The effect of methanogenesis inhibition,inoculum and substrate concentration on hydrogen and carboxylic acids production from cassava wastewater

Manipueira is a carbohydrate-rich agro-industrial waste from cassava processing. It is considered well suitable for biotechnological processes, such as hydrogen and carboxylic acids production, due to the high content of easily degradable organic matter. However, the proper methanogenesis inhibition method, inoculum type, and organic loads are factors still limiting the processes. The objective in this work was to evaluate the effects of such factors on byproducts production in anaerobic reactors. Batch experiments were conducted with 2.3-L flasks during two operational phases. In the first phase (P1), inhibition of methanogens in the sludge was evaluated using acetylene (1% v/v of headspace) and heat treatment (120 °C, 1 atm for 30 min). In the second phase (P2), three inoculum types obtained from common anaerobic sludges (bovine rumen and sludges from municipal and textile industrial wastewater treatment plants) were individually assayed. P2 aimed to identify the best inoculum, based on hydrogen production ability, which was tested for three initial concentrations of manipueira in terms of chemical oxygen demand (COD) (10, 20 and 40 g O₂/L). Results of P1 indicated that either acetylene or heat treatment efficiently inhibited methanogenesis, with no methane production. However, the maximum H₂ production potential by applying heat treatment (~ 563 mL) was more than twice compared with that by acetylene treatment (~ 257 mL); and butyrate was the main carboxylic acid by-product (~ 3 g/L). In P2 experiments after sludge heat treatment, the highest hydrogen yield (1.66 ± 0.07 mol H₂/mol glucose) and caproic acid production (~ 2 g/L) were observed at 20 g O₂/L of manipueira COD, when bovine rumen was the inoculum. The primary metabolic degradation products in all P2 experiments were ethanol, acetic, butyric, propionic and caproic acids. The finding of caproic acid detection indicated that the applied conditions in manipueira anaerobic degradation favored carbon chain elongation over methanogenesis.     

Pretreatment of municipal waste activated sludge for volatile sulfur compounds control in anaerobic digestion

The effect of combination of mechanical and chemical pretreatment of municipal waste activated sludge (WAS) prior to anaerobic digestion was studied using a laboratory scale system with an objective to decrease volatile sulfur compounds in biogas and digested sludge. Mechanical pretreatment was conducted using depressurization of WAS through a valve from a batch pretreatment reactor pressurized at 75 ± 1 psi, while combined pretreatments were conducted using six different dosages of hydrogen peroxide (H₂O₂) and ferrous chloride (FeCl₂) along with mechanical pretreatment. About 37-46% removal of H₂S in biogas occurred for different combined pretreatment conditions. Sludge solubilization achieved due to the mechanical pretreatment increased total cumulative methane production by 8-10% after 30 days during the biochemical methane potential (BMP) test. The pretreatment also improved dewaterability in terms of time to filter (TTF), and decreased methyl mercaptan generation potential of the digested sludge.     

Two-stage biogas production by co-digesting molasses wastewater and sewage sludge

We evaluated the feasibility of co-digesting molasses wastewater and sewage sludge in a two-stage hydrogen- and methane-producing system. The highest energy was recovered at the 21-h hydraulic retention time (HRT) of the first hydrogenic reactor and at 56-h HRT of the secondary methanogenic reactor. Hence, the two-stage system recovered 1,822 kJ from 1 L of the mixed wastes (19.7: hydrogenic reactor plus, 1,802 kJ L^?1: methanogenic reactor). Despite the overloaded VFA-run with a short HRT of 56 h, the GAC-CH₄ reactor increased methane production rate and yields due to enhanced pH buffer capacity. An RNA-based community analysis showed that the Ethanoligenens and Methanosaeta dominated the hydrogen and methane bioreactor, respectively. The two-stage system of co-digesting molasses and sewage sludge is particularly cost-effective due to non-pretreatment of sewage sludge.     

