Influence of moisture content and cultivation duration on Clostridium thermocellum 27405 end-product formation in solid substrate cultivation on Avicel

Avicel serves as a model microcrystalline cellulose substrate for investigations of cellulolytic microbial performance and cellulase enzyme systems in submerged liquid cultures. Clostridium thermocellum is a thermophilic, anaerobic bacterium capable of degrading lignocellulose and fermenting it to ethanol and other products, suggesting the native growth environment is similar to that supported by solid substrate cultivation. Few studies have examined the effects of process parameters on the metabolism of thermophilic anaerobes in solid substrate cultivation, however. The effects of solid substrate cultivation (SSC) substrate moisture content (30%, 50% and 70% wet-basis) and cultivation duration (2, 4 and 8 days) on the metabolic activity of C. thermocellum 27405 on Avicel was studied. The 70% substrate moisture content SSC culture yielded total end-product concentrations that were comparable to submerged liquid cultures. The SSC cultivation conditions with the highest end-product formation on Avicel were the combination of 70% substrate moisture content and cultivation duration period of 4 days, producing approximately 100mM of total end-products. The ethanol and lactate concentrations were fairly constant and did not change significantly over time in SSC. Acetate production was more dependent on the cultivation conditions in SSC and was significant for both the 70% substrate moisture content SSC and liquid cultivation experiments, making up on average 56% and 86% of total end-products, respectively. Performance of C. thermocellum 27405 in SSC was more dependent on the kinetic properties rather than the thermodynamic properties of substrate moisture content. High substrate loadings in C. thermocellum cultivation affected product ratios, resulting in the higher observed acetate production. In addition, cessation of metabolism was observed prior to complete Avicel conversion; the mechanisms involved need further investigation.     

Screening of thermophilic anaerobic bacteria for solid substrate cultivation on lignocellulosic substrates

Interest in solid substrate cultivation (SSC) techniques is gaining for biochemical production from renewable resources; however, heat and mass transfer problems may limit application of this technique. The use of anaerobic thermophiles in SSC offers a unique solution to overcoming these challenges. The production potential of nine thermophilic anaerobic bacteria was examined on corn stover, sugar cane bagasse, paper pulp sludge, and wheat bran in submerged liquid cultivation (SmC) and SSC. Production of acetate, ethanol, and lactate was measured over a 10 day period, and total product concentrations were used to compare the performance of different organism-substrate combinations using the two cultivation methods. Overall microbial activity in SmC and SSC was dependent on the organism and growth substrate. Clostridium thermocellum strains JW20, LQRI, and 27405 performed significantly better in SSC when grown on sugar cane bagasse and paper pulp sludge, producing at least 70 and 170 mM of total products, respectively. Growth of C. thermocellum strains in SSC on paper pulp sludge proved to be most favorable, generating at least twice the concentration of total products produced in SmC (p-value < 0.05). Clostridium thermolacticum TC21 demonstrated growth on all substrates producing 30-80 and 60-116 mM of total product in SmC and SSC, respectively. Bacterial species with optimal growth temperatures of 70 degrees C grew best on wheat bran in SmC, producing total product concentrations of 45-75 mM. For some of the organism-substrate combinations total end product concentrations in SSC exceeded those in SmC, indicating that SSC may be a promising alternative for microbial activity and value-added biochemical production.     

Microbial pretreatment of cotton stalks by solid state cultivation of Phanerochaete chrysosporium

White rot fungi degrade lignin and have biotechnological applications in conversion of lignocellulose to valuable products. Pretreatment is an important processing step to increase the accessibility of cellulosic material in plant biomass, impacting efficiency of subsequent hydrolysis and fermentation. This study investigated microbial pretreatment of cotton stalks by solid state cultivation (SSC) using Phanerochaete chrysosporium to facilitate the conversion into ethanol. The effects of substrate moisture content (M.C.; 65%, 75% and 80% wet-basis), inorganic salt concentration (no salts, modified salts without Mn(2+), modified salts with Mn(2+)) and culture time (0-14 days) on lignin degradation (LD), solids recovery (SR) and availability of carbohydrates (AOC) were examined. Moisture content significantly affected lignin degradation, with 75% and 80% M.C. degrading approximately 6% more lignin than 65% M.C. after 14 days. Within the same moisture content, treatments supplemented with salts were not statistically different than those without salts for LD and AOC. Within the 14day pretreatment, additional time resulted in greater lignin degradation, but indicated a decrease in SR and AOC. Considering cost, solid state cultivation at 75% M.C. without salts was the most preferable pretreatment resulting in 27.6% lignin degradation, 71.1% solids recovery and 41.6% availability of carbohydrates over a period of 14 days. Microbial pretreatment by solid state cultivation has the potential to be a low cost, environmentally friendly alternative to chemical approaches. Moisture relationships will be significant to the design of an effective microbial pretreatment process using SSC technology.     

