Stoichiometry and homeostasis of terrestrial fungi obtained near Irvine, California

Stoichiometry and homeostasis of terrestrial fungi obtained near Irvine,

California

Nick Kelley

with Allison Moreno

Drs. Adam Martiny and Anthony Amend

Stoichiometry and homeostasis of terrestrial fungi 2

Abstract

The carbon, nitrogen, and phosphorus (C:N:P) ratios of 43 terrestrial fungal isolates

acquired near Irvine, California, were analyzed and compared to the Redfield ratio (106:16:1)

and global soil microbial biomass (60:7:1). To evaluate level of homeostasis or plasticity (non-

homeostasis), three of the isolates (Davidiella, Mucor flavus, Helotiales) were grown in malt-

yeast extract treatments with modified nutrient ratios. Carbon and nitrogen levels were measured

using combustion analysis. Soluble organic phosphorus was extracted using heated HCL and

analyzed with a molybdenum reagent indicator and spectrophotometer. The average C:N:P of the

42 fungal strains was 153:31:1. The three isolates grown in nutrient-modified media exhibited

weak homeostasis with respect to C:N, weak plasticity with respect to N:P, and strong plasticity

with respect to C:P. These results suggest that the local terrestrial fungi around Irvine are

controlled by environmental conditions, and their growth is both nitrogen- and phosphorus-

limited.

Introduction

The Redfield ratio has become canonical in the study of marine biogeochemistry. Alfred

Redfield found that marine primary producers were composed most notably of three elements:

carbon (C), nitrogen (N), and phosphorus (P); and that these elements formed common atomic

ratios. In plankton, Redfield observed the average ratio of these three elements to be 106 carbon

atoms and 16 nitrogen atoms for every 1 atom of phosphorus. This ratio was reflected in the

plankton's environment as well; the ocean water where the plankton lived also averaged carbon,

nitrogen, and phosphorus in a ratio of 106:16:1 (Redfield 1958).


Organisms commonly handle their internal nutrients in one of two ways. The ability of an

organism to maintain consistent internal nutrient ratios independent of its environment is known

as homeostasis. It is generally regarded that most heterotrophic organisms are homeostatic, while

most autotrophs are non-homeostatic, or plastic; that is, they can alter their internal ratios to

match their environment. The importance of the ability to maintain homeostasis or to be plastic is

the effect that such organisms have in the ecosystem, primarily how they affect nutrient cycling.

The metabolism requirements of each type of organism affect environmental nutrient content

both by what the organism eats while it is alive, and what it releases when it dies. Trophic level

also comes in to play as heterotrophs and autotrophs consume nutrients in different ways.

While the convention of homeostatic or non-homeostatic can be useful in determining the

role of an organism in its ecosystem, it does not necessarily hold true for all members of a certain

trophic type. Persson et al. 18 found that many of the autotrophs included in their analyses

exhibited strong homeostasis in regards to N:C and N:P, going against the convention of

autotrophs being plastic. Another study also found that in (primarily freshwater) phytoplankton,

N:P ratios remained consistent with that of the Redfield ratio (N:P of 16) despite wide ranges of

N:P supply ratios, revealing the phytoplankton to be more homeostatic 10.

In the oceans, nutrient supply ratios may vary from Redfield, going as high as 100:1 for

N:P in some areas, or holding an average of 37:1 while the local plankton retain Redfield's 16:1

6. These observations suggest that marine autotrophs may be more homeostatic than we think,

and that they have limits to how far their internal nutrient ratios can go but that their range is

wider than it is for heterotrophs.

While the Redfield ratio has helped us expand our understanding of marine

biogeochemical cycles and the significance that nutrient levels play in the ecosystem, the


terrestrial analog is not as well understood. Given the non-homogeneous nature of soil, it could

be assumed that nutrient ratios of soil and soil microbes may vary significantly from one location

to the next depending on soil type, vegetation, climate, etc. Nutrient ratios of soils and microbial

biomass compiled by Cleveland and Liptzin 4 resulted in a global terrestrial "Redfield ratio" of

186:13:1 for soil, and 60:7:1 for soil microbes. And nutrient ratios of aboveground foliage and

litter were found to be 1212:28:1 and 3007:45:1, respectively 14.