Hydrogen production of a salt tolerant strain Bacillus sp. B2 from marine intertidal sludge

To isolate a salt tolerant hydrogen-producing bacterium, we used the sludge from the intertidal zone of a bathing beach in Tianjin as inoculum to enrich hydrogen-producing bacteria. The sludge was treated by heat-shock pretreatment with three different temperature (80, 100 and 121°C) respectively. A hydrogen-producing bacterium was isolated from the sludge pretreated at 80°C by sandwich plate technique and identified using microscopic examination and 16S rDNA gene sequence analysis. The isolated bacterium was named as Bacillus sp. B2. The present study examined the hydrogen-producing ability of Bacillus sp. B2. The strain was able to produce hydrogen over a wide range of initial pH from 5.0 to 10.0, with an optimum at pH 7.0. The level of hydrogen production was also affected by the salt concentration. Strain B2 has unique capability to adapt high salt concentration. It could produce hydrogen at the salt concentration from 4 to 60‰. The maximum of hydrogen-producing yield of strain B2 was 1.65 ± 0.04 mol H₂/mol glucose (mean ± SE) at an initial pH value of 7.0 in marine culture conditions. Hydrogen production under fresh culture conditions reached a higher level than that in marine ones. As a result, it is likely that Bacillus sp. B2 could be applied to biohydrogen production using both marine and fresh organic waste.     

Start‐up of Anaerobic Hydrogen Producing Reactors Seeded with Sewage Sludge

The procedure for starting‐up continuously stirred tank reactors (CSTR) for acclimating anaerobic hydrogen‐producing microorganisms with sewage sludge was investigated. Initially, feeding with glucose and sucrose as well as mixing were carried out in semicontinuous mode; hydraulic retention time (HRT) was in an order of 20, 15, 10, 5, 2.5 and 2 days. When the pH declined to its lowest value (pH 5.18), it was adjusted to 6.7 using sodium hydroxide (1 N). At the same time, the semi‐continuous operation was changed to a continuous one. Finally, the pH was continuously regulated at approximately 6.7. The results indicate that this procedure can be used to cultivate seed sludge for hydrogen production from sewage sludge resulting in a large hydrogen production in less than 60 days. When the substrate was glucose, a hydrogen yield of 1.63 mol H₂/mol glucose and a specific hydrogen production rate of 321 mmol H₂/g VSS day at an HRT of 13.3 h was achieved. When the substrate was sucrose with the same HTR, a hydrogen yield of 4.45 mol H2/mol sucrose and a specific hydrogen production rate of 707 mmol H₂/g VSS day was obtained.     

Intensive bioconversion of sewage sludge and food waste by Bacillus thermoamylovorans

The main aim of this work was to intensify conventional composting of a mixture of sewage sludge and solid food wastes by a one-stage thermophilic bioconversion of these wastes into an organic fertilizer. An intensive process was carried out in a closed system, with or without addition of a starter culture of Bacillus thermoamylovorans. The most effective thermophilic bioconversion of the mixture of food waste and sewage sludge, with addition of starter culture, was when the pH was buffered with calcium carbonate, or the pH drop in the material was prevented by preliminary removal of sulphides from sewage sludge by hydrogen peroxide.     

Effect of Coronatine on Antioxidant Enzyme Response of Chickpea Roots to Combination of PEG-Induced Osmotic Stress and Heat Stress

Abiotic stresses, such as high temperature and drought, are major limiting factors of crop production and growth. Coronatine (COR), a structural and functional analog of jasmonates, is suggested to have a role in abiotic stress tolerance. The aim of our study was to examine whether pretreatment with COR enhances the tolerance of chickpea (Cicer arietinum L. cv ICC 4958) roots to PEG-induced osmotic stress, heat stress, and their combination. Therefore, seedlings raised hydroponically in a growth chamber for 15 days were pretreated with or without COR at 0.01 μM for 24 h and then exposed to 6 % PEG 6000-induced osmotic stress or heat (starting at 35 °C and then gradually increased 1 °C every 15 min and kept at 44 °C for 1 h) stress for 3 days. After different treatment periods, the changes in relative growth rate (RGR); malondialdehyde (MDA), proline (Pro), and hydrogen peroxide (H₂O₂) contents; and the activities of antioxidant enzymes/isoenzymes in roots of chickpea seedlings with or without 0.01 μM COR application were studied. RGR in roots was increased by COR application. Under all stress conditions, H₂O₂, MDA, and Pro levels increased sharply, but pretreatment with COR significantly reduced them. Moreover, COR increased the activities of H₂O₂ scavenger enzymes such as catalase (CAT) under heat stress, ascorbate peroxidase (POX) under PEG stress, and CAT and POX under combined stresses. Therefore, COR might alleviate adverse effects of PEG stress and heat stress and combined stresses on roots of chickpea by reduction of H₂O₂ production, enhancing or keeping the existent activity of antioxidant enzymes, thereby preventing membrane peroxidation.     