构树的污泥适应性及养分和重金属吸收累积特征

构树是我国重要速生经济树种,具有适应性强、生物量大和重金属富集能力强等优点,而污泥中含有大量养分和重金属,在污泥中种植构树有望同时实现污泥生态修复和构树资源生产。本研究通过盆栽试验,分析在对照(赤红壤)、50%污泥(污泥、赤红壤混合基质,重量比各50%)和100%污泥基质中构树生长及不同部位(根、茎、叶)养分和重金属吸收累积特征,并通过主成分分析和隶属度函数对吸收累积能力进行综合评价。结果表明: 构树在50%和100%污泥中均可正常生长且株高、生物量显著高于对照,在100%污泥中长势最好,质量指数(1.02)分别是对照和50%污泥处理的4.3和2.4倍。50%和100%污泥处理构树各部位N含量和茎P含量显著高于对照,100%污泥处理构树茎、叶K含量显著低于对照。构树对Cu、Zn、Pb、Cd、Ni的吸收部位以根为主,根系重金属含量与污泥比例呈正相关,叶Pb、Cd含量符合《饲料卫生标准》(GB 13078—2017)。构树对Cd的吸收累积效果好于其他重金属元素。与对照相比,50%和100%污泥处理构树根部Zn、Pb、Cd滞留率显著提高(57.8%~85.8%),100%污泥处理构树根部Cu、Ni滞留率显著提高(67.5%和74.8%)。污泥处理全株养分和重金属累积量均显著大于对照,其中100%污泥处理显著大于50%污泥处理。与50%污泥处理相比,100%污泥处理构树各部位及全株养分和重金属累积量大幅提高。不同处理下构树污泥适应性和元素吸收累积的综合评价得分为100%污泥(0.848)>50%污泥(0.344)>对照(0.080)。构树对污泥具有良好的适应性,在纯污泥中能够正常生长并具有较强的吸收累积养分和重金属能力,可在修复污泥的同时进行构树资源生产。     

Investigation of the use of agricultural byproducts for fungal protein production

Cellulose and nutrient salts as well as potato pulp and potato protein liquor (PPL), were used as substrates for the cultivation of Chaetomium cellulolyticum in batch and repeated-batch operations. Using cellulose as the substrate a linear relationship existed between the rates of cell mass formation and acid production. The repeated-batch process was controlled by NaOH consumption using a simple computer model. When the production of cell mass and acid stopped because of a lack of substrate cellulose was fed into the reactor. This occurred within 10 min at which point no NaOH-feed was needed to maintain a constant pH. Repeated-batch operations yielded higher cell concentrations and productivities than batch operations. The relationship between the NaOH and H₂SO₄ consumed, and the fungal mass concentration was complex in cultivation media containing potato pulp and PPL, because various substrates were consumed by the fungus simultaneously and successively. Therefore, for repeated-batch cultivation a constant time interval was used. Repeated-batch cultivation of the fungus on potato pulp and PPL did not yield higher cell concentrations and productivities than did batch cultivation. With the optimal pulp-to-PPL ratio a maximum specific growth rate of 0.61 h¹ was obtained. These investigations indicate, that potato pulp and PPL are well suited to fungal protein production by Chaetomium cellulolyticum for fodder supplement.     

