses to give xylose and glucose‐containing liquors (suitable to make fermentation media for lactic acid production with Lactobacillus pentosus) and a solid phase containing cellulose and lignin. The solid residues from prehydrolysis were treated with NaOH in order to increase their cellulase digestibility. In the alkaline treatments, the effects of temperature (in the range, 50–130 °C), reaction time (30–120 min) and NaOH concentration (4–12 wt% of solution) on the composition and susceptibility to enzymatic hydrolysis of solid residues were assessed by means of an experimental plan with factorial structure. The lignin content decreased, whereas the susceptibility towards the enzymatic hydrolysis increased with temperature, reaction time and NaOH concentration within the tested range. Using the cellulosic residues achieved under the harsher conditions, favorable fermentation kinetics during simultaneous saccharification and fermentation carried out by L rhamnosus for lactic acid production were observed. The nutrients employed were the complete MRS broth and a cheaper medium developed using viticulture lees coming from the white wine making technology. In all cases the final lactic acid concentration achieved was similar, although the volumetric productivity was lower when using lees due to inhibitory effects over the enzymatic hydrolysis. Copyright © 2004 Society of Chemical Industry     

Simultaneous lactic acid and xylitol production from vine trimming wastes

Hemicellulosic hydrolyzates from vineshoot trimmings obtained by dilute sulfuric acid hydrolysis were evaluated for xylitol production by Debaryomyces hansenii NRRL Y‐7426. Bioconversion was not efficient, however, since a mixture of products (mainly ethanol) was achieved. Taking into account that hexoses (such as glucose or mannose) can inhibit xylose metabolism by repression and inactivation of the xylose transport system or catabolic enzymes and that these hemicellulosic hydrolyzates are characterized by a high glucose concentration, a novel technology was developed, sequentially transforming glucose into lactic acid by Lactobacillus rhamnosus followed by fermentation of xylose into xylitol by Debaryomyces hansenii after L. rhamnosus removal by microfiltration. Optimal conditions were achieved using detoxified concentrated hemicellulosic hydrolyzates, after CaCO₃ addition in both stages of fermentation and using nitrogen purges after sampling in order to reduce the oxygen dissolved. Under these conditions 31.5 g lactic acid L^?1 (Q_LA = 1.312 g L^?1 h^?1 and Y_LA/S = 0.93 g g^?1) and 27.5 g xylitol L^?1 (Q_Xylitol = 0.458 g L^?1 h^?1 and Y_Xylitol/S = 0.53 g g^?1) were produced. Finally, lactic acid was selectively recovered using the resin Amberlite IRA 400 (0.0374 g of lactic acid g^?1 of dry resin), allowing a further recovery of xylitol by sequential stages of adsorption, concentration, ethanol precipitation, concentration and crystallization to obtain food‐grade xylitol according to a developed process. Copyright © 2007 Society of Chemical Industry     

Biovalorization of brewers’ spent grain for the production of laccase and polyphenols

Brewers’ spent grain (BSG) is the main solid by‐product of the brewing process and is typically disposed of as cattle feed. In this study, BSG was evaluated as a substrate for the production of polyphenols and the lignin‐degrading enzyme laccase using fungal solid‐state fermentation by Trametes versicolor. Laccases are finding increasing applications in the food industry and polyphenols have benefits for human health. After 14 days of fermentation with T. versicolor, there was a 3.4‐fold increase in the extraction of total polyphenols compared with untreated BSG. Using BSG as the sole source of carbon and nitrogen, maximum laccase activity was achieved after seven days of treatment with an activity of 560 U/L. Based on these results, BSG is suggested to be a good lignocellulose waste material to produce value‐added products such as the enzyme laccase and polyphenols. Copyright © 2018 The Institute of Brewing & Distilling     

Increase of xylitol productivity by cell-recycle fermentation of Candida tropicalis using submerged membrane bioreactor

Candida tropicalis, an osmophilic strain isolated from honeycomb, produced xylitol at a maximal volumetric productivity of 3.5 g l(-1) h(-1) from an initial xylose concentration of 200 g l(-1). Even at a very high xylose concentration, e.g., 350 g l(-1), this strain produced xylitol at a moderate rate of 2.07 g l(-1) h(-1). In a fed-batch fermentation of xylose and glucose, 260 g l(-1) xylose was added, and the xylitol production was 234 g l(-1) for 48 h, corresponding to a rate of 4.88 g l(-1) h(-1). To increase xylitol productivity, cells were recycled in a submerged membrane bioreactor with suction pressure and air sparging. For each recycle round in cell-recycle fermentation, the average concentration of xylitol produced, fermentation time, volumetric productivity, and product yield were 180 g l(-1), 19.5 h, 8.5 g l(-1) h(-1), and 85%, respectively. When cell-recycle fermentation was started with the cell mass concentrated twofold after batch fermentation and performed for 10 recycle rounds, we achieved a very high productivity of 12 g l(-1) h(-1). The productivity and total amount of xylitol in cell-recycle fermentation were 3.4- and 11.0-fold higher than those in batch fermentation, respectively.     