Dark-Fermentative Biological Hydrogen Production from Mixed Biowastes Using Defined Mixed Cultures

Biological hydrogen (H₂) production from the biowastes is widely recognized as a suitable alternative approach to utilize low cost feed instead of costly individual sugars. In the present investigation, pure and mixed biowastes were fermented by defined sets of mixed cultures for hydrolysis and H₂ production. Under batch conditions, up to 65, 67 and 70 L H₂/kg total solids (2%, TS) were evolved from apple pomace (AP), onion peels (OP) and potato peels (PP) using a combination of hydrolytic mixed culture (MHC5) and mixed microbial cultures (MMC4 or MMC6), respectively. Among the different combinations of mixed biowastes including AP, OP, PP and pea-shells, the combination of OP and PP exhibited maximum H₂ production of 73 and 84 L/kg TS with MMC4 and MMC6, respectively. This study suggested that H₂ production can be effectively regulated by using defined sets of mixed cultures for hydrolysis and H₂ production from pure and mixed biowastes as feed even under unsterile conditions.     

Integrative Biological Hydrogen Production: An Overview

Biological hydrogen (H₂) production by dark and photo-fermentative organisms is a promising area of research for generating bioenergy. A large number of organisms have been widely studied for producing H₂ from diverse feeds, both as pure and as mixed cultures. However, their H₂ producing efficiencies have been found to vary (from 3 to 8 mol/mol hexose) with physiological conditions, type of organisms and composition of feed (starchy waste from sweet potato, wheat, cassava and algal biomass). The present review deals with the possibilities of enhancing H₂ production by integrating metabolic pathways of different organisms-dark fermentative bacteria (from cattle dung, activated sludge, Caldicellulosiruptor, Clostridium, Enterobacter, Lactobacillus, and Vibrio) and photo-fermentative bacteria (such as Rhodobacter, Rhodobium and Rhodopseudomonas). The emphasis has been laid on systems which are driven by undefined dark-fermentative cultures in combination with pure photo-fermentative bacterial cultures using biowaste as feed. Such an integrative approach may prove suitable for commercial applications on a large scale.     

Sewage sludge addition to food waste synergistically enhances hydrogen fermentation performance

The effect of sewage sludge (SWS) addition on the H₂ fermentation of food waste (FW) was investigated. It was found that a slight addition of SWS (10:1 = FW:SWS on a COD basis) significantly enhanced the H₂ fermentation performance, not only increasing the total amount of H₂ produced but accelerating the whole reaction, shortening the lag period, and increasing the H₂ production rate. Substrate degradation and microbial germination were also facilitated by SWS addition. A simple calculation reveals that the increased amount of H₂ production derived mostly from FW, indicating that SWS addition synergistically enhanced H₂ fermentation performance. This was attributed to the existence of Fe and Ca at much higher concentrations in the SWS compared to the FW. The batch process treating a mixture of FW and SWS was repeated and showed an average H₂ yield of 2.11 ± 0.20 mol H₂/mol hexose_added, which was 13% higher than that of FW treated alone.     

Co-fermentation of cassava waste pulp hydrolysate with molasses to ethanol for economic optimization

Cassava waste pulp (CWP)–enzymatic hydrolysate was co-fermented with molasses (CWP-EH/molasses mixture) with the aim to optimize ethanol production by Saccharomyces cerevisiae TISTR 5606 (SC 90). The optimal fermentation conditions for ethanol production using this mixture were 245 g/L initial total sugar supplemented with KH₂PO₄ (8 g/L), at 30 °C for 48 h of fermentation under an oxygen-limited condition with agitation at 100 rpm, producing an ethanol concentration of 70.60 g/L (0.31 g ethanol/g total sugar). The addition of cassava tuber fiber (solid residue of CWP after enzymatic hydrolysis) at 30 g/L dry weight to the CWP-EH/molasses mixture increased ethanol production to 74.36 g/L (0.32 g ethanol/g total sugar). Co-fermentation of CWP-EH with molasses had the advantage of not requiring any supplementation of the fermentation mixture with reduced nitrogen.     

Optimization of a two-step biodiesel production process comprised of lipid extraction from blended sewage sludge and subsequent lipid transesterification