Methanogenic toxicity in anaerobic digesters treating municipal wastewater

In upflow anaerobic sludge blanket (UASB) digesters treating raw sewage at low temperatures, the sludge progressively lost methanogenic activity, indicating the possibility of methanogenic activity inhibition caused by wastewater constituents. To check this fact, batch and semi-continuous methanogenic toxicity assays were carried out with raw and centrifuged sewage. Permanent methanogenic toxicity on anaerobic sludge of approximately 50% was found when the sludge exposure to wastewater was renewed in a semi-continuous way. A stronger methanogenic inhibition of about 70-100% was observed when an active anaerobic sludge was exposed to mixed liquor from the UASB digester treating municipal wastewater. Suspended solids removal from sewage slightly reduced methanogenic toxicity. Effective concentration of municipal wastewater that caused a 50% reduction in methanogenic activity was estimated to be in the range of 150-200 mg CODl(-1). As methanogenic inhibition appeared to be related to remaining COD, higher methanogenic toxicity in digesters operating with low conversion efficiency will be expected.     

Characterization of food waste as feedstock for anaerobic digestion

Food waste collected in the City of San Francisco, California, was characterized for its potential for use as a feedstock for anaerobic digestion processes. The daily and weekly variations of food waste composition over a two-month period were measured. The anaerobic digestibility and biogas and methane yields of the food waste were evaluated using batch anaerobic digestion tests performed at 50 degrees C. The daily average moisture content (MC) and the ratio of volatile solids to total solids (VS/TS) determined from a week-long sampling were 70% and 83%, respectively, while the weekly average MC and VS/TS were 74% and 87%, respectively. The nutrient content analysis showed that the food waste contained well balanced nutrients for anaerobic microorganisms. The methane yield was determined to be 348 and 435 mL/gVS, respectively, after 10 and 28 days of digestion. The average methane content of biogas was 73%. The average VS destruction was 81% at the end of the 28-day digestion test. The results of this study indicate that the food waste is a highly desirable substrate for anaerobic digesters with regards to its high biodegradability and methane yield.     

An untapped bacterial cellulolytic community enriched from coastal marine sediment under anaerobic and thermophilic conditions

A bacterial community with strong cellulose [filter paper (FP) and microcrystalline cellulose] degradation ability was isolated from the coastal marine environment. They were isolated under thermophilic (60?°C) and anaerobic cultivation conditions. The library of 16S rRNA gene clones revealed a total of 16 operational taxonomic units after 50 clones were surveyed. Sixty percent of the clones were most related to the type strain of Clostridium thermocellum with 16S rRNA gene identity around 87-89%. All of them showed extremely low sequence similarities and were novel at least in species level. The gene clone libraries of glycosyl hydrolase family 48 showed low gene and amino acid sequence similarities around 70-72%. The results indicated that the cellulose degradation systems in the specific environment have not been well studied. The enrichment could disrupt FP within 3?days in a basal medium. The cellulase activity of the community was comparable to that of C.?thermocellum LQR1. The main fermentation products were ethanol, acetic acid and butyric acid. This work identified a novel microbial resource with a potential in lignocellulose conversion and biofuel production.     

水肥处理对黄瓜土壤养分、酶及微生物多样性的影响

以津优1号黄瓜为试材,设3个土壤相对含水量水平（50%～60%、70%～80%、90%～100%）和2个肥料追施量（600 kg N·hm^-2和420 kg P₂O₅·hm^-2,420 kg N·hm^-2和294 kg P₂O₅·hm^-2）处理,研究了不同水肥供应对日光温室黄瓜土壤养分、酶活性及微生物多样性的影响.结果表明：土壤中NH₄⁺-N含量随施肥量的增加而提高,随土壤相对含水量的增加而降低;水肥供给的增加有利于提高土壤中速效磷含量和蔗糖酶活性;肥料增加使土壤中蛋白酶活性降低,而水分降低使土壤中脲酶活性提高.土壤中微生物多样性与土壤中养分含量无显著相关性,与土壤脲酶活性呈显著正相关,与蔗糖酶活性呈显著负相关.土壤相对含水量70%～80%、氮肥追施量600 kg N·hm^-2和420 kg P₂O₅·hm^-2处理的土壤养分含量、蔗糖酶、磷酸酶和脲酶活性较高,且土壤中微生物多样性和均匀度显著高于其他处理,土壤生产潜力最优.     