The extent to which soil microbes affect soil nutrients is not as well understood as the

roles of marine microbes, but undoubtedly these organisms do play significant roles in soil

nutrient cycling. However, one key difference between the terrestrial and marine realms is that

most terrestrial microbes are heterotrophs, while most marine microbes are autotrophs. So does

the environment dictate what kinds of organisms can live there, or do organisms shape their

environments by altering nutrient supplies to suit their needs? Our goals were to derive a

"Redfield ratio" for local terrestrial fungi, compare that to pre-established nutrient ratios,

specifically the actual Redfield ratio, and determine the level of homeostasis of the fungi. How

similar or dissimilar are the nutrient ratios of specific terrestrial microbes compared to marine

microbes? Being heterotrophs, fungi should be homeostatic, but how well does the convention

actually hold? And to what extent is the environment controlling the nutrient content of fungi?

Materials and Methods

We obtained 43 strains of terrestrial fungi (Table 1) that were collected from soils near

Irvine, California. The fungi were grown in liquid malt-yeast extract (MYE) medium (2.5 g malt

extract, 2.5g yeast extract, 500 mL water) on a shaker at room temperature for ten days. After ten

days, the cultures were stored in a refrigerator at 0 C to limit any further growth.


Ten percent dilutions of the fungal cultures were created using 1 mL taken from the

original cultures added to 9 mL of fresh MYE medium. We prepared 6 diluted replicates for each

fungal strain - 3 replicates to be used for carbon and nitrogen (C/N) analysis, and 3 replicates for

organic phosphorus (POP) analysis – resulting in a total of 252 replicates.

5 mL from each diluted replicate was then filtered using low vacuum filtration. The

filters used were 25 mm GF/F glass microfiber circle filters that had been precombusted in a

muffle furnace at 500 C for 5 hours. Each filter was rinsed with 0.2 M HCl and Milli-Q water

before filtering. The filters to be used for C/N analysis were also weighed before and after

filtering to find the filtered biomass. After filtration, the filters were placed in labeled 4-well

petri dishes and then stored in a freezer at -20 C. Before analyzing, the filters were dried in an

oven overnight at 60 C. Filters to be analyzed for C/N were weighed after drying to obtain total

filtered mass. They were then analyzed using a CHN analyzer.

The filters to be analyzed for organic phosphorus (POP) were transferred from the 4-well

petri dishes and placed in glass scintillation vials that had been washed with phosphorus-free

soap and soaked in an HCl bath, and precombusted in a muffle furnace at 450 C for 5 hours. Six

standards were also prepared for each set of fungal replicates using KH2PO4 in concentrations of

0, 10, 25, 50, 75, and 100 microliters. 2 mL of 0.017 M MgSO4 was added to each vial and then

capped with foil and placed in an oven to dry at 80-90 C. After drying, the vials were recapped

with precombusted foil and placed in a muffle furnace for 2.5 hours at 450 C. After cooling, 5

mL of 0.2 M HCl was added to each vial and the vials placed in an oven at 80-90 C for 30

minutes. After cooling again, the HCl was decanted into 50 mL tubes. The vials were rinsed with

5 mL of dH20, which was also decanted into the 50 mL tubes. An extra 20 mL of dH20 was

added to the 50 mL tubes for the first set of replicates (set A), and 30 mL of dH20 was added to


the second two sets of replicates (set B, set C). 1 mL of molybdenum blue reagent was added to

each tube and mixed. The molybdenum blue reagent consisted of 0.025 M ammonium molybdate

tetrahydrate, 5 N sulfuric acid, 0.0023 M potassium antimonyl tartrate solution, and 0.307 M

ascorbic acid, mixed in the ratio of 2:5:1:2.

After adding and mixing the molybdenum blue reagent to the samples, 11 mL of solution

was transferred to a glass centrifuge tube and centrifuged for 1 minute. Each centrifuge tube was

then placed in a rack covered, covered with foil and let to sit for 20 minutes after which time the

contents were analyzed using a UV-Vis spectrophotometer. The spectrophotometer was zeroed

using a 50/50 solution of 0.2 M HCl and dH20, and the samples were read using a wavelength of

885 nm.