As a preliminary research for the development of feasible and economical biodiesel production using blended sewage sludge (BSS), a sustainable and non-edible feedstock, the two-step process comprised of lipid extraction (first step) and subsequent transesterification of the lipid with methanol (second step) was optimized. The total lipid content of the free fatty acid (FFA) containing BSS was determined to be 14.5% using the Blight and Dyer method with ultrasonication pretreatment, where 40.8% of the total lipid content was FFAs. The highest lipid yield of 13.5% (g-lipid/g-dry sludge), corresponding to 92.9% extraction efficiency, was obtained using 20 mL-solvent/g-dry sludge of the total solvent mixture with a 2/1 (v/v) ratio of chloroform and methanol. In the transesterification step, an acidic catalyst (H₂SO₄) exhibited significantly higher performance than an alkaline catalyst (NaOH). Thus, the optimal reaction conditions were 0.2% (g/g-lipid) H₂SO₄, 20 mL-methanol/g-lipid, 70°C and 8 h, respectively. Although the reaction temperature was increased from 50 to 70°C, we could save H₂SO₄, methanol, and a reaction time by 75, 50 and 66.7%, respectively compared with previous optimal conditions suggest by others’ research. Under our optimal conditions, a biodiesel yield of 39.0% (g-biodiesel/g-lipid) and an overall yield (i.e., extraction and transesterification) of 5.3% (g-biodiesel/g-BSS) were achieved, which are substantially higher than those from others’ research.     

Acclimation Strategy of a Biohydrogen Producing Population in a Continuous‐Flow Reactor with Carbohydrate Fermentation

Poor startup of biological hydrogen production systems can cause an ineffective hydrogen production rate and poor biomass growth at a high hydraulic retention time (HRT), or cause a prolonged period of acclimation. In this paper a new startup strategy was developed in order to improve the enrichment of the hydrogen‐producing population and the efficiency of hydrogen production. A continuously‐stirred tank reactor (CSTR) and molasses were used to evaluate the hydrogen productivity of the sewage sludge microflora at a temperature of 35 °C. The experimental results indicated that the feed to microorganism ratio (F/M ratio) was a key parameter for the enrichment of hydrogen producing sludge in a continuous‐flow reactor. When the initial biomass was inoculated with 6.24 g of volatile suspended solids (VSS)/L, an HRT of 6 h, an initial organic loading rate (OLR) of 7.0 kg chemical oxygen demand (COD)/(m³ × d) and an feed to microorganism ratio (F/M) ratio of about 2–3 g COD/(g of volatile suspended solids (VSS) per day) were maintained during startup. Under these conditions, a hydrogen producing population at an equilibrium state could be established within 30 days. The main liquid fermentation products were acetate and ethanol. Biogas was composed of H₂ and CO₂. The hydrogen content in the biogas amounted to 47.5 %. The average hydrogen yield was 2.01 mol/mol hexose consumed. It was also observed that a special hydrogen producing population was formed when this startup strategy was used. It is supposed that the population may have had some special metabolic pathways to produce hydrogen along with ethanol as the main fermentation products.     

Beyond the Theoretical Yields of Dark-Fermentative Biohydrogen

Theoretical hydrogen (H₂) yield by dark fermentative route is 12 mol/mol of glucose. Biological H₂ production yields of 3.8 mol/mol of glucose by microbes have been reported. Transient gene inactivation in combination with adaptive laboratory evolution strategy has enabled the H₂ yield to exceed the stoichiometric production values.     

Eliminating methanogenic activity in hydrogen reactor to improve biogas production in a two-stage anaerobic digestion process co-digesting municipal food waste and sewage sludge

Laboratory scale two-stage anaerobic digestion process model was operated for 280 days to investigate the feasibility to produce both hydrogen and methane from a mixture feedstock (1:1 (v/v)) of municipal food waste and sewage sludge. The maximum hydrogen and methane yields obtained in the two stages were 0.93 and 9.5 mL/mL feedstock. To eliminate methanogenic activity and obtain substantial hydrogen production in the hydrogen reactor, both feedstock and mixed liquor required treatment. The heat treatment (100 °C, 10 min) for feedstock and a periodical treatment (every 2-5 weeks, either heating, removal of biomass particles or flushing with air) for mixed liquor were effective in different extent. The methane production in the second stage was significantly improved by the hydrogen production in the first stage. The maximum methane production obtained in the period of high hydrogen production was more than 2-fold of that observed in the low hydrogen production period.     

On-site removal of H<Subscript>2</Subscript>S from biogas produced by food waste using an aerobic sludge biofilter for steam reforming processing