水分对有机基质栽培番茄生理特性、品质及产量的影响

以"齐粉"番茄品种为试材,研究了不同相对含水量对有机基质栽培番茄生长、生理特性、产量、品质和水分利用率的影响.结果表明:随着有机基质相对含水量的升高,番茄株高、茎粗、节间长和单株叶面积极显著增加,叶片色素含量、水势、渗透势及根系活力和果实产量显著增加,而番茄果实品质和水分利用率显著降低.从果实产量、品质和水分利用率方面综合考虑,80%基质相对含水量处理的果实产量达26 kg.m-2以上,可作为番茄有机基质栽培水分管理的量化指标;如果仅考虑果实品质,则50%基质相对含水量可以作为水分管理指标.     

Factors affecting the bioconversion of Philippine tung seed by black soldier fly larvae for the production of protein and oil-rich biomass

A systematic study on the use of Philippine tung (Reutealis trisperma) seed as a substrate for the cultivation of black soldier fly larvae (Hermetia illucens) was performed. The characteristics of Reutealis trisperma seed from two different locations: West Java and Papua, were determined. The seed has a relatively high oil (37.6–39.2%, dry weight) and protein content (14.9–28.2%, dry weight). The effect of cake content in the substrate (0–20%, wet weight), moisture content in the substrate (50–70%, wet weight), feeding rate (50–100?mg/larva/d), lighting condition (dark-light) and substrate depth in a rearing container (4–10?cm) was performed. An optimum prepupal biomass productivity of 123.4?g/m²/d was obtained (20%, wet weight of cake content in the substrate, 60%, wet weight of moisture content in the substrate, 100?mg/larva/d, dark, 6?cm substrate depth). The protein and oil content of the biomass were also determined to evaluate the effect of Reutealis trisperma seed as a substrate for the cultivation of black soldier fly larvae to produce protein and oil-rich biomass. The oil content in the biomass was also extracted and the fatty acid composition was identified. The prepupal biomass has a relatively high amount of protein (45%, dry weight) and oil content (26.6%, dry weight) and is suitable for cattle feed application.     

Phytase production byAspergillus ficuum on semisolid substrate

Summary Phytase production byAspergillus ficuum was studied using solid state cultivation on several cereal grains and legume seeds. The microbial phytase was used to hydrolyze the phytate in soybean meal and cotton seed meal. Wheat bran, soybean meal, cottonseed meal and corn meal supported good fungal growth and yielded a high level of phytase when an adequate amount of moisture was present. The level of phytase production on solid substrate was higher than that obtained by submerged liquid fermentation. Higher levels of phosphorus (more than 10 mg Pi/100 g substrate) in the growth medium (static culture) inhibited phytase synthesis, and the degree of phosphorus inhibition was less apparent in semisolid medium than in liquid medium. A static cultivation on semisolid substrate produced a higher level of phytase (2-20-fold) than that obtained by agitated cultivation. The minimal amount of water required for growth and enzyme production on those substrates was about 15%, while the optimum level for phytase production was between 25 and 35% and that for cell growth was above 50%. Optimum pH for phytase production was between 4 and 6.A ficuum grew well on raw (unheated) substrate containing a minimal amount of water and produced as much phytase as on heated substrate. About half of the phytic acid in soybean meal and cottonseed meal was hydrolyzed by treatment withA. ficuum phytase.     

SSF production of lactic acid from cellulosic biosludges

The use of cellulosic biosludges generated in a Kraft pulp mill was investigated as substrate for lactic acid production by simultaneous saccharification and fermentation (SSF). The effect of the operation mode (batch or fedbatch), the initial liquid to solid ratio (12 or 30 g/g) and the nutrient supplementation (MRS components or none) on several parameters including lactic acid concentration, volumetric productivity and product yields, were evaluated. When the operation was carried out in fedbatch mode with nutrient supplementation and using a LSR(0)=12 g/g, a broth containing 42 g/L was obtained after 48 h with a volumetric productivity of 0.87 g/L h and a product yield of 37.8 g lactic acid/100 g biosludges. In a similar experiment carried out without nutrient supplementation, a lactic acid concentration of 39.4 g/L was obtained after 48 h with a volumetric productivity of 0.82 g/L h and a product yield of 35.5 g L-lactic acid/100 g biosludges.     