For the homeostasis experiment, modified MYE treatments were prepared including

carbon-limited, nitrogen-limited, and phosphorus-limited treatments. All treatments were based

on a mixture of 0.15 g malt extract and 0.15 g yeast extract in 300 mL of dH20, which was also

used as the control treatment. The carbon-limited treatment included the addition of 0.544 g

KH2PO4 and 8.544 g NaNO3. The nitrogen-limited treatment included the addition of 0.068 g

KH2PO4 and 1.802 g dextrose. The phosphorus-limited treatment included the addition of 0.383

g NaNO3 and 0.643 g dextrose. All treatments were autoclaved before use.

From the full set of 43 fungal cultures, three strains representing two of the main fungal

phyla were selected to test homeostasis. The three strains chosen were the ascomycetes

Davidiella and Helotiales, and the zygomycete Mucor flavus. Three replicates of each treatment

were prepared for each strain resulting in 36 total replicates. Using sterile technique, glass test

tubes were filled with 24 mL of growth medium and inoculated with 0.5 mL of liquid fungal

culture. After inoculation, each tube was vortexed and set in a rack to incubate at room


temperature for up to 12 days. 1 mL samples were taken from each replicate 5 days after

inoculation and 12 days after inoculation. The samples were analyzed for C/N and POP

following the same protocol as for the nutrient ratio samples.

TABLE 1. Terrestrial fungal cultures obtained from soils near Irvine, CaliforniaAlternaria Sordariomycete Tetracladium PleosporalesHypocrea koningii Sordariomycetes Mucor flavus CryptococcusHypocreales Camarosporium brabeji Mucor racemosus Rhodotorula minutaRhodotorula Rhodotorula Lewia Rhodotorula minutaDavidiella Sordariomycetes Neofusicoccium RhodotorulaPleosporales Penicillium Dothideomycetes ExophialaExophiala Myrothecium roridum Helotiales PleurophomaCercophorum Cryptococcus Dothidea PhaeosphaeriaAlternaria Aureobasidium Cryptococcus HelotialesMyrothecium roridum Rhodotorula Phaeomoniella Epicoccum nigrumCryptococcus Cryptococcus Cryptococcus

Results

C:N:P ratios

Six replicates for each of the 43 fungal cultures were analyzed; three replicates for carbon

and nitrogen (C/N) analysis, and three replicates for organic phosphorus (POP) analysis.

Table 2 shows the mean and median molar amounts of carbon, nitrogen, and phosphorus

for the total set of fungal cultures. Mean and median nutrient ratios were also calculated (C:P,

N:P, C:N, C:N:P), along with standard deviations for molar carbon, nitrogen, and phosphorus.

The average C:N:P ratio for the entire set of fungal cultures was found to be 152:33:1. Figures

A1 – A3 show average C:P, N:P, and C:N values for each fungal strain compared to the

corresponding ratio of the Redfield ratio.

Homeostasis


During the homeostasis experiment, the treatments were sampled twice; 5 days after

inoculation and 12 days after inoculation. These time periods were chosen because they fit into

the experiment schedule while still allowing time to analyze the samples. Table B1 contains the

mean values of molar carbon, nitrogen, and phosphorus of the three cultures during the first

sampling point, and Table B2 shows values from the second sampling point. Nutrient ratios for

both the fungi and the growth medium of the various treatments were calculated.

Graphs of homeostasis were created by log-transforming the nutrient ratios of the fungi

and the nutrient ratios of the growth medium and plotting them against each other (Fig C1 – C3).

Nutrient ratios of the fungal cultures were taken as the average of the three replicates for each

treatment type at each sampling point. This resulted in two data sets per graph, one for each

sampling point. Linear trend lines were applied to the data sets and the slopes of the trend lines

were calculated. An average of the two slopes was used to find the level of homeostasis for that

specific culture and ratio type (e.g., C:P, N:P, C:N).

