Effects of Temperature on Methanogenesis in a Thermophilic (58°C) Anaerobic Digestor

The short-term effects of temperature on methanogenesis from acetate or CO₂ in a thermophilic (58°C) anaerobic digestor were studied by incubating digestor sludge at different temperatures with ¹⁴C-labeled methane precursors (¹⁴CH₃COO⁻ or ¹⁴CO₂). During a period when Methanosarcina sp. was numerous in the sludge, methanogenesis from acetate was optimal at 55 to 60°C and was completely inhibited at 65°C. A Methanosarcina culture isolated from the digestor grew optimally on acetate at 55 to 58°C and did not grow or produce methane at 65°C. An accidental shift of digestor temperature from 58 to 64°C during this period caused a sharp decrease in gas production and a large increase in acetate concentration within 24 h, indicating that the aceticlastic methanogens in the digestor were the population most susceptible to this temperature increase. During a later period when Methanothrix sp. was numerous in the digestor, methanogenesis from ¹⁴CH₃COO⁻ was optimal at 65°C and completely inhibited at 75°C. A partially purified Methanothrix enrichment culture derived from the digestor had a maximum growth temperature near 70°C. Methanogenesis from ¹⁴CO₂ in the sludge was optimal at 65°C and still proceeded at 75°C. A CO₂-reducing Methanobacterium sp. isolated from the digestor was capable of methanogenesis at 75°C. During the period when Methanothix sp. was apparently dominant, sludge incubated for 24 h at 65°C produced more methane than sludge incubated at 60°C, and no acetate accumulated at 65°C. Methanogenesis was severely inhibited in sludge incubated at 70°C, but since neither acetate nor H₂ accumulated, production of these methanogenic substrates by fermentative bacteria was probably the most temperature-sensitive process. Thus, there was a correlation between digestor performance at different temperatures and responses to temperature by cultures of methanogens believed to play important roles in the digestor.     

Pressure and Temperature Effects on Growth and Methane Production of the Extreme Thermophile Methanococcus jannaschii

The marine archaebacterium Methanococcus jannaschii was studied at high temperatures and hyperbaric pressures of helium to investigate the effect of pressure on the behavior of a deep-sea thermophile. Methanogenesis and growth (as measured by protein production) at both 86 and 90°C were accelerated by pressure up to 750 atm (1 atm = 101.29kPa), but growth was not observed above 90°C at either 7.8 or 250 atm. However, growth and methanogenesis were uncoupled above 90°C, and the high-temperature limit for methanogenesis was increased by pressure. Substantial methane formation was evident at 98°C and 250 atm, whereas no methane formation was observed at 94°C and 7.8 atm. In contrast, when argon was substituted for helium as the pressurizing gas at 250 atm, no methane was produced at 86°C. Methanogenesis was also suppressed at 86°C and 250 atm when the culture was pressurized with a 4:1 mix of H₂ and CO₂, although limited methanogenesis did occur when the culture was pressurized with H₂.     

C. elegans and H. sapiens mRNAs with edited 3' UTRs are present on polysomes

Adenosine deaminases that act on RNA (ADARs) are editing enzymes that convert adenosine to inosine in double-stranded RNA (dsRNA). ADARs sometimes target codons so that a single mRNA yields multiple protein isoforms. However, ADARs most often target noncoding regions of mRNAs, such as untranslated regions (UTRs). To understand the function of extensive double-stranded 3′ UTR structures, and the inosines within them, we monitored the fate of reporter and endogenous mRNAs that include structured 3′ UTRs in wild-type Caenorhabditis elegans and in strains with mutations in the ADAR genes. In general, we saw little effect of editing on stability or translatability of mRNA, although in one case an ADR-1 dependent effect was observed. Importantly, whereas previous studies indicate that inosine-containing RNAs are retained in the nucleus, we show that both C. elegans and Homo sapiens mRNAs with edited, structured 3′ UTRs are present on translating ribosomes.     