Inoculum source for anaerobic fermentation of coffee pulp

Summary An experiment was conducted to find out the correct seeding (inoculum) material for the start up of anaerobic digestion using coffee pulp as substrate. Three different inocula viz., slurry from cowdung biogas plant, sewage sludge and rumen fluid were tried for their efficiency in producing maximum methanogenic activities. The result revealed that the sludge from a biogas plant is the best source of inoculum, which enhanced microbial activities, substrate utilization and gas yield. The various physico-chemical changes that occurred during fermentation were also discussed in detail.     

Biodegradation of estrogens by facultative anaerobic iron-reducing bacteria

Natural estrogens such as estrone, 17β-estradiol, estriol, and the synthetic component of contraceptive pills, 17α-ethinylestradiol, enter the municipal wastewater treatment plant via human excretions. A significant portion of these substances is found to remain in reject water produced after anaerobic digestion of activated sludge. In this study, the effect of the oxidant, Fe(III), and facultative anaerobic strain of iron-reducing bacteria on the anaerobic degradation of estrogens in reject water was investigated. Synthetic 17α-ethinylestradiol remained resistant to anaerobic biodegradation by iron-reducing bacteria, while natural estrogens such as 17β-estradiol, estriol, and estrone were removed by 92%, 60% and 27%, respectively, after 15 days of batch cultivation of iron-reducing bacteria in reject water with the addition of all estrogens to concentrations 100 μg l⁻¹ each. The ability of facultative anaerobic iron-reducing bacteria to degrade estrogens can be used for the anaerobic removal of trace organics from reject water in municipal wastewater treatment plant.     

General method for determining anaerobic biodegradation potential

A simple, generalized method was refined and validated to test whether an organic chemical was susceptible to anaerobic degradation to CH4 + CO2. The method used digested sewage sludge diluted to 10% and incubated anaerobically in 160-ml serum bottles with 50 micrograms of C per ml of test chemical. Biodegradation was determined by the net increase in gas pressure in bottles with test chemicals over the pressure in nonamended sludge bottles. Gas production was measured by gas chromatography and by a pressure transducer. The latter method is recommended because of its speed, accuracy, and low cost. Sewage sludge from municipal digesters with 15- to 30-day retention times was found to be suitable. The sludge could be stored anaerobically at 4 degrees C for up to 4 weeks with satisfactory test results. p-Cresol, phthalic acid, and ethanol are suggested as reference chemicals to confirm sludge activity and method reliability. A revised anaerobic salts medium was developed which minimizes problems of a biological gas production (CO2), avoids precipitation, and meets the requirements of the anaerobic microbiota. When greater than 75% of the theoretical gas production was observed, the chemical was judged to be degradable, and when 30 to 75% of the expected gas was produced, it was termed partially degradable. This method has been tested on more than 100 chemicals of various physical properties and found to reproducibly determine anaerobic biodegradation potential. Of the chemicals tested, 46 were found to be anaerobically degraded. Sludges from nine different municipal treatment plants were surveyed for their ability to degrade nine chemicals which differed in susceptibility to degradation.(ABSTRACT TRUNCATED AT 250 WORDS)     

General method for determining anaerobic biodegradation potential.

A simple, generalized method was refined and validated to test whether an organic chemical was susceptible to anaerobic degradation to CH4 + CO2. The method used digested sewage sludge diluted to 10% and incubated anaerobically in 160-ml serum bottles with 50 micrograms of C per ml of test chemical. Biodegradation was determined by the net increase in gas pressure in bottles with test chemicals over the pressure in nonamended sludge bottles. Gas production was measured by gas chromatography and by a pressure transducer. The latter method is recommended because of its speed, accuracy, and low cost. Sewage sludge from municipal digesters with 15- to 30-day retention times was found to be suitable. The sludge could be stored anaerobically at 4 degrees C for up to 4 weeks with satisfactory test results. p-Cresol, phthalic acid, and ethanol are suggested as reference chemicals to confirm sludge activity and method reliability. A revised anaerobic salts medium was developed which minimizes problems of a biological gas production (CO2), avoids precipitation, and meets the requirements of the anaerobic microbiota. When greater than 75% of the theoretical gas production was observed, the chemical was judged to be degradable, and when 30 to 75% of the expected gas was produced, it was termed partially degradable. This method has been tested on more than 100 chemicals of various physical properties and found to reproducibly determine anaerobic biodegradation potential. Of the chemicals tested, 46 were found to be anaerobically degraded. Sludges from nine different municipal treatment plants were surveyed for their ability to degrade nine chemicals which differed in susceptibility to degradation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of varying levels and frequencies of irrigation on growth,yield, nutrient uptake and water use efficiency of maize and cowpeas on a sandy loam ultisol