The slope of the linear trend lines was used to find the level of homeostasis, as the slope

is equivalent to the value 1/H, where H equals the homeostatic coefficient of the organism. The

calculated value was left as 1/H and categorized using a scale from 0 to 1, where 0 indicates the

organism is ‘strictly homeostatic’, while 1 indicates a strict level of non-homeostasis 18. This

range was further broken down to four categories to give more specific levels of homeostasis as

follows: 0 < 1/H < 0.25 = strictly homeostatic; 0.25 < 1/H < 0.5 = weakly homeostatic; 0.5 < 1/H

< 0.75 = weakly plastic; 0.75 < 1/H < 1 = strictly plastic.

With regards to C:P (Fig. C1), the following 1/H values were obtained: 1.100

(Davidiella), 0.898 (M. flavus), and 1.298 (Helotiales). For N:P (Fig. C2), the values were: 0.631


(Davidiella), 0.515 (M. flavus), and 0.655 (Helotiales). For C:N (Fig. C3), 1/H values were:

0.476 (Davidiella), 0.373 (M. flavus), and 0.450 (Helotiales).

TABLE 2. Statistical values of nutrients in experimental fungi that were obtained from soils near Irvine, California.

Carbon (micromoles

)Nitrogen

(micromoles)Phosphorus

(micromoles)C:P N:P C:N C:N:P

Mean 69.85 14.95 0.46 152 33 5 152:33:1Median 69.81 15.89 0.49 142 32 4 142:32:1

S.D. 29.52 7.49 0.46 43 11 1Minimum 22.47 2.98 0.19Maximum 144.98 35.79 0.81

Discussion

Total fungal C:N:P ratios

Differences can be seen when comparing the average nutrient ratio of the observed fungal

cultures to that of more established ratios. The overall C:N:P ratio for this experiment was

152:33:1, compared to 106:16:1 for marine phytoplankton (Redfield ratio), and 60:7:1 for global

soil microbial biomass. Comparing C:P and N:P among the three major ratios, it could be

inferred that the observed fungi have less phosphorus than marine phytoplankton and other soil

microbes due to the higher ratios of both carbon and nitrogen compared to phosphorus. Overall

N:P for the fungi is double that of the Redfield ratio (33:1 for fungi, 16:1 for Redfield) and

remains higher on average for most of the observed cultures (Fig. A1). Overall N:P is also almost

five times higher than soil microbial biomass, which had an N:P of 7:1. The C:N ratios however,

show the opposite, with the observed fungi having the lowest C:N of the three major ratios (5:1

for fungi, 7:1 for Redfield, 9:1 for soil microbes) (Fig. A3). The lower C:N for the observed


fungi may indicate that they have a higher nitrogen demand than other soil microbes and marine

phytoplankton, while the higher N:P may indicate a phosphorus limitation in their environment.

It is interesting to note that the observed fungi have much higher nutrient ratios than the

global soil microbial biomass (with the exception of C:N). Fungi were assumed to be a part of

the data set assembled by Cleveland and Liptzin 4, though fungi were not separated from bacteria

as the data were collected through literature review, focusing on chloroform fumigation and

extraction (FE) technique. The FE technique analyzes soil samples for C, N, and P and does not

discriminate for individual organisms (and may also extract non-microbial biomass as well).

There are several possible explanations for the seemingly large difference in these

nutrient ratios. Fungal biomass in the data collected for the soil microbial biomass study may not

have been large enough compared to microbial biomass to raise the average ratio closer to that

obtained in this experiment. Bacteria are also predominantly unicellular while fungi are

multicellular. The differences in cellular structure and function could also attribute to differences

in organismal nutrient ratios, as well as how the organisms obtain and metabolize nutrients.

Another significant difference is that fungal nutrient ratios obtained in this experiment are

representative only of the area from which they were collected, as well as the time of year. In

contrast, the soil microbial biomass study performed by Cleveland and Liptzin 4 used data from a

multitude of locations, climates, soil, and vegetation types. This larger sampling would probably

be closer to the Redfield ratio in that it is a global average representing a variety of locations and

organisms, overshadowing the fewer locations that might have significantly different nutrient

ratios.

Fungal homeostasis


The results of this homeostasis experiment indicate that the three observed fungal

cultures (Davidiella, M. flavus, Helotiales) appear to be more plastic (non-homeostatic) than

homeostatic (Fig. C1 – C3). The average 1/H values of the cultures were in the range of being

strictly plastic for C:P (1.100, 0.898, 1.298 for Davidiella, M. flavus, and Helotiales,

respectively); weakly plastic for N:P (0.631, 0.515, and 0.655); and weakly homeostatic for C:N

(0.476, 0.373, and 0.450). If these values carry over to the other fungal cultures, then these fungi

appear to be more plastic than homeostatic, contradicting the previous assumption that all

heterotrophs are homeostatic. However, there has not been significant study into the homeostasis

of heterotrophs, so whether or not the majority of heterotrophs are indeed homeostatic has yet to

be determined.

There is the concept of homeostatic gradient, where some organisms have been found to

be strictly one way or the other, but other organisms have been found to be more in the middle,

not being entirely homeostatic or non-homeostatic 8. There is also the possibility that

homeostasis levels may be reflective of certain growth conditions. Some species of

phytoplankton reveal differing internal nutrient ratios from their environment, giving way to

generalized growth strategies, such as the “survivalist” which can sustain growth even during

periods of low environmental resources 2. This concept could carry over to fungi; perhaps fungi

grow differently during early growth periods than compared to more established fungi with

significant biomass. Larger fungal masses may level off their internal nutrient ratios and become

more homeostatic. There is also the issue of how an organism grown in a liquid medium

compares to how it would grow in soil, as the liquid is more dynamic and gives greater access to

nutrients than soil.


Interestingly, the fungal C:N:P ratios of the P-limited treatments 5 days after inoculation

closely resemble the Redfield ratio, (Table B1). The C:NP ratios of Davidiella, M. flavus, and

Helotiales were 129:19:1, 116:14:1, and 117:26:1, respectively. However, these ratios showed

some change during the second sampling point - 128:22:1, 80:12:1, 182:36:1 (Table B2). This

may support the previous suggestion that fungi exhibit differing characteristics throughout

growth. Figures C1 – C3 show the trends of the two sampling points for each of the three fungal

cultures. Most of the data sets remain relatively equal or proportional, supporting a steady level

of homeostasis through the two sampling points. However, deviations may be seen in a few of

the graphs, particularly in C:P for Helotiales, and in C:N for Davidiella and M. flavus. These

deviations may be insignificant, indicating a standard variation in the internal nutrients of a

growing organism. Or they may be indicative of a larger trend leading towards a change in

homeostasis at different growth periods.

Future directions

Nutrient cycling in terrestrial soil microbes, specifically fungi, is not as well understood

as it is in marine phytoplankton. Further research should be performed to better understand

possible correlations between marine and terrestrial chemical cycling, as well as the role and

significance of microorganisms in biogeochemical cycles. Knowledge in terrestrial

biogeochemical cycling maintains importance in many areas such as environmental and

ecological health, and applications in agriculture and bioremediation. The homeostasis of

terrestrial microorganisms also plays an important role in furthering our understanding

biogeochemical cycles. Knowledge of how these microorganisms grow and metabolize can aid

in mapping evolutionary lineages between terrestrial and marine microbes, even giving light to


the chemical composition of prehistoric environments. On a larger scale, this information can

lead to an increased ability and accuracy of modeling past climates. Microorganisms already play

a large role in shaping our environment and our lives. More clearly understanding how exactly

they behave will allow us to further our applications and appreciation for these crucial

organisms.


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Figure A1. Average carbon to phosphorus (C:P) value of each fungal culture. Average values

were obtained during the nutrient ratio experiment. These values were used to calculate the

average C:P for the entire set of fungal cultures that were obtained from soils near Irvine,

California. Values are unitless ratios and are the average of three replicates for each culture.

Standard deviations are shown as I-bars. The black horizontal line represents the C:P value of the

Redfield ratio.


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Figure A2. Average carbon to phosphorus (N:P) value of each fungal culture. Average values


average N:P for the entire set of fungal cultures that were obtained from soils near Irvine,


Standard deviations are shown as I-bars. The black horizontal line represents the N:P value of the

Redfield ratio.


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Fungi C:N

Figure A3. Average carbon to phosphorus (C:N) value of each fungal culture. Average values


average C:N for the entire set of fungal cultures that were obtained from soils near Irvine,


Standard deviations are shown as I-bars. The black horizontal line represents the C:N value of

the Redfield ratio.


Appendix B – Nutrient values of homeostasis experiment

TABLE B1. Nutrient values of homeostasis experiment 5 days after inoculating modified growth medium treatments with fungal cultures obtained from soils near Irvine, California.

Strain TreatmentCarbon (μmol)

Nitrogen (μmol)

Phosphorus (μmol)

C:P N:P C:N C:N:P

Davidiella Control 7.98 2.03 0.16 51.34 13.03 3.94 51:13:1C-Limited 4.38 19.64 1.24 3.54 15.86 0.22 4:16:1N-Limited 15.54 1.42 0.37 42.46 3.87 10.96 42:4:1P-Limited 8.64 1.24 0.07 129.33 18.60 6.95 129:19:1

M. flavus Control 7.21 1.43 0.13 53.46 10.61 5.04 53:11:1C-Limited 4.06 21.39 1.20 3.38 17.81 0.19 3:18:1N-Limited 15.69 1.32 0.38 41.57 3.51 11.85 42:4:1P-Limited 8.30 1.00 0.07 116.04 13.94 8.33 116:14:1

Helotiales Control 6.26 1.43 0.14 44.22 10.10 4.38 44:10:01C-Limited 3.95 20.39 1.12 3.51 18.16 0.19 4:18:01N-Limited 16.48 1.69 0.39 41.77 4.29 9.74 42:04:01P-Limited 8.51 1.87 0.07 116.57 25.65 4.54 117:26:01

Growth Control 119.56 25.13 4.76 120:25:1Medium C-Limited 3.10 25.13 0.12 3:25:1

N-Limited 119.56 4.40 27.17 120:4:1P-Limited 320.53 67.37 4.76 321:67:1


TABLE B2. Nutrient values of homeostasis experiment 12 days after inoculating modified growth medium treatments with fungal cultures obtained from soils near Irvine, California.

Strain TreatmentCarbon (μmol)

Nitrogen (μmol)

Phosphorus (μmol)

C:P N:P C:N C:N:P

Davidiella Control 8.54 2.38 0.21 40.95 11.41 3.59 41:11:1C-Limited 3.48 21.90 1.09 3.19 20.07 0.16 3:20:1N-Limited 16.42 1.49 0.41 40.12 3.65 11.00 40:4:1P-Limited 7.00 1.23 0.05 127.61 22.37 5.70 128:22:1

M. flavus Control 8.45 1.90 0.20 41.34 9.29 4.45 41:9:1C-Limited 3.01 18.59 1.13 2.67 16.49 0.16 3:16:1N-Limited 17.34 1.59 0.44 39.24 3.59 10.92 39:4:1P-Limited 7.73 1.11 0.10 80.20 11.57 6.93 80:12:1

Helotiales Control 8.19 2.15 0.27 30.06 7.90 3.80 30:8:1C-Limited 2.25 13.36 1.19 1.90 11.26 0.17 2:11:1N-Limited 17.79 1.79 0.38 47.05 4.73 9.94 47:5:1P-Limited 8.27 1.62 0.05 182.27 35.67 5.11 182:36:1

Growth Control 119.56 25.13 4.76 120:25:1Medium C-Limited 3.10 25.13 0.12 3:25:1

N-Limited 119.56 4.40 27.17 120:4:1P-Limited 320.53 67.37 4.76 321:67:1


Appendix C - Fungi nutrient ratios versus growth medium

FIGURE C1. Homeostasis relative to C:P of fungi obtained from soils near Irvine, California.

FIGURE C1. Fungal C:P ratios versus growth medium C:P ratios for the fungal cultures

Davidiella, M. flavus, and Helotiales. Equations and R2 values of trend lines are shown, as is the

1:1 line (diagonal solid line) representing a perfect 1 to 1 ratio.


FIGURE C2. Homeostasis relative to N:P of fungi obtained from soils near Irvine, California.

FIGURE C2. Fungal N:P ratios versus growth medium N:P ratios for the fungal cultures




FIGURE C3. Homeostasis relative to C:N of fungi obtained from soils near Irvine, California.

FIGURE C3. Fungal C:N ratios versus growth medium C:N ratios for the fungal cultures



Stoichiometry and homeostasis of terrestrial fungi obtained near Irvine, California

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