Effect of Temperature on Anaerobic Ethanol Oxidation and Methanogenesis in Acidic Peat from a Northern Wetland

The effects of temperature on rates and pathways of CH₄ production and on the abundance and structure of the archaeal community were investigated in acidic peat from a mire in northern Scandinavia (68°N). We monitored the production of CH₄ and CO₂ over time and measured the turnover of Fe(II), ethanol, and organic acids. All experiments were performed with and without specific inhibitors (2-bromoethanesulfonate [BES] for methanogenesis and CH₃F for acetoclastic methanogenesis). The optimum temperature for methanogenesis was 25°C (2.3 μmol CH₄ · g [dry weight]⁻¹ · day⁻¹), but the activity was relatively high even at 4°C (0.25 μmol CH₄ · g [dry weight]⁻¹ · day⁻¹). The theoretical lower limit for methanogenesis was calculated to be at −5°C. The optimum temperature for growth as revealed by real-time PCR was 25°C for both archaea and bacteria. The population structure of archaea was studied by terminal restriction fragment length polymorphism analysis and remained constant over a wide temperature range. Hydrogenotrophic methanogenesis accounted for about 80% of the total methanogenesis. Most 16S rRNA gene sequences that were affiliated with methanogens and all McrA sequences clustered with the exclusively hydrogenotrophic order Methanobacteriales, correlating with the prevalence of hydrogenotrophic methanogenesis. Fe reduction occurred parallel to methanogenesis and was inhibited by BES, suggesting that methanogens were involved in Fe reduction. Based upon the observed balance of substrates and thermodynamic calculations, we concluded that the ethanol pool was oxidized to acetate by the following two processes: syntrophic oxidation with methanogenesis (i) as an H₂ sink and (ii) as a reductant for Fe(III). Acetate accumulated, but a considerable fraction was converted to butyrate, making volatile fatty acids important end products of anaerobic metabolism.     

A Functional Approach To Uncover the Low-Temperature Adaptation Strategies of the Archaeon Methanosarcina barkeri

Low-temperature anaerobic digestion (LTAD) technology is underpinned by a diverse microbial community. The methanogenic archaea represent a key functional group in these consortia, undertaking CO₂ reduction as well as acetate and methylated C₁ metabolism with subsequent biogas (40 to 60% CH₄ and 30 to 50% CO₂) formation. However, the cold adaptation strategies, which allow methanogens to function efficiently in LTAD, remain unclear. Here, a pure-culture proteomic approach was employed to study the functional characteristics of Methanosarcina barkeri (optimum growth temperature, 37°C), which has been detected in LTAD bioreactors. Two experimental approaches were undertaken. The first approach aimed to characterize a low-temperature shock response (LTSR) of M. barkeri DSMZ 800^T grown at 37°C with a temperature drop to 15°C, while the second experimental approach aimed to examine the low-temperature adaptation strategies (LTAS) of the same strain when it was grown at 15°C. The latter experiment employed cell viability and growth measurements (optical density at 600 nm [OD₆₀₀]), which directly compared M. barkeri cells grown at 15°C with those grown at 37°C. During the LTSR experiment, a total of 127 proteins were detected in 37°C and 15°C samples, with 20 proteins differentially expressed with respect to temperature, while in the LTAS experiment 39% of proteins identified were differentially expressed between phases of growth. Functional categories included methanogenesis, cellular information processing, and chaperones. By applying a polyphasic approach (proteomics and growth studies), insights into the low-temperature adaptation capacity of this mesophilically characterized methanogen were obtained which suggest that the metabolically diverse Methanosarcinaceae could be functionally relevant for LTAD systems.     

RNase activity of polynucleotide phosphorylase is critical at low temperature in Escherichia coli and is complemented by RNase II

In Escherichia coli, the cold shock response is exerted upon a temperature change from 37°C to 15°C and is characterized by induction of several cold shock proteins, including polynucleotide phosphorylase (PNPase), during acclimation phase. In E. coli, PNPase is essential for growth at low temperatures; however, its exact role in this essential function has not been fully elucidated. PNPase is a 3′-to-5′ exoribonuclease and promotes the processive degradation of RNA. Our screening of an E. coli genomic library for an in vivo counterpart of PNPase that can compensate for its absence at low temperature revealed only one protein, another 3′-to-5′ exonuclease, RNase II. Here we show that the RNase PH domains 1 and 2 of PNPase are important for its cold shock function, suggesting that the RNase activity of PNPase is critical for its essential function at low temperature. We also show that its polymerization activity is dispensable in its cold shock function. Interestingly, the third 3′-to-5′ processing exoribonuclease, RNase R of E. coli, which is cold inducible, cannot complement the cold shock function of PNPase. We further show that this difference is due to the different targets of these enzymes and stabilization of some of the PNPase-sensitive mRNAs, like fis, in the Δpnp cells has consequences, such as accumulation of ribosomal subunits in the Δpnp cells, which may play a role in the cold sensitivity of this strain.     

Identification of elements in human long 3′ UTRs that inhibit nonsense-mediated decay

The nonsense-mediated mRNA decay (NMD) pathway serves an important role in gene expression by targeting aberrant mRNAs that have acquired premature termination codons (PTCs) as well as a subset of normally processed endogenous mRNAs. One determinant for the targeting of mRNAs by NMD is the occurrence of translation termination distal to the poly(A) tail. Yet, a large subset of naturally occurring mRNAs contain long 3′ UTRs, many of which, according to global studies, are insensitive to NMD. This raises the possibility that such mRNAs have evolved mechanisms for NMD evasion. Here, we analyzed a set of human long 3′ UTR mRNAs and found that many are indeed resistant to NMD. By dissecting the 3′ UTR of one such mRNA, TRAM1 mRNA, we identified a cis element located within the first 200 nt that inhibits NMD when positioned in downstream proximity of the translation termination codon and is sufficient for repressing NMD of a heterologous reporter mRNA. Investigation of other NMD-evading long 3′ UTR mRNAs revealed a subset that, similar to TRAM1 mRNA, contains NMD-inhibiting cis elements in the first 200 nt. A smaller subset of long 3′ UTR mRNAs evades NMD by a different mechanism that appears to be independent of a termination-proximal cis element. Our study suggests that different mechanisms have evolved to ensure NMD evasion of human mRNAs with long 3′ UTRs.     

Microbiology of Methanogenesis in Thermal, Volcanic Environments

Microbial methanogenesis was examined in thermal waters, muds, and decomposing algal-bacterial mats associated with volcanic activity in Yellowstone National Park. Radioactive tracer studies with [¹⁴C]glucose, acetate, or carbonate and enrichment culture techniques demonstrated that methanogenesis occurred at temperatures near 70°C but below 80°C and correlated with hydrogen production from either geothermal processes or microbial fermentation. Three Methanobacterium thermoautotrophicum strains (YT1, YTA, and YTC) isolated from diverse volcanic habitats differed from the neotype sewage strain ΔH in deoxyribonucleic acid guanosine-plus-cytosine content and immunological properties. Microbial methanogenesis was characterized in more detail at a 65°C site in the Octopus Spring algal-bacterial mat ecosystem. Here methanogenesis was active, was associated with anaerobic microbial decomposition of biomass, occurred concomitantly with detectable microbial hydrogen formation, and displayed a temperature activity optimum near 65°C. Enumeration studies estimated more than 10⁹ chemoorganotrophic hydrolytic bacteria and 10⁶ chemolithotrophic methanogenic bacteria per g (dry weight) of algal-bacterial mat. Enumeration, enrichment, and isolation studies revealed that the microbial population was predominantly rod shaped and asporogenous. A prevalent chemoorganotrophic organism in the mat that was isolated from an end dilution tube was a taxonomically undescribed gram-negative obligate anaerobe (strain HTB2), whereas a prevalent chemolithotrophic methanogen isolated from an end dilution tube was identified as M. thermoautotrophicum (strain YTB). Taxonomically recognizable obligate anaerobes that were isolated from glucose and xylose enrichment cultures included Thermoanaerobium brockii strain HTB and Clostridium thermohydrosulfuricum strain 39E. The nutritional properties, growth temperature optima, growth rates, and fermentation products of thermophilic bacterial strains 39E, HTB2, and YTB were determined.     

Effects of Temperature on Bacterial Communities and Metabolites during Fermentation of Myeolchi-Aekjeot,a Traditional Korean Fermented Anchovy Sauce

Myeolchi-aekjeot (MA) in Korea is produced outdoors without temperature controls, which is a major obstacle to produce commercial MA products with uniform quality. To investigate the effects of temperature on MA fermentation, pH, bacterial abundance and community, and metabolites were monitored during fermentation at 15°C, 20°C, 25°C, and 30°C. Initial pH values were approximately 6.0, and pH values increased after approximately 42 days, with faster increases at higher temperatures. Bacterial abundances increased rapidly in all MA samples after quick initial decreases during early fermentation and then they again steadily decreased after reaching their maxima, which were significantly greater at higher temperatures. Bacterial community analysis revealed that Proteobacteria and Tenericutes were predominant in all initial MA samples, but they were rapidly displaced by Firmicutes as fermentation progressed. Photobacterium and Mycoplasma belonging to Proteobacteria and Tenericutes, respectively, which may include potentially pathogenic strains, were dominant in initial MA, but decreased with the growth of Chromohalobacter, which occurred faster at higher temperatures––they were dominant until 273 and 100 days at 15°C and 20°C, respectively, but not detected after 30 days at 25°C and 30°C. Chromohalobacter also decreased with the appearance of subsequent genera belonging to Firmicutes in all MA samples. Tetragenococcus, halophilic lactic acid bacteria, appeared predominantly at 20°C, 25°C, and 30°C; they were most abundant at 30°C, but not detected at 15°C. Alkalibacillus and Lentibacillus appeared as dominant genera with the decrease of Tetragenococcus at 25°C and 30°C, but only Lentibacillus was dominant at 15°C and 20°C. Metabolite analysis showed that amino acids related to tastes were major metabolites and their concentrations were relatively higher at high temperatures. This study suggests that high temperatures (approximately 30°C) may be appropriate in MA fermentation, in the light of faster disappearance of potentially pathogenic genera, higher amino acids, growth of Tetragenococcus, and faster fermentation.     

Activation of cap-independent translation by variant eukaryotic initiation factor 4G in vivo

Protein synthesis is tightly controlled by assembly of an intricate ribonucleoprotein complex at the m⁷GTP-cap on eukaryotic mRNAs. Ensuing linear scanning of the 5′ untranslated region (UTR) is believed to transfer the preinitiation complex to the initiation codon. Eukaryotic mRNAs are characterized by significant 5′ UTR heterogeneity, raising the possibility of differential control of translation initiation rate at individual mRNAs. Curiously, many mRNAs with unconventional, highly structured 5′ UTRs encode proteins with central biological roles in growth control, metabolism, or stress response. The 5′ UTRs of such mRNAs may influence protein synthesis rate in multiple ways, but most significantly they have been implicated in mediating alternative means of translation initiation. Cap-independent initiation bypasses strict control over the formation of initiation intermediates at the m⁷GTP cap. However, the molecular mechanisms that favor alternative means of ribosome recruitment are not understood. Here we provide evidence that eukaryotic initiation factor (eIF) 4G controls cap-independent translation initiation at the c-myc and vascular endothelial growth factor (VEGF) 5′ UTRs in vivo. Cap-independent translation was investigated in tetracycline-inducible cell lines expressing either full-length eIF4G or a C-terminal fragment (Ct) lacking interaction with eIF4E and poly(A) binding protein. Expression of Ct, but not intact eIF4G, potently stimulated cap-independent initiation at the c-myc/VEGF 5′ UTRs. In vitro RNA-binding assays suggest that stimulation of cap-independent translation initiation by Ct is due to direct association with the c-myc/VEGF 5′ UTR, enabling 43S preinitiation complex recruitment. Our work demonstrates that variant translation initiation factors enable unconventional translation initiation at mRNA subsets with distinct structural features.     

Conserved regulation of MAP kinase expression by PUF RNA-binding proteins

Mitogen-activated protein kinase (MAPK) and PUF (for Pumilio and FBF [fem-3 binding factor]) RNA-binding proteins control many cellular processes critical for animal development and tissue homeostasis. In the present work, we report that PUF proteins act directly on MAPK/ERK-encoding mRNAs to downregulate their expression in both the Caenorhabditis elegans germline and human embryonic stem cells. In C. elegans, FBF/PUF binds regulatory elements in the mpk-1 3′ untranslated region (3′ UTR) and coprecipitates with mpk-1 mRNA; moreover, mpk-1 expression increases dramatically in FBF mutants. In human embryonic stem cells, PUM2/PUF binds 3′UTR elements in both Erk2 and p38α mRNAs, and PUM2 represses reporter constructs carrying either Erk2 or p38α 3′ UTRs. Therefore, the PUF control of MAPK expression is conserved. Its biological function was explored in nematodes, where FBF promotes the self-renewal of germline stem cells, and MPK-1 promotes oocyte maturation and germ cell apoptosis. We found that FBF acts redundantly with LIP-1, the C. elegans homolog of MAPK phosphatase (MKP), to restrict MAPK activity and prevent apoptosis. In mammals, activated MAPK can promote apoptosis of cancer cells and restrict stem cell self-renewal, and MKP is upregulated in cancer cells. We propose that the dual negative regulation of MAPK by both PUF repression and MKP inhibition may be a conserved mechanism that influences both stem cell maintenance and tumor progression.     

Temperature Effects on Development of Meloidogyne Enterolobii and M. Floridensis

Meloidogyne enterolobii and M. floridensis are virulent species that can overcome root-knot nematode resistance in economically important crops. Our objectives were to determine the effects of temperature on the infectivity of second-stage juveniles (J2) of these two species and determine differences in duration and thermal-time requirements (degree-days [DD]) to complete their developmental cycle. Florida isolates of M. enterolobii and M. floridensis were compared to M. incognita race 3. Tomato cv. BHN 589 seedlings following inoculation were placed in growth chambers set at constant temperatures of 25°C, and 30°C, and alternating temperatures of 30°C to 25°C (day–night). Root infection by the three nematode species was higher at 30°C than at 25°C, and intermediate at 30°C to 25°C, with 33%, 15%, and 24% infection rates, respectively. There was no difference, however, in the percentages of J2 that infected roots among species at each temperature. Developmental time from infective J2 to reproductive stage for the three species was shorter at 30°C than at 25°C, and 30°C to 25°C. The shortest time and DD to egg production for the three species were 13 days after inoculation (DAI) and 285.7 DD, respectively. During the experimental timeframe of 29 d, a single generation was completed at 30°C for all three species, whereas only M. floridensis completed a generation at 30°C to 25°C. The number of days and accumulated DD for completing the life cycle (from J2 to J2) were 23 d and 506.9 DD for M. enterolobii, and 25 d and 552.3 DD for M. floridensis and M. incognita, respectively. Exposure to lower (25°C) and intermediate temperatures (30°C to 25°C) decreased root penetration and slowed the developmental cycle of M. enterolobii and M. floridensis compared with 30°C.     

Role of Glucose in Enhancing the Temperature-Dependent Growth Inhibition of Escherichia coli O157:H7 ATCC 43895 by a Pseudomonas sp.

Growth of Escherichia coli O157:H7 strain ATCC 43895 was monitored at 5, 10, 15, and 25°C in both pure and mixed (1:1) cultures with a gluconate-producing Pseudomonas sp. found in meat to evaluate the effect of the absence and presence of 1% glucose in broth on temperature-dependent competition. The number of colonies of the Pseudomonas strain exceeded 9 log CFU/ml under all conditions tested. The pathogen grew better as the temperature increased from 10 to 15 and 25°C and grew better in pure culture than in mixed cultures. Pseudomonas sp. inhibited E. coli O157:H7 in cocultures with glucose at 10°C, while at 15°C the pathogen exhibited a biphasic pattern of growth with an intermediate inactivation period. Pathogen inhibition was much weaker in cocultures grown without glucose at 10 to 15°C and, irrespective of glucose, at 25°C. These results indicate that glucose enhances the growth inhibition of E. coli O157:H7 by some Pseudomonas spp., potentially due to its rapid uptake and conversion to gluconate, at low (≤15°C) temperatures.     

Novel RNA-binding Protein P311 Binds Eukaryotic Translation Initiation Factor 3 Subunit b (eIF3b) to Promote Translation of Transforming Growth Factor β1-3 (TGF-β1-3)

P311, a conserved 8-kDa intracellular protein expressed in brain, smooth muscle, regenerating tissues, and malignant glioblastomas, represents the first documented stimulator of TGF-β1-3 translation in vitro and in vivo. Here we initiated efforts to define the mechanism underlying P311 function. PONDR® (Predictor Of Naturally Disordered Regions) analysis suggested and CD confirmed that P311 is an intrinsically disordered protein, therefore requiring an interacting partner to acquire tertiary structure and function. Immunoprecipitation coupled with mass spectroscopy identified eIF3 subunit b (eIF3b) as a novel P311 binding partner. Immunohistochemical colocalization, GST pulldown, and surface plasmon resonance studies revealed that P311-eIF3b interaction is direct and has a K_d of 1.26 μm. Binding sites were mapped to the non-canonical RNA recognition motif of eIF3b and a central 11-amino acid-long region of P311, here referred to as eIF3b binding motif. Disruption of P311-eIF3b binding inhibited translation of TGF-β1, 2, and 3, as indicated by luciferase reporter assays, polysome fractionation studies, and Western blot analysis. RNA precipitation assays after UV cross-linking and RNA-protein EMSA demonstrated that P311 binds directly to TGF-β 5′UTRs mRNAs through a previously unidentified RNA recognition motif-like motif. Our results demonstrate that P311 is a novel RNA-binding protein that, by interacting with TGF-βs 5′UTRs and eIF3b, stimulates the translation of TGF-β1, 2, and 3.     

Clamping down on weak terminal base pairs: oligonucleotides with molecular caps as fidelity-enhancing elements at the 5'- and 3'-terminal residues

The base-pairing fidelity of oligonucleotides depends on the identity of the nucleobases involved and the position of matched or mismatched base pairs in the duplex. Nucleobases forming weak base pairs, as well as a terminal position favor mispairing. We have searched for 5′-appended acylamido caps that enhance the stability and base-pairing fidelity of oligonucleotides with a 5′-terminal 2′-deoxyadenosine residue using combinatorial synthesis and MALDI-monitored nuclease selections. This provided the residue of 4-(pyren-1-yl)butyric acid as a lead. Lead optimization gave (S)-N-(pyren-1-ylmethyl)pyrrolidine-3-phosphate as a cap that increases duplex stability and base-pairing fidelity. For the duplex of 5′-AGGTTGAC-3′ with its fully complementary target, this cap gives an increase in the UV melting point T_m of +10.9°C. The T_m is 6.3–8.3°C lower when a mismatched nucleobase faces the 5′-terminal dA residue. The optimized cap can be introduced via automated DNA synthesis. It was combined with an anthraquinone carboxylic acid residue as a cap for the 3′-terminal residue. A doubly capped dodecamer thus prepared gives a melting point decrease for double-terminal mismatches that is 5.7–5.9°C greater than that for the unmodified control duplex.     

Optimization of Ultrasonic-Assisted Extraction and Radical-Scavenging Capacity of Phenols and Flavonoids from Clerodendrum cyrtophyllum Turcz Leaves

Ultrasonic-assisted extraction (UAE) was developed to extract phenolic and flavonoid antioxidants from Clerodendrum cyrtophyllum Turcz leaves. The optimal experimental parameters for antioxidant extraction from C. cyrtophyllum leaves were measured using single-factor experimentation combined with response surface methodology (RSM). Total phenolic content (TPC) and total flavonoid content (TFC) assays were used to quantify antioxidant compounds. Next, antioxidant radical scavenging capacity was measured using 2,2′-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′ -azino-bis(3-ethylbenzothiazoline-6-sulphonicacid) (ABTS) radicals. Optimized extraction conditions for UAE from C. cyrtophyllum leaves were as follows: 60.9% ethanol, 85.4 min, and 63.3°C for maximal TPC extraction (16.8±0.2 mg GAE/g DW); 67.7% ethanol, 82.9 min, and 63.0°C for maximal TFC extraction (49.3±0.4 mg RT/g DW); 48.8% ethanol, 85.1 min, and 63.9°C for maximal DPPH radical-scavenging capacity (86.8±0.2%); and 50.6% ethanol, 81.3 min, and 63.4°C for maximal ABTS radical-scavenging capacity (92.9±0.5%). Ethanol concentration was the most important factor in the extraction process. Our work offers optimal extraction conditions for C. cyrtophyllum as a potential source of natural antioxidants.     

Effects of Abiotic Factors on Acetylene Reduction by Cyanobacteria Epiphytic on Moss at a Subantarctic Island

Acetylene reduction (AR) rates by cyanobacteria epiphytic on a moss at Marion Island (46°54′ S, 37°45′ E) increased from −5°C to a maximum at 25 to 27°C. Q₁₀ values between 0 and 25°C were between 2.3 and 2.9, depending on photosynthetic photon flux density. AR rates declined sharply at temperatures above the optimum and were lower at 35°C than at 0°C. Photosynthetic photon flux density at low levels markedly influenced AR, and half of the maximum rate occurred at 84 μmol m⁻² s⁻¹, saturation occurring at ca. 1,000 μmol m⁻² s⁻¹. Higher photosynthetic photon flux density levels decreased AR rates. AR increased up to the highest sample moisture content investigated (3,405%), and the pH optimum was between 5.9 and 6.2. The addition of P, Co, and Mo, individually or together, depressed AR.