Fermented sugarcane and pineapple beverage produced using Saccharomyces cerevisiae and non‐Saccharomyces yeast

The potential of Saccharomyces cerevisiae (strains UFLA CA11 and UFLA FW15) and Pichia caribbica (UFLA CAF733) to produce a fermented sugarcane and pineapple drink was evaluated. Co‐ and pure cultures using different proportions of sugarcane juice and pineapple pulp (80:20, 70:30 and 60:40) were prepared. The sugar concentration of the must was adjusted to 16° Brix and was inoculated with approximately 7 log CFU/mL. After a preliminary test and based on higher concentrations of desirable volatile components, low production of acetic acid, high production of ethanol, and kinetic parameters, P. caribbica was chosen to perform the fermentation in 5 L batches. The fermentation performed with P. caribbica in the proportion of 60:40 showed a yield in ethanol of 0.45 g/g, an ethanol productivity of 1.32 g/L/h and a fermentation efficiency of 88.22%. The maximum ethanol concentration was 79.78 g/L and P. caribbica increased concentrations of desirable volatile compounds, such as 2‐phenyethanol, 2‐methyl‐1‐propanol, 3‐methyl‐1‐butanol, ethyl acetate and phenylethyl acetate. Copyright © 2015 The Institute of Brewing & Distilling     

Efficient production of L‐lactic acid by Crabtree‐negative yeast Candida boidinii

Industrial production of L ‐lactic acid, which in polymerized form as poly‐lactic acid is widely used as a biodegradable plastic, has been attracting world‐wide attention. By genetic engineering we constructed a strain of the Crabtree‐negative yeast Candida boidinii that efficiently produced a large amount of L ‐lactic acid. The alcohol fermentation pathway of C. boidinii was altered by disruption of the PDC1 gene encoding pyruvate decarboxylase, resulting in an ethanol production that was reduced to 17% of the wild‐type strain. The alcohol fermentation pathway of the PDC1 deletion strain was then successfully utilized for the synthesis of L ‐lactic acid by placing the bovine L ‐lactate dehydrogenase‐encoding gene under the control of the PDC1 promoter by targeted integration. Optimizing the conditions for batch culture in a 5 l jar‐fermenter resulted in an L ‐lactic acid production reaching 85.9 g/l within 48 h. This productivity (1.79 g/l/h) is the highest thus far reported for L ‐lactic acid‐producing yeasts. DDBJ/EMBL/GenBank nucleotide database with Accession Nos. AB440630 and AB440631. Copyright © 2009 John Wiley & Sons, Ltd.     

Klebsiella pneumoniae在不同发酵罐中发酵甘油制1,3-丙二醇的控制条件研究

对克雷伯氏肺炎杆菌间歇发酵甘油产生1,3-丙二醇的发酵条件进行了优化,确定了50L搅拌式发酵罐和15L气升式发酵罐发酵的最佳发酵条件,得到的1,3-丙二醇的浓度、生产率和甘油摩尔转化率分别是80.97g/L、1.69 g/(h·L)、0.57 mol/mol和74.81 g/L、1.56 g/(h·L)、0.50 mol/mol;对比了不同类型发酵罐的发酵结果,表明50 L搅拌式发酵罐发酵结果优于15L气升式发酵罐,前者相对于后者来说,1,3-丙二醇的浓度和甘油摩尔转化率分别提高了8.23%和14.91%;确定了最适底物浓度(以初始甘油浓度计)在25 g/L左右。     

渗透汽化原位分离耦合拜氏梭菌丁醇发酵的研究

以筛选得到的聚二甲基硅氧烷（polydimethylsiloxane,PDMS）-聚偏氟乙烯（polyvinylidene fluoride,PVDF）复合膜为分离用膜,开展了拜氏梭菌（Clostridium beijerinckii）ZL01丁醇发酵与渗透汽化原位分离耦合的研究,结果表明：分批发酵-渗透汽化原位分离耦合与分批发酵相比,初始葡萄糖质量浓度从50 g/L提高至90 g/L;在90 g/L的初始葡萄糖质量浓度下,发酵结束时发酵液和渗透液中的丁醇总产量从13.2 g/L提高到16.9 g/L,总溶剂（丙酮（acetone）、丁醇（butanol）、乙醇（ethanol）,简称ABE）产量从17.8 g/L提高到24.3 g/L,葡萄糖利用率从59.4%提高到95.7%。另外,分离过程中膜的总渗透通量平均为705 g/（m2·h）,丁醇分离因子平均为19.0;经渗透汽化分离,渗透液可直接进入下一步蒸馏阶段,其中丁醇和总溶剂ABE质量浓度分别为178 g/L和292 g/L,与分批发酵工艺中发酵液直接进入蒸馏塔相比,丁醇和总溶剂ABE质量浓度分别提高了10.9 倍和14.1 倍,可大大降低蒸馏能耗。     

Cashew Apple Juice as Substrate for Lactic Acid Production

The aim of the present work was to investigate the use of cashew apple juice as a low cost substrate for Lactobacillus casei B-442 cultivation and lactic acid production. Ammonium sulfate was employed as the only exogenous nitrogen source. The effect of cashew apple juice reducing sugars and ammonium sulfate concentration and the fermentation pH and temperature on biomass formation, lactic acid production, and productivity were evaluated. The highest productivity (2.36 g/L.h) was obtained applying 50 g/L of reducing sugar from the cashew apple juice supplemented with 6 g/L of ammonium sulfate. The process yield was about 95% when fermentation was carried out at 37 °C with pH controlled at pH 6.5 using NaOH (120 g/L).