H₂S in biogas was removed by sludge-loaded biofiltration, rendering the biogas suitable for catalytic reforming into a mixture of CO and H₂ syngas that was then applied for the generation of electricity using a solid oxide fuel cell or for the chemical synthesis of methanol. The biogas was anaerobically produced in a 2 m³ bioreactor at 35°C for 2 years using restaurant food waste from Korea Advanced Institute of Science and Technology (KAIST), and the concentration of H₂S in the biogas ranged from 612 to 1,500 ppmv (Avg. 1,060 ppmv). Two immobilized cell bioreactors 0.2 and 8.5 L in volume were loaded with aerobic sludge and used to study characteristics of H₂S removal from biogas. At a retention time of 400 sec, the removal efficiency of H₂S was over 99% following initial stabilization for 7 days in the 8.5 L bioreactor installed at the on-site biogas facility. The maximum rate of H₂S removal in this study was 359 g-H₂S/m³/h with an average mass loading rate of 14.7 g-H₂S/m³/h (kinetic analysis: V _m = 842.6 g-H₂S/m³/h and K _s = 2.2 mg/L). Therefore, purified biogas with a negligible concentration H₂S was efficiently reformed to syngas. This study demonstrates the feasibility of biogas purification as a part of high-quality syngas production.     

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

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

Production of biobutanol from acid-pretreated corncob using Clostridium beijerinckii TISTR 1461: Process optimization studies

Corncob is a potential feedstock in Thailand that can be used for fermentable sugar production through dilute sulfuric acid pretreatment and enzymatic hydrolysis. To recover high amounts of monomeric sugars from corncob, the sulfuric pretreatment conditions were optimized by using response surface methodology with three independent variables: sulfuric acid concentration, temperature, and time. The highest response of total sugars, 48.84 g/L, was found at 122.78°C, 4.65 min, and 2.82% (v/v) H₂SO₄. With these conditions, total sugars from the confirmation experiment were 46.29 g/L, with 5.51% error from the predicted value. The hydrolysate was used as a substrate for acetone–butanol–ethanol fermentation to evaluate its potential for microbial growth. The simultaneous saccharification and fermentation (SSF) showed that C. beijerinckii TISTR 1461 can generate acetone–butanol–ethanol products at 11.64 g/L (5.29 g/L acetone, 6.26 g/L butanol, and 0.09 g/L ethanol) instantly using sugars from the hydrolysed corncob with Novozymes 50013 cellulase enzyme without an overliming process.     

Bioethanol production from brown seaweed, Undaria pinnatifida, using NaCl acclimated yeast

Strain improvement of Pichia angophorae KCTC 17574 was successfully carried out for bioethanol fermentation of seaweed slurry with high salt concentration. P. angophorae KCTC 17574 was cultured under increasing salinity from five practical salinity unit (psu, ‰) to as high as 100 psu for 723 h. The seaweed, Undaria pinnatifida (sea mustard, Miyuk), was fermented to produce bioethanol using high-salt acclimated yeast. The pretreatment of U. pinnatifida was optimized using thermal acid hydrolysis to obtain a high monosaccharide yield. Optimal pretreatment conditions of 75 mM H₂SO₄ and 13 % (w/v) slurry at 121 °C for 60 min were determined using response surface methodology. A maximum monosaccharide content of 28.65 g/L and the viscosity of 33.19 cP were obtained. The yeasts cultured under various salinity concentrations were collected and inoculated to the pretreated seaweed slurry after the neutralization using 5 N NaOH. The pretreated slurry was fermented with the inoculation of 0.1 g dcw/L of P. angophorae KCTC 17574 strain obtained at 90 psu. The maximum ethanol concentration of 9.42 g/L with 27 % yield of theoretical case of ethanol production from total carbohydrate of U. pinnatifida was obtained.     

Nickel in soil and maize plants grown on an oxisol treated over a long time with sewage sludge

Abstract

The major limitation for the use of sewage sludge in agriculture is the risk of soil contamination with heavy metals, and their possible transference to man via the food chain. The objective of this study was to evaluate the content of nickel (Ni) in soil by the two methods of digestion (HNO₃ + H₂O₂ + HCl and HClO₄ + HF), and in different parts of maize plants grown on a tropical soil classified as Typic Eutrorthox, that had been treated with sewage sludge for nine consecutive years, and the effects on dry matter and grain production. The experiment was carried out under field conditions in Jaboticabal-SP, using a randomized block design with four treatments and five replicates. Treatments consisted of: 0.0 (control, mineral fertilization), 45.0, 90.0 and 127.5t ha^?1 sewage sludge (dry basis), accumulated during nine years. Sewage sludge was manually applied to the soil and incorporated at 0.1 m depth before sowing the maize. Soil Ni evaluated by Jackson’s method was 76.8% higher than evaluated by the United States Environmental Protection Agency method that digests the samples by heating with concentrated HNO³, H²O² and HCl. Sewage sludge rates did not affect Ni content in the soil. Ni was accumulated in leaf and stem but was not detected in grain. Sewage sludge and mineral fertilization applied to soil for a long time caused similar effects on dry matter and grain production.