Summary A greenhouse study was carried out on an Nsukka sandy loam Ultisol having low soil moisture retention capacity to investigate the soil moisture regime and irrigation frequency required for optimum growth, yield, nutrient uptake and water use efficiency of maize (Zea mays L.) and cowpeas (Vigna unguiculata L. Walp). Four irrigation amounts (400 cm³, 300 cm³, 200 cm³ and 100 cm³ equivalent to 100, 75, 50 and 25% of field capacity, respectively) and four irrigation frequencies (daily, 2-day, 3-day and 4-day intervals) were tested in a factorial randomized design with three replications.Growth of maize was best when irrigation with water equivalent to 75% field capacity at daily interval but the optimum yields and nutrient uptake of both crops as well as cowpea nodulation were obtained when irrigating with water equivalent to 100% field capacity at daily or 2-day interval. The optimum water use efficiency was, however, achieved when irragating with amount equivalent to 100% field capacity at a 2-day interval. Irrigation with water equivalent to 50 or 25% field capacity at any interval resulted in various degrees of moisture stress which manifested in poor crop performance.     

Effect of initial COD concentration, nutrient addition, temperature and microbial acclimation on anaerobic treatability of broiler and cattle manure

In this study, anaerobic treatability and biogas generation potential of broiler and cattle manure were investigated. For this purpose, seven sets of anaerobic batch reactor experiments were performed using broiler and cattle manure and their mixtures in five different ratios (100% broiler; 75% broiler, 25% cattle; 50% broiler, 50% cattle; 25% broiler, 75% cattle; 100% cattle). These manure mixtures had two different initial chemical oxygen demand (COD) (12,000 and 53,500 mg/l) concentrations. The effects of initial COD concentration, nutrient and trace metal supplementation, microbial acclimation and digestion temperature were investigated. Results revealed that the efficiency of total COD removal was 32.0-43.3% and 37.9-50% for initial COD concentrations of 12,000 and 53,500 mg/l, respectively. The biogas yields observed for initial COD concentrations of 12,000 and 53,500 mg/l were 180-270 and 223-368 ml gas/g COD added, respectively. A decrease in biogas yield was observed as the fraction of broiler manure increased in mixture of broiler and cattle manure at initial COD values of 53,500 mg/l.     

Pretreatment of pulp mill secondary sludge for high-rate anaerobic conversion to biogas

Three pretreatment methods were compared based on their ability to increase the extent and rate of anaerobic bioconversion of pulp mill secondary sludge to biogas. The pretreatment technologies used in these experiments were: (i) thermal pretreatment performed at 170 °C; (ii) thermochemical (caustic) pretreatment performed at pH 12 and 140 °C; and (iii) sonication performed at 20 kHz and 1 W mL⁻¹. Sludge samples were obtained from a sulfite and a kraft pulp mill, and biochemical methane potential (BMP) assays were performed using microbial granules obtained from a high-rate anaerobic digester operating at a pulp mill. Biogas production from untreated sludge was 0.05 mL mg⁻¹ of measured chemical oxygen demand (COD) and 0.20 mL mg⁻¹ COD for kraft and sulfite sludge, respectively. Thermal pretreatment had the highest impact on sludge biodegradability. In this case, biogas yield and production rate from sulfite sludge increased by 50% and 10 times, respectively, while biogas yield and production rate from kraft sludge increased by 280% and 300 times, respectively. Biogas yield correlated to soluble carbohydrate content better than soluble COD.