Fungi to bacteria ratio: Historical misinterpretations and potential implications

Acta Oecologica 95 (2019) 1–11

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Fungi to bacteria ratio: Historical misinterpretations and potential implications

T

Xiaoli Wanga,d,1, Weixin Zhangb,1, Yuanhu Shaob, Jie Zhaoc, Lixia Zhoud, Xiaoming Zoue, Shenglei Fub,∗ a

State Key Laboratory of Plateau Ecology and Agriculture, Qinghai Academy of Animal and Veterinary Sciences, Qinghai University, Xining, 810016, PR China Key Laboratory of Geospatial Technology for the Middle and Lower Yellow River Regions, College of Environment and Planning, Henan University, Kaifeng, 475004, PR China c Key Laboratory of Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, 410125, PR China d Key Laboratory of Vegetation Restoration and Management of Degraded Ecosystems, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, 510650, PR China e Department of Environmental Sciences, University of Puerto Rico, P. O. Box 70377, San Juan, PR, 00936-8377, USA b

ARTICLE INFO

ABSTRACT

Keywords: Fungi to bacteria ratio Fungal-based ecosystems Bacterial-based ecosystems Microbial turnover rate Assimilation Energy channel

Bacteria and fungi are the primary consumers and, thus, the decomposition pathways are described accordingly as bacterial-based or fungal-based energy channels. However, the fungi to bacteria ratios (F: B), which indicated either by microbial biomass, respiration, or growth, represents only a snapshot of the whole energy channel during a given period rather than the cumulative contribution. Even the energy channel biomass only takes into account one dimension without considering the time. We believe that the F: B ratio has been misinterpreted in an ecological sense due to a lack of a clear definition. Here, we estimated the F: B biomass ratios, production ratios (microbial biomass multiplied by the turnover rate) and assimilation ratios (the sum of microbial production and respiration) using a dataset from 192 relevant studies. The F: B biomass ratios varied from 0.106 to 9.080, depending on the methods used. Based on direct microscopy method, the fungal/(fungal + bacterial) production and assimilation ratios ranged from 0.39 to 54.78% and 0.25–45.05%, respectively; while, those ratios based on phospholipids fatty acids (PLFAs) method were 0.06–5.51% and 0.04–0.66%, respectively. We conclude that bacteria contributes greater to the energy flow in terrestrial ecosystems compared with fungi based on the F: B assimilation. The relative contribution of bacteria and fungi can be better evaluated using the F: B assimilation ratio, rather than the biomass ratio or production ratio. Nevertheless, there are still uncertainties in the estimations of microbial production and assimilation due to their complicated responses to soil fauna activities. The regulation of soil fauna on microbial biomass, turnover rate and respiration, and associated changes in the energy allocations in the soil food web should be emphasised in future studies.

1. Introduction Soil microbial communities, as principle decomposers, play critical roles in the regulation of nutrient cycling, energy flow and ecosystem productivity (Wardle, 1998; Jackson et al., 2007; Rinklebe and Langer, 2013). Soil fungi and bacteria are major microbial components with distinct physiological and ecological characteristics (van der Wal, 2006; Wang et al., 2016). For instance, bacterial biomass has a lower carbon to nitrogen (C: N) ratio (3–6), whilst fungal biomass has a higher C: N ratio (5–15) (McGill et al., 1981). Accordingly, a higher soil C: N ratio may increase the relative abundance of fungi due to stoichiometric

constraints, as bacteria are generally believed to require more N per unit of biomass C accumulation than fungi (Elliott et al., 1983; Fierer et al., 2009). Therefore, the ratio of fungi to bacteria (F: B), as an indicator of microbial community structure, has important ecological significance in soil ecology (Bailey et al., 2002), and reflects the capacity of ecosystem self-regulation (Bardgett and McAlister, 1999; de Vries et al., 2006). A higher biomass ratio of fungi to bacteria is believed to indicate a more sustainable agro-ecosystem (de Vries et al., 2006). It had also been reported that a clear relationship between increasing plant productivity and bacterial versus fungal dominance (Ingham and Slaughter, 2004; de Ruiter et al., 2005).

Corresponding author. College of Environment and Planning, Henan University, Kaifeng, 475004, PR China. E-mail addresses: [email protected], [email protected] (S. Fu). 1 These authors contributed equally to this work. ∗

https://doi.org/10.1016/j.actao.2018.10.003 Received 5 February 2018; Received in revised form 11 October 2018; Accepted 13 October 2018 1146-609X/ © 2018 Elsevier Masson SAS. All rights reserved.

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Table 1 The categories of fungal to bacterial ratios (F: B) in the current studies. Classify

Characterised

Methods

References

F: B biomass ratio F: B biomass ratio

The ratio of fugal colonies to bacterial colonies The length and diameter of the fungal hyphae were measured by the membrane filter method, using optical microscopy and phenol aniline blue as a stain. Bacteria and fungi have different fatty acid compositions in their phospholipids. PLFA 18:2ω6, 9 as a measure of fungal biomass and the sum of 13 bacteria-specific PLFAs as a measure of bacterial biomass. Using the streptomycin and cycloheximide antibiotics were applied alone or in combination to the soil to determine the fungal and bacterial respiration It is based on DNA and estimated using quantitative polymerase chain reaction Fungi growth was assessed using the Ac-in-erg method. Bacteria growth was assessed using the thymidine and leucine incorporation. That can directly estimate of microbial growth rates under natural soil conditions exist. The relative flow of energy and nutrients through the bacterial and fungal channel can be assessed by the fungal to bacterial feeder ratio (The ratios of fungal to bacterial feeding nematodes) fungal-energy channel was fungi and fungal-feeding fauna biomass, and bacterial-energy channel was bacteria and bacterial-feeding fauna biomass (The bacterial-feeding fauna included the enchytraeids, flagellates, amoebae, bacterivorous nematodes, omn. + pred. nematodes, nematophagous mites, pred. mites + collembola. The fungal-feeding fauna included enchytraeids, fungivorous nematodes, microbi-detritivores, omn. + pred. nematodes, nematophagous mites, pred.mites + collembola)

Plate count method Direct microscopy method

Olsen and Bakken (1987) Shield et al., 1973; Hanssen et al. (1974); Hobbie et al. (1977); Nishio (1983) Frostegård and Bååth (1996)

F: B biomass ratio F: B respiratory ratio F: B gene ratio F: B growth ratio F: B feeder ratio F: B channel biomass ratio

Phospholipid fatty acid (PLFAs) Substrate-induced respiration (SIR) qPCR (16S RNA/18S RNA)

Anderson and Domsch (1975); Bailey et al. (2002) Fierer et al. (2005)

Acetate into ergosterol incorporation and the thymidine and leucine incorporation Microscopic examination

Rousk and Bååth (2007a); 2007b; Bapiri et al. (2010) Ruess and Ferris (2004)

Based on the soil food web and the feeding preference of the predators

Holtkamp et al. (2008); de Vries et al., 2012a

bacterial component generally occur in N-rich soils that contain readily decomposable substrates. Zhao and Neher (2014) analysed 67 raw data sets of nematode genera from the three types of ecosystems and concluded that energy pathways are bacterial-dominated in all ecosystems whether expressed as nematode abundance or biomass. Furthermore, both bacteria and fungi support their soil fauna predator in the corresponding food chain (de Ruiter et al., 1993; Wardle and Lavelle, 1997). Hence, it was proposed that bacterial channel biomass is the sum of bacteria and bacterial-feeding fauna, and fungal channel biomass is the sum of fungi and fungal-feeding fauna (Holtkamp et al., 2008; de Vries et al., 2012b). In the abovementioned studies, the F: B biomass ratios were sometimes greater than 1.0 (Ingham et al., 1989; Ohtonen et al., 1999). Also, the F: B channel biomass ratios were sometimes greater than 1.0, indicating there are fungal-based ecosystems in grassland (de Vries et al., 2012b, 2012c). However, it may not be appropriate to determine the relative fungal or bacterial dominance only by the F: B standing biomass ratio. Soil microbial biomass is controlled by two distinct processes: changes in biomass production in biomass destruction (e.g., predation, physical disruption or microbial death); thus, the standing biomass is only a snapshot of the outcome of these processes. In contrast, the microbial metabolic quotient (respiration: biomass ratio) can reflect the ecological efficiency of fungi and bacteria (Beare et al., 1992; Blagodatskaya and Anderson, 1998). As a result, the microbial biomass markers may be largely unrelated to their contributions to processes such as carbon mineralisation and nutrient cycling (Bapiri et al., 2010). In other words, standing microbial biomass may account for much less proportions of the total energy flow due to the ignorance of microbial turnover and respiration. Overall, a wide variety of conceptual terms regarding the F: B ratios have gradually emerged, and we believe that the F: B ratio has been misinterpreted in an ecological sense due to a lack of a clear definition. For a better understanding of the F: B ratios, we summarised and explain the various terms of F: B ratios, e.g., F: B biomass ratio, F: B activity or respiration ratio, F: B gene ratio, F: B growth ratio and F: B channel biomass ratio (Table 1). Given that assimilation may reflect the contribution of fungi or bacteria to the energy flow in ecosystems (Odum, 1957), we used F: B production, as well as F: B assimilation ratios, to quantify the relative dominance of fungi or bacteria. We collected data on the F: B ratios in terms of biomass, production and assimilation from literature as well as from our own experiments, and

The earliest descriptions of fungal-dominated and bacterial-dominated microbial communities appeared 40 years ago, where microbial communities in streams were reported to shift from fungal-dominated to bacterial-dominated as litter decomposition proceeded (Bärlocher and Kendrick, 1974; Suberkropp and Klug, 1976). Also, compared with fungi, a greater population of soil bacteria was observed in rotation regimes (Golebiowska and Ryszkowski, 1977). Thereafter, a large amount of researches on the effects of the different tillage systems on microorganisms has been conducted. A conventionally tilled agro-ecosystem was reported to be characterised by a bacterial-based food web; in contrast, a no-tillage system by a fungi-based food web (Hendrix et al., 1986). Meanwhile, soil food web structures amongst different ecosystems during natural succession attracted significant attention. It was found to change gradually from the bacterial-based grassland ecosystem to the fungal-based forest ecosystem (Ingham et al., 1986a, 1986b; 1989; Ingham and Thies, 1996). A large number of plants with high-quality (high nitrogen and low tannin) litter is beneficial to the growth of bacteria in the early succession, but in late stage of succession, plants with low-quality (low nitrogen and high tannin) litter generate and then promote the growth of fungi (Ohtonen et al., 1999). Note that these previous studies were based on the dilution plate or direct microscopy counting method, and the reported F: B ratios were normally > 1.0 which may overemphasize the contribution of fungi. F: B ratios were then extensively studied with the gradual development of methodology for microbial studies, such as phospholipid fatty acids (PLFAs), substrate-induced respiration (SIR) and 16S or 18S DNA sequencing (Frostegård and Bååth, 1996; Bailey et al., 2002; Fierer et al., 2009). In addition, the concept of bacterial channel or fungal channel was introduced to consider the biomasses of both bacteria and fungi and their feeders (Ruess and Ferris, 2004). Strong associations between nematodes and their fungal or bacterial food sources were revealed. The bacterial-dominated soils have many bacterial predators (e.g., protozoa and bacterial-feeding nematodes), whilst fungal-dominated soils have many fungal predators (e.g., fungal-feeding nematodes and fungal-feeding microarthropods) (Ingham et al., 1989). Subsequently, Ruess and Ferris (2004) proposed that consumer organisms affect the rates of energy and nutrient release. The nature and abundance of available resources can be monitored by faunal analysis of fungal- and bacterial-feeding nematodes. Pathways with a strong 2

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Table 2 Bacterial and fungi turnover time (days), bacterial and fungi production (the accumulated biomass in per day) in different ecosystems and types of forests.A (Bloem et al., 1992),B (Uhlířová and Săntrùčková, 2003),C (Rousk and Bååth, 2007b). The data of F: B biomass were all based on the PLFAs method. Ecosystems

Bacterial turnover time (days)

Fungal turnover time (days)

F: B turnover time

F: B biomass (PLFAs)

F: B production

Agro-ecosystem Grassland Forest Broadleaved forest Coniferous forest Mixed forest

0.75A 1.05B 30.35B 30.35 30.35 30.35

140C 140 140 140 140 140

186.67 133.33 4.63 4.63 4.63 4.63

0.2141 0.2868 0.2696 0.3289 0.2779 0.2086

0.0011 0.0022 0.0582 0.0711 0.0600 0.0451

summarised the patterns of F: B ratios in different ecosystems. The specific steps are as follows: 1) collecting the F: B ratios in term of biomass from literature; 2) collecting the turnover time of bacteria and fungi in different ecosystem, then the F: B biomass was multiplied by the turnover time to obtain the F: B production ratio per unit of time (Carter and Suberkropp, 2004; Appendix S3). 3) collecting the ratio of production to respiration for bacteria and fungi (Holland and Coleman, 1987), then calculating the assimilation of bacteria or fungi per unit of time equaled the sum of the production and the respiration of bacteria or fungi at a trophic level (Odum, 1957). Our objectives were to 1) demonstrate the effect of microbial turnover, production and respiration on the F: B ratios in different ecosystems; 2) compare various expressions of F: B ratios and differences of their interpretations; and 3) estimate the relative contribution of fungi and bacteria to energy flow at a trophic level.

and replication numbers in each ecosystem were determined and reported in all data sets. 2.2. Modified estimation of F: B ratio 2.2.1. Ratio of fungal to bacterial production Generally, the bacterial turnover time is measured using thymidine and leucine incorporation into cold-acid-insoluble material (Thomas et al., 1974). Fungal growth rate is typically measured using acetatein-ergosterol incorporation, which is based on the incorporation of radioactively labelled acetate into the fungal-specific lipid ergosterol (Newell and Fallon, 1991; Pennanen et al., 1998; Bååth, 2001). Rousk and Bååth (2011) wrote a review named “Growth of saprotrophic fungi and bacteria in soil” and they listed the estimated bacterial and fungal turnover times in soil (Please see the detail from the following diagrams). We reference the data of bacterial and fungal turnover time. More concretely, bacterial turnover times were 7–53.7 days, 1.0–1.1 days and 0.5–1.0 day in the forest, grassland and farmland ecosystems, respectively (Bloem et al., 1992; Uhlířová and Săntrùčková, 2003). Fungal turnover times ranged from 130 to 150 days (Rousk and Bååth, 2007b). The F: B turnover rate was also examined (Table 2). The F: B biomass was multiplied by the turnover time to obtain the F: B production ratio per unit of time (Carter and Suberkropp, 2004; Appendix S3). Based on the turnover rate data, we calculated the biomass accumulated by bacteria or fungi per unit of time (Formula (1)). Considering that the proportions are more mathematically stable, we calculated the ratio of fungi/(fungi + bacteria) rather than fungi/bacteria (Sohlenius et al., 1988) (Formula (2)).

2. Materials and methods 2.1. Conventional estimation of F: B ratio 2.1.1. Ratio of fungal to bacterial biomass In the present study, we focused on field studies in which the ratio of fungal to bacterial biomass was measured for different ecosystems either by dilution plate culture, direct microscopy or phospholipid fatty acid (PLFA) analysis. Google Scholar and the ISI Web of Knowledge were used to select literature that met the inclusion criteria. The following were used as search terms: “the ratio of fungal to bacterial biomass”, “fungal and bacterial biomass”, “soil microbial community structure”, “fungaldominated and bacterial-dominated microbial community”, “pathways”, “channels ”, “budgets ”, “food chain”, or ”trophic group ratio”. We directly acquired the ratios of fungal to bacterial biomass from the figures or tables in the selected studies. If only the biomasses of fungi and bacteria were reported, we calculated the F: B ratios using the raw data. When only the means and errors were presented in a graph, the data were digitised using an open source Engauge Digitizer 4.1 software (Informer Technologies, Inc., Bilbao, Spain). Three types of data sets were established according to the measurement methods. Specifically, we collected the F: B ratio data from 57 studies using the dilution plate culture method (Appendix S1), 130 studies using PLFA analysis (Appendix S2), and 8 studies using the direct microscopy method (Appendix S3). It is worth noting that the F: B ratio based on the plate counts represented the F: B relative ratio. Despite well-known bias, such as the overwhelming number of non-cultivable microorganisms which are not considered by such techniques, the method on more or less specific solid media are still used and informative (Janvier et al., 2007). The media for enumeration of bacteria is “beef-peptone agar” and the media for enumeration of fungi is “Martin's rose Bengal streptomycin agar” (Martin, 1950). Additionally, we collected 41 studies on soil microbial communities and calculated the F: B ratios of agro-ecosystems with of conventional tillage (CT) or no-tillage (NT) treatments (Appendix S4). Furthermore, we used “fungal to bacterial channel biomass” and “fungal channel biomass and bacterial channel biomass” as the search terms and found four related studies (Appendix S5). The means, standard errors (SEs)

BF × PF = PB BB ×

T TF T TB

=

BF × TB BB × TF

(1)

T

BF × T PF F = T PB + PF BB × T + BF × B

T TF

=

1 BB × TF BF × TB

+1

(2)

where PF and PB represent the production of soil fungi and bacteria, respectively; BF and BB represent the biomass of soil fungi and bacteria, respectively; TF and TB represent the turnover time of soil fungi and bacteria, respectively; and T represents a year as a unit. 2.2.2. Ratio of fungal to bacterial assimilation In the model of energy flow, the assimilation of bacteria or fungi per unit of time equaled the sum of the production and the respiration of bacteria or fungi at a trophic level (Odum, 1957). We obtained the total energy per unit of time that flowed through fungi or bacteria to multiple trophic levels in the soil food web. Substrate-induced respiration (SIR) procedure for measuring the soil microbial biomass was first presented by the Anderson and Domsch (1975). It was an attempt to directly measure the bacterial and fungal contributions to the total metabolic activity of the soil based on the selective inhibition of protein synthesis in prokaryotic and eukaryotic cells by the antibiotics streptomycin and actidione. Considering that the respiration data from SIR represent the initial maximal respiration rate induced by glucose rather 3

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AF PF + RF = = AB + AF (PB + RB) + (PF + RF)

PF+PF /

(P

B

+ PB/

()

P R F

( ) ) + (P P R B

( ))

P F+PF / R F

1

= 1+

PB PF

×

() ( RP )B ×

() P 1+( ) R F

P P × 1+ R F R B

(4)

where AF and AB represent the assimilation of soil fungi and bacteria, respectively; and RF and RB represent the respiration of soil fungi and P bacteria, respectively. P + R represents the ratio of fungal production

( )

F

( )

( )

to total fungi-metabolised C and the value of P + R was 52%. P + R B F represents the ratio of bacterial production to total bacteria-metaboP lised C and the value of P + R was 33%. The two carbon assimilation

( )

P

P

B

efficiencies were the recalculated mean values using the data set in Holland and Coleman (1987). More concretely, there were two methods to determinate of yield coefficients (carbon assimilation efficiencies), the 14C-labelled substrates and direct measurement of microbial biomass. Using the 14Clabelled substrates assumes all the substrate is used, and includes metabolite production as biomass. Alternatively, yield coefficients may be determined by direct measurement of microbial biomass (sizes and numbers), and the assumption that substrate used is the sum of biomass-C plus respired-C, is defined here as the amount of biomass-C produced per unit substrate-C used exclusive of extracellular metabolites produced. This value was calculated as Y = biomass-C/(biomassC + Cumulative CO2eC) (Elliott et al., 1983). Finally, we summed the energy model that could be used to estimate the relative contribution of fungi and bacteria to energy flow within the soil food web. Given that bacterial and fungal turnover and respiration represent the major proportions of the unaccounted energy flow in the soil food web, we ignored the turnover and respiration of soil fauna. Fungalfeeder and bacterial-feeder have their turnover and respiration rate in the complex soil food web, so we did not calculated the specific data and just gave the model. The fungal assimilation and fungal-feeder assimilation (Afungal-feeder) together equaled the assimilation of the fungal-based food chain; the bacterial assimilation and bacterial-feeder assimilation (Abacterial-feeder) together equaled the assimilation of the bacterial-based food chain (Formula (5)). The equation is simplified as follows:

Afungal based food chain AF + Afungal CF = = CB Abacterial based food chain AB + A bacterial

Fig. 1. Soil fungi to bacteria ratios in different ecosystems: (A) The data of forest, grassland and farmland from Appendix S1 based on the method of dilution plate culture; (B) The data of forest, grassland and farmland from Appendix S2 based on the method of PLFAs (Phospholipid fatty acids); (C) The data of forest, grassland and farmland from Appendix S3 based on the method of direct microscopy.

() () P

B

P = F × PB

feeder

(5)

where CF and CB represent the channel energy of soil fungi and bacteria, respectively. All values are expressed as kilocalories per m2 per year. 2.3. Statistical analysis

than the carbon assimilation efficiencies of bacteria and fungi. So, we did not use the respiration data based on the method of SIR. We use the carbon assimilation efficiencies referring to the (P/(P + R)) (the production/(production + respiration)) to calculate the assimilation rather than the respiration of fungi and bacteria. Carbon assimilation efficiencies means the (retained in biomass)/(retained in biomass + respired as CO2). The reported fungal carbon assimilation efficiencies tend to be significantly higher than those for bacteria, i.e., a higher proportion of the carbon metabolised by fungi is retained in biomass instead of respired as CO2 (Holland and Coleman, 1987). Then we calculated F: B assimilation by adding production and respiration (Formula (3) and Formula (4)).

PF+PF / R AF P + RF F = F = P AB PB + RB PB + PB / R

feeder

We analysed the data from different ecosystems using one-way ANOVA. The least significance difference (LSD, the variance was homogeneous) or Tamhane's T2 (the variance was not homogeneous) was performed for the three groups of ecosystem data sets and the three types of forest data sets. We analysed the data from CT and NT farmlands using a t-test and detected the differences amongst the CT, reduced tillage (RT) and N by one-way ANOVA. All statistical analyses were performed using SPSS 18.0 software (SPSS Inc., Chicago, IL). Statistical significance was determined at P < 0.05. 3. Results

()

× 1+

()

3.1. F: B biomass ratios

()

× 1+

()

Based on the dilution plate method, there was a significant difference in the F: B colony ratios amongst the three types of ecosystems (one-way ANOVA; P = 0.010). Specifically, the F: B ratio in farmland

P R B P R F

P R F

P R B

(3) 4

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Fig. 2. Soil fungi to bacteria ratios in different types of forests: (A) The data of broadleaved forest, coniferous forest and mixed forest from Appendix S2 based on the method of PLFAs (Phospholipid fatty acids); (B) The data of conventional tillage and no tillage from Appendix S4 based on the method of PLFAs; (C) The data of broadleaved forest, coniferous forest and mixed forest from Appendix S3 based on the method of direct microscopy; (D) The data of conventional tillage, reduced tillage, and no tillage from Appendix S3 based on direct microscopy.

farmland (0.004) (Fig. 3). In farmland, grassland and forests ecosystems, the F: B production ratios were in the range of 0.004–1.269 and those in forests were significantly greater than that in farmland (P = 0.000) and grassland (P = 0.000) (Fig. 3A). The F: B production ratio was 1.960, 1.063 and 0.247 in broad-leaved, coniferous and mixed forests, respectively. It was significantly greater in broad-leaved forests than in mixed forests (P = 0.006) (Fig. 3B). The F: B production ratio following RT (0.011) was significantly greater than that following NT (0.004) treatment (P = 0.009). There was no significant difference between RT and CT (P = 0.367) (Fig. 3C). In summary, F: B production ratios can be greater than 1.0 in forests but are always less than 1.0 in farmland or grassland based on the direct microscopy method.

was significantly greater than that in grassland (P = 0.050) (Fig. 1A). Based on the phospholipid fatty acids (PLFAs) method, there was no significant difference in the F: B ratios amongst the three types of ecosystems (one-way ANOVA; P = 0.360) (Fig. 1B). However, based on the direct microscopy method, the F: B ratios increased up to 5.873 in forests, which was significantly greater than that in farmland (one-way ANOVA; P = 0.016) (Fig. 1C). Although the F: B biomass ratios appear to be the highest in broadleaved forests, there were no significant differences in the F: B biomass ratios amongst the three types of forests using the PLFA method (P = 0.680; Fig. 2A). Additionally, the F: B biomass ratios in the CT and NT were 0.11 and 0.18, respectively, and there was no significant difference between the two treatments (t-test; P = 0.330; Fig. 2B). Importantly, all F: B ratios in the terrestrial ecosystems were lower than 1.0 based on either the dilution plate culture or PLFA analysis (Fig. 1A and B and Fig. 2A and B). In contrast, based on the direct microscopy method, the F: B ratios ranged from 9.075 to 1.144 in the three types of forests. The differences amongst the three types of forests were not significant albeit exhibited large variations (P = 0.297) (Fig. 2C). The F: B ratio following RT treatment was significantly higher than that following CT treatment (P = 0.009) (Fig. 2D). Overall, the F: B ratios in the terrestrial ecosystems were greater than 1.0 in most cases based on direct microscopy (Figs. 1C and 2C, D).

3.3. F: B assimilation ratios The even smaller F: B assimilation ratios were all less than 0.005 in all three ecosystems based on the PLFA method. The F: B assimilation ratios were down to almost 0.000 in farmland and grasslands, and they were significant smaller than that (F: B assimilation ratio = 0.003) in forests (P = 0.000). The ratios were 0.003, 0.003 and 0.002 in coniferous, broad-leaved and mixed forests, respectively. And there was no significant difference among the three types of forests (P = 0.615). Then the ratios were almost 0.000 in NT and CT treatments (Table 3). However, the F: B assimilation ratios ranged from 0.003 to 0.926 in the three types of ecosystems based on the direct microscopy method. Specifically, the ratios were 0.529, 0.003, and 0.013 in forest, farmland, and grassland, respectively. It was significantly greater in forests than that in farmland (P = 0.020). Then the F: B assimilation ratios were 0.441, 0.920, and 0.176 in coniferous, broad-leaved, and mixed forests, respectively. There was no significant difference among the three types of forests (P = 0.297). The F: B assimilation ratio was 0.005 following CT, and it following NT (0.003) was significantly smaller than that following RT (0.005) treatment (P = 0.009). In a word, F: B assimilation ratios were always less than 1.0 based on the direct microscopy method (Table 3).

3.2. F: B production ratios Based on the PLFA method, the F: B production ratios were all less than 1.0 in all three ecosystems. The F: B production ratios were 0.001, 0.002 and 0.058 in farmlands, grasslands and forests, respectively (Table 2). The ratio in forests was significant greater than those in farmland and grassland (P = 0.000). The F: B production ratios were 0.071, 0.060 and 0.045 in broad-leaved forests, coniferous forests and mixed forests, respectively (Table 2). Based on direct microscopy method, the maximum F: B production ratio was in broad-leaved forests (1.960) and the minimum was in 5

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the ranges of F/(F + B) biomass, production and assimilation in the different ecosystems were 39.85%–66.96%, 0.39%–40.07% and 0.25%–32.35%, respectively. The ranges of F/(F + B) biomass, production and assimilation in different forests were 51.55%–81.95%, 19.49%–54.78% and 13.38%–45.05%, respectively. In the different tillage managements, the ranges of F/(F + B) biomass, production and assimilation were 40.63%–65.51%, 0.43%–1.03% and 0.27%–0.66%, respectively. There was a lower proportion of fungi based on the PLFA method compared to direct microscopy. The ranges of F/(F + B) biomass, production and assimilation in these ecosystems were only 8.35%–19.86%, 0.06%–5.51% and 0.04%–3.66%, respectively (Table 4). Furthermore, we re-analysed the F: B PLFA biomass and F: B channel biomass data from the two case studies (de Vries et al., 2012a; Holtkamp et al., 2008) and calculated the corresponding F: B assimilation ratios. The F: B PLFAs were in the range of 0.059–0.140 and the F: B channel biomasses were in the range of 0.790–2.230 in the treatments of wheat field-control, wheat field-drought, grassland-control and grassland-drought. However, the F: B assimilation ratios were in the range of 0.006–0.016. Because these ratios are too low, the data are presented as (fungi/(fungi + bacteria)) × 100% (Fig. 4). 4. Discussion 4.1. Incompatibility of F: B biomass ratio as an indicator of the ecosystem energy channel 4.1.1. Biomass: a snapshot of the ecosystem energy channel Biomass is the mass of living organisms in a given area or ecosystem at a given time (McNaught and Wilkinson, 1997). The processes of consumption and decomposition are considered ecologically as systemlevel metabolism, and the primary decomposition agents are bacteria and fungi, which are often referred to as “microbial biomass” (Coleman et al., 1983; Coleman and Crossley, 1996). However, the microbial biomass in soil may be largely unrelated to its real contributions to related processes (Bapiri et al., 2010). As mentioned above, microbial biomass is only a snapshot of energy flow and represents only one trophic level in the soil food web. Moore (1994) reported that a species with low biomass and a high turnover may have a greater impact on the functioning and stability of the food web compared to that with high biomass and a low turnover. However, the transfer of energy from the primary producers into organisms at higher trophic levels supports a wide range of heterotrophs (Coleman and Crossley, 1996). On the other hand, a standing microbial biomass fails to evaluate the proportion of active fraction of microbial community (Klein and Paschke, 2000) and, thus is appropriate to be used to reflect the influences of active microorganisms on biogeochemical processes. Hence, analyses of the active and potentially active fractions are essential in

Fig. 3. Fungal to bacterial production based on the biomass data using the direct microscopy method from Appendix S3. (A) Three types of ecosystems: Forests, Farmland and Grassland; (B) Three types of forests: Broadleaved forests, Coniferous forests and Mixed forests; (C) Three types of management treatment: Conventional tillage (CT), Reduced tillage (RT) and No tillage (NT).

3.4. The proportion of fungi in microbial biomass, production and assimilation Based on direct microscopy, the greatest proportion of fungi was in broad-leaved forests and the lowest in farmland (Table 4). Specifically,

Table 3 The ratio of fungal biomass to bacterial biomass, the ratio of fungal production to bacterial production, the ration of fungal assimilation to bacterial assimilation in different ecosystems, forests and farmlands using the method of PLFAs and the direct microscopy. “CF, BF, CBF, NT, RT, CT “represent “coniferous forests, broadleaved forests, coniferous and broadleaved forests, no tillage, reduced tillage and conventional tillage”. P (production) = B (biomass) *T (turnover time), the data of T (turnover time) from Table 2. Treatments

Forest Farmland Grassland CF BF CBF NT RT CT

The date based on direct microscopy method

The date based on the PLFAs method

F: B biomass

F: B production

F: B assimilation

F: B biomass

F: B production

F: B assimilation

2.026 0.663 1.824 1.552 4.539 1.064 0.684 1.900 0.821

0.669 0.004 0.018 0.548 1.211 0.242 0.004 0.010 0.007

0.529 0.003 0.013 0.441 0.920 0.176 0.003 0.008 0.005

0.223 0.174 0.181 0.248 0.204 0.185 0.122 N/A 0.091

0.054 0.001 0.002 0.058 0.057 0.044 0.001 N/A 0.001

0.003 0.000 0.000 0.003 0.003 0.002 0.000 N/A 0.000

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Table 4 The proportion of fungi in microbial biomass, production and assimilation ((Fungi/(Fungi + Bacteria)) %) in different ecosystems, forests and farmlands using the method of PLFAs and the direct microscopy. “CF, BF, CBF, NT, RT, CT “represent “coniferous forests, broadleaved forests, coniferous and broadleaved forests, no tillage, reduced tillage and conventional tillage”. Treatments

Forest Farmland Grassland CF BF CBF NT RT CT

The date based on direct microscopy method (in %)

The date based on the PLFAs method (in %)

F/(F + B) biomass

F/(F + B) production

F/(F + B) assimilation

F/(F + B) biomass

F/(F + B) production

F/(F + B) assimilation

66.96 39.85 64.58 60.82 81.95 51.55 40.63 65.51 45.09

40.07 0.39 1.76 35.41 54.78 19.49 0.43 1.03 0.68

32.35 0.25 1.12 28.63 45.05 13.38 0.27 0.66 0.43

18.23 14.82 15.36 19.86 16.94 15.60 10.86 N/A 8.35

5.14 0.11 0.22 5.51 5.35 4.19 0.10 N/A 0.06

3.38 0.07 0.14 3.60 3.66 2.72 0.06 N/A 0.04

1998). As a result, the fungal-dominated pathway was considered to be responsible for carbon decomposition in many natural ecosystems (Berg et al., 1998; Myers et al., 2001; Leckie, 2005). However, the most significant source of error in the direct microscopic method of estimating fungal biomass in soil is observer subjectivity (Stahl et al., 1995). Importantly, this method tends to overestimate the fungal biomass because a large part of fungal hypha visible through a microscope could be inactive or dead (Bardgett et al., 1993; Six et al., 2005). The epifluorescence microscopy technique was developed to determine the fungal biomass considering the active fungal hyphae (Babiuk and Paul, 1970; Ingham and Klein, 1984; Lodge and Ingham, 1991; Binnerup et al., 1993) and confocal laser scanning microscopy reduced the labour associated with direct microscopy (Bloem et al., 1995). These modified microscopy methods may reduce the uncertainties associated with and improve the efficiency of fungal biomass estimation. As an alternative, the agar film direct count in the Jones-Mollison technique is often used to estimate fungal and bacterial biomass in samples, but it is considered labourious and requires manual dexterity (Jones and Mollison, 1948). Note that these direct count or microscopy method are likely to overestimate fungal biomass due to the large part of inactive or dead fungal hypha (Bardgett et al., 1993; Six et al., 2005). Another wide used approach of microbial biomass estimation is the PLFA method. The PLFA method has an advantage that both living fungi and bacteria can be determined in the same sample using the same technique (Frostegård and Bååth, 1996). In general, PLFA analysis can provide a general fingerprint of the microbial community over direct microscopy or DNA methods due to the high precision between replicate samples (Drenovsky et al., 2004, 2010; Ramsey et al., 2006; Frostegård et al., 2011; Contosta et al., 2015). Most importantly, PLFA analysis may have the lowest Type II error rate of common methods used to detect changes in microbial community composition with experimental treatments (Ramsey et al., 2006). Therefore, PLFAs, as essential structural components of living cell membranes, have been applied to assess microbial biomass, community composition and physiological status in environmental samples (White et al., 1996). In contrast, our results demonstrate that the PLFA method is likely to underestimate fungal biomass and results in a F: B biomass ratio of < 1.0, in all the examined ecosystems. This may results from two reasons: 1) the two major components of fungal cell walls are the polymers of melanin and of chitin, whilst the main component of bacterial membranes is phospholipid (Adu and Oades, 1978) and 2) bacterial cells have a higher surface-to-volume ratio than fungal hyphae and contain more PLFAs per unit of biomass (de Vries et al., 2012a). Overall, F: B biomass ratios can either greater or less than 1.0 due to the method biases, which causes inconsistent understanding on the relative contributions of fungi and bacteria in ecosystem energy flow. However, it is more important to emphasize that the metabolised microbial biomass, instead of the standing microbial biomass, is the major components of the cumulative biomass during a given period. In other

Fig. 4. The comparisons among F: B PLFAs biomass, F: B channel biomass (details in Table 1), and F: B assimilation based on dataset from two previous studies. In this study, we calculated the F: B assimilation and presented all the three types of data in a (Fungi/(Fungi + Bacteria)) × 100%. The calculation of assimilation refer to Formula (4) based on channel biomass. The data above the bars were the mean values of the four treatments. (A): F: B data based on de Vries et al. (2012) and (B): F: B data based on Holtkamp et al. (2008).

studies focused on soil functions (Blagodatskaya and Kuzyakov, 2013). Therefore, F: B biomass ratio may not be a suitable indicator to justify whether an ecosystem is a bacterial- or fungal-based energy channel. An alternative approach is to take the microbial biomass turnover rate into account, which may illustrate the characteristics of microbial-based energy channel more clearly. 4.1.2. Methodology limitations In this study, we found that F: B ratios differ considerably when different methods were used. Based on the direct microscopy method the F: B biomass ratios were higher than 1.0 in most ecosystems except for farmland. Indeed, many earlier studies demonstrated a higher fungal biomass relative to bacteria based on this method (Ingham et al., 1986a, b; Schnurer et al., 1986; Sakamoto and Oba, 1994; Berg et al., 7

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words, the F: B biomass ratio that including different biomass turnover rates should be calculated to show the real roles of fungi and bacteria in ecological processes.

et al., 2000). Then, microbial assimilation would be affected due to changing of microbial production and respiration. Therefore, there are many uncertainties regarding microbial production and assimilation under the predation pressure of soil fauna.

4.2. Potential of the F: B assimilation ratio as an indicator of the ecosystem energy channel

4.3. Ecological implications of the expanded fungal and bacterial ratios

4.2.1. Production and assimilation: the full process of the ecosystem energy channel Microbial production was calculated by multiplying the microbial biomass by turnover rate (Carter and Suberkropp, 2004) and microbial assimilation was calculated by adding the production to respiration in the model of energy flow (Odum, 1957). In the present study, we summarised not only the ratios of F: B biomass (BF: BB) but also those of F: B production (PF: PB) and F: B assimilation (AF: AB). Our results revealed that the ratios of F/(F + B) production and assimilation were almost less than 50% in the studied terrestrial ecosystems. Moreover, bacterial and fungal energy channels have distinct functions, representing ‘fast’ and ‘slow’ cycles of nutrient availability, respectively (Coleman et al., 1983; Moore and Hunt, 1988). Fungi with a slower turnover rate usually immobilised more substances for their growth and encountered the scarcity of energy for predators in higher trophic levels (Scheu and Setälä, 2002). Generally, fungi are credited with a greater growth efficiency compared to bacteria (Holland and Coleman, 1987; Six et al., 2005; Lipson et al., 2009). In other words, the percentage of soil carbon mineralised by bacteria is far higher than that by fungi in the soil systems. Bacterial-derived carbon, originating from both above- and below-ground plant inputs, is very important for sustaining soil food webs and is channelled to higher trophic levels, including predators (Pollierer et al., 2012). Compared to bacteria, fungi have more recalcitrant cell walls and incorporate more carbon into biomass. More importantly, fungi can facilitate carbon stabilisation by enhancing soil aggregation (Thiet et al., 2006). Overall, the F: B production and assimilation ratio may represent the full process of the ecosystem energy channel with the following uncertainties.

The ratio of fungi to bacteria (F: B) is likely to be related to ecosystem processes such as decomposition, nutrient cycling, C-sequestration potential and ecosystem self-regulation. Microorganisms have developed different mechanisms for the uptake and assimilation of mineral and organic forms of N, enabling them to utilise a wide range of organic and mineral compounds (Merrick and Edwards, 1995; Marzluf, 1997; Geisseler et al., 2010). In general, bacterial-based ecosystems are characterised by having high-nutrient availability and low amounts of nutrient-rich organic matter, resulting in elevated biological activity, whereas fungal-based channels often occur in acidic soils of low-nutrient availability and high organic matter content (Bardgett and Wardle, 2010). In the soil ecosystem, bacterial decomposer pathways with high turnover rates prefer to decompose easily available substrates, whilst slower fungal decomposer pathways prefer to support the decomposition of more complex organic materials (Wardle et al., 2004; de Graaff et al., 2010; de Vries et al., 2012a). There are exceptions; for example, there were no significant differences between detrital food webs dominated by bacteria compared to those dominated by fungi (Rousk and Frey, 2015). Consequently, there is a need to revise our basic understanding of microbial communities and the processes that they regulate in soil. When determining the F: B ratio, as a significance indicator in ecosystem processes, one should keep in mind that 1) the F: B ratio cannot be used to determine the contribution of fungi compared to bacteria in the same ecosystem, but rather to determine the proportionate change in fungi or bacteria in different ecosystems; 2) when the F: B assimilation ratio is less than 1.0, it does not indicate that the contribution of fungi is less important than bacteria (normally, recalcitrant substances must be decomposed by fungi before they are utilised by bacteria in the given ecosystem, such as coniferous forests; therefore, both fungi and bacteria are indispensable and dependent on each other in the decomposition process); and 3) the F: B assimilation ratio may be more suitable as an indicator to determine the fungal or bacterial contribution to the ecosystem energy flow.

4.2.2. Uncertainties associated with microbial production and assimilation As mentioned above, microbial biomass is only a snapshot of some ecological processes involving microbial communities, such as nutrient cycling. However, it is difficult to estimate the biomass of all soil organisms due to the high biodiversity. The rates of material flow amongst organisms to generate additional patterns in the soil food web are constantly changing. For example, collembolan and oribatid mites feed selectively on certain soil fungi (Klironomos and Kendrick, 1996; Maraun et al., 1998) and therefore it is likely that the fungal community in the field is subject to selective feeding pressure by soil micro-arthropods. The selective feeding pressure may either reduce fungal biomass or stimulate the growth of the less preferred species due to reduced competition between fungal species (Fitter and Sanders, 1992; Bonkowski et al., 2000). Furthermore, it is difficult to relate laboratory findings to the field situation. Microbial production represents the accumulated biomass over a period of time. Excluding microbial respiration, production is the fixed energy or production of organic materials by microorganisms per unit of time (Oades, 1967). For example, daily fungal production is calculated by multiplying the fungal biomass by the growth rate (Carter and Suberkropp, 2004). The turnover rate and production of the microorganisms are not constant and the rate is influenced positively or negatively by animals in the soil food web. The turnover of soil organisms results not only from the natural death caused by unfavourable abiotic conditions but also from the predation by organisms at higher hierarchical levels. The roles of soil animals (e.g., nematodes, protozoa, mesofauna and earthworms) in decomposing organic matter operate via controlling the turnover and production of soil microorganisms through trophic interactions (Scheu and Parkinson, 1994; Bonkowski et al., 2000; Fu et al., 2000, 2005; Hedlund and Sjögren-Öhrn, 2000; Zhang

4.4. Concluding remarks and perspective In the present study, we proposed an improved calculation to determine the F: B ratio considering the catabolism and anabolism of the microbial community. Based on the microbial biomass, production and assimilation, we estimated the relative functional contribution of bacteria and fungi to energy flow within the soil food web. First, the F: B biomass ratios, as a snapshot, were either greater than 1.0 or less than 1.0 depending on the method(s) used. The microbial biomass at any given point of time only accounts for a small proportion of the accumulated biomass during any period. Second, based on the microbial biomass and turnover time, the fungal/(fungal + bacteria) production ratios were mostly less than 50% depending on the ecosystem. However, this ratio will still underestimate the contribution of bacteria to the total energy flux because the per unit biomass of bacteria usually respires out more CO2 than fungi (Six et al., 2005). Third, based on the microbial turnover time and respiration, the fungal/(fungal + bacteria) assimilation ratios were mostly less than 50% in the studied ecosystems. Hence, we propose that bacteria have a proportionately greater contribution over fungi to the energy flow in soil food webs. Our findings challenge the traditional view in the field of soil microbial ecology. Nevertheless, the lower F: B ratios do not indicate that fungi are less important than bacteria because they play different roles and are usually non-substitutable in ecosystems. 8

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Moreover, there are still many uncertainties and unknowns in the estimation of microbial assimilation. Both microbial turnover rates and respiration may change with soil faunal activities (i.e., trophic and nontrophic effects) and their environmental conditions. Fungi and bacteria respond differently to these factors, which may further reduce the estimation precision of F: B ratios. Given that bacteria and fungi may metabolize with contrast rates and respond to biotic and/or abiotic disturbances differently, there are enormous challenges in assessing the relative contribution of bacteria and fungi to ecosystem processes.

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Author contributions Xiaoli Wang, Weixin Zhang and Shenglei Fu designed the experiment. Xiaoli Wang, Yuanhu Shao, Jie Zhao and Lixia Zhou collected the studies on soil microbial communities and Fungi: Bacteria ratios. Xiaoli Wang and Weixin Zhang analysed the data and drew the figures. Xiaoming Zou condensed the theme of article and modified the English language. Xiaoli Wang and Shenglei Fu wrote the manuscript. Acknowledgements This study was financially supported by the National Science Foundation for Young Scientists of China (Grant No.31700454 and Grant No. 41501268), the Natural Science Foundation of Qinghai Province (Grant No.2018-ZJ-939Q), the Chinese Academy of Sciences (CAS)/State Administration of Foreign Experts Affairs (SAFEA) International Partnership Programme for Creative Research Teams, the National Natural Science Foundation of Major International (Regional) Joint Research Project (Grant No. 31210103920), the National Science Foundation of China (NSFC)-Guangdong Provincial Government Joint Project (U1131001), the South China Botanical Garden Programme for Foster Featured Institute for Young Scholars (Y561011001), the Knowledge Innovation Programme of Chinese Academy of Sciences (KSCX2-EW-Z-6), and the program of the Youth Innovation Promotion Association of Chinese Academy of Sciences (2015303). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.actao.2018.10.003. References Adu, J.K., Oades, J.M., 1978. Utilization of organic materials in soil aggregates by bacteria and fungi. Soil Biol. Biochem. 10, 117–122. https://doi.org/10.1016/00380717(78)90081-0. Anderson, J.P.E., Domsch, K.H., 1975. Measurement of bacterial and fungal contributions to respiration of selected agricultural and forest soils. Can. J. Microbiol. 21, 314–322. https://doi.org/10.1139/m75-045. Bååth, E., 2001. Estimation of fungal growth rates in soil using 14C-acetate incorporation into ergosterol. Soil Biol. Biochem. 33, 2011–2018. https://doi.org/10.1016/S00380717(01)00137-7. Babiuk, L.A., Paul, E.A., 1970. The use of fluorescein isothiocyanate in the determination of the bacterial biomass of grassland soil. Can. J. Microbiol. 16, 57. https://doi.org/ 10.1139/m70-011. Bailey, V.L., Smith, J.L., Bolton Jr., H., 2002. Fungal-to-bacteria ratios in soils investigated for enhanced C sequestration. Soil Biol. Biochem. 34, 997–1007. https:// doi.org/10.1016/S0038-0717(02)00033-0. Bapiri, A., Bååth, E., Rousk, J., 2010. Drying-rewetting cycles affect fungal and bacterial growth differently in an arable soil. Microb. Ecol. 60, 419–428. https://doi.org/10. 1007/s00248-010-9723-5. Bardgett, R.D., McAlister, E., 1999. The measurement of soil fungal: bacterial biomass ratios as an indicator of ecosystem self-regulation in temperate meadow grasslands. Biol. Fertil. Soils 29, 282–290. https://doi.org/10.1007/s003740050554. Bardgett, R.D., Wardle, D.A., 2010. Aboveground-belowground Linkages: Biotic Interactions, Ecosystem Processes, and Global Change. Oxford University Press, Oxford. Bardgett, R.D., Frankland, J.C., Whittaker, J.B., 1993. The effects of agricultural management on the soil biota of some upland grasslands. Agric. Ecosyst. Environ. 45, 25–45. https://doi.org/10.1016/0167-8809(93)90057-V. Bärlocher, F., Kendrick, B., 1974. Dynamics of the fungal population on leaves